The DAP Pathway to Lysine as a Target for Antimicrobial Agents Russell J. Cox School of Chemistry University of Bristol Cantock's Close Bristol BS8 1TS UK Reviewing the literature published up to September 1995 1 Introduction The Diaminopimelate Pathway to L-Lysine 1.1 Enzymes of the Diaminopimelate Pathway 2.0 L-Dihydrodipicolinate Synthase EC 4.2.1.52 dapA 2.1 L-Dihydrodipicolinate Reductase EC 1.3.1.26 dapB 2.2 Succinyl CoA L-Tetrahydrodipicolinate N-Succinyl- transferase dapD 2.3 N-Succinyl-LL-DAP Aminotransferase EC 2.6.1.17 dapC 2.4 N-Succinyl-LL-DAP Desuccinylase EC 3.5.1.18 dapE 2.5 meso-DAP Epimerase EC 5.1.1.7 dapF 2.6 meso-DAP Dehydrogenase EC 1.4.1.16 ddh 2.7 meso-DAP Decarboxylase EC 4.1.1.20 lysA 3 Antibiotic Properties of DAP Pathway Inhibitors 4 Overview 5 References 1 Introduction The Diaminopimelate Pathway to L-Lysine The enzymes which catalyse the synthesis of L-lysine in plants and bacteria have attracted'interest from two directions; firstly from those interested in inhibiting lysine biosynthesis as a (1a) R' strategy for the development of novel antibiotic or herbicidal n compounds (vide infra);' and secondly from those wishing to enhance lysine yields in over-producing organisms.2-4 Mammals are deficient in lysine biosynthesis and thus require a dietary source of this amino acid for protein ~ynthesis.~ In plants and bacteria lysine is synthesized de novo and is utilized for protein biosynthesis.Crucially however most bacteria also require either lysine or its biosynthetic precursor diaminopimelate (DAP),6 as a component of the peptidoglycan (la) layer of the cell wall. This polymeric compound consists of chains of alternating N-acetylglucosamine and N-acetylmuramic acid cross linked by short pep tide^.^ For almost all bacteria the cross links are formed by diamines; either lysine or DAP.8-10 The cross links give the cell wall the strength to resist lysis caused by high intracellular osmotic pressures.ll Inhibitors of lysine or DAP biosynthesis could therefore be very effective antibiotics like other successful drugs targeted towards cell wall bio- synthesis such as p-lactams and g1ycopeptides.l. l2 The absence of the DAP pathway in mammals offers the potential for the development of novel antibiotics with low mammalian toxici- ties.Peptidoglycan monomers such as the potent cytotoxin (1 b) from Bordetella pertussis and Neisseria g~norrhoeae,'~ and related DAP-containing peptides possess a range of biological effects including cytoto~icity,~~ antitumour activities15 and angio tensin converting enzyme (ACE) inhibition. l6 The investigations into lysine biosynthesis have classically focused on the enzymes themselves their properties mech- anisms and modes of reg~1ation.l~ More recently much more information has come from genetic studies. This has allowed (7a,b) comparisons of protein sequences from diverse organisms and has ultimately resulted in cloning of the biosynthetic genes and their over-expression.The availability of lysine biosynthetic proteins in substantial quantities means that the enzymes are becoming amenable to more widespread study. It is the intention of this review to highlight opportunities and stimulate AcHN O4 HNY O*NH &02H A' = D-ala-mDAP-oglu-L-ala-(MurNAc-GlcNAc),; R2= D-ala; = polymer (1b) R' = H; R2 = OH; n = monomer efforts for the further study of enzyme mechanism and inhibition in this area. The later steps of peptidoglycan biosynthesis have received attention e1sewhere.l' l8 1.1 Enzymes of the Diaminopimelate Pathway The early steps of the pathway (reduction of L-aspartate to L-aspartate semialdehyde) are common with methionine and threonine biosynthesis and will not be dealt with here." The first step unique to lysine biosynthesis (Scheme 1) is the condensation of pyruvate (2) with L-aspartate semialdehyde (L- ASA) (3) to form ~-2,3-dihydrodipicolinate(L-DHDP) (4) catalysed by L-DHDP synthase.20 The heterocyclic product is reduced by L-DHDP reductase to form ~-2,3,4,5-tetra-hydrodipicolinate (L-THDP) (5).At this point the pathway splits and two main routes have been identified in different bacterial species.21 The longer of the two routes proceeds via acylation of L-THDP (3,yielding acyl-blocked cc-amino-e- ketopimelate (6a,b). In many bacterial species including Escherichia coli the blocking group is succinate while in many species of Bacillus acetate is utilized.8 The e-keto group thus freed is subject to transamination by a pyridoxal phosphate (PLP) dependent aminotransferase utilizing glutamate as the amino-donor.2z Removal23 of the blocking acyl-group from then affords LL-DAP (8a) which is subsequently epimerized to meso-DAP (8b).24 In the shorter pathway rneso-DAP (8b) is produced directly from L-THDP (5) by meso-DAP dehydrogenase; this enzyme is less widespread but is found in Bacillus sphaericus for example.Dual pathways are present in some bacterial strains ;for example both the succinyl-blocked and dehydrogenase pathways are presentz5 and operative26 in 29 NATURAL PRODUCT REPORTS 1996 mco2H-H02C'0 ;3CO2H NH2 NH2 H2NOICO*H H02CQCO2" (2)pyruvate Ii (3) L-ASA (9) L-Lysine viii (3) L-ASA (4) L-DHDP 02pH 7.41 t H02C 0C02H H02CQC02H HOzCY-+7C02H (104 NH2 NH2 (4) L-DHDP (8b) meso.DAP NAD(P)H I J,ii vii vi n H02C' (5) L-THDP (8a) LL-DAP RSCoA t ROH V CoASH 1iii 1 L-glU a-kg JLL.H02GWCO2H Ho2C~Cu2H iv 0 NHR NH2 NHR (6a) R = COCH2CH2C02H (7a) R = COCH2CH2C02H (6b) R=COMe (7b)R=COMe The DAP pathway to L-lysine. Enzymes and corresponding genetic loci i Dihydrodipicolinate Synthase EC 4.2.1.52 dupA ;ii Dihydro- dipicolinate Reductase EC 1.3.1.26 dupB; iii Acyl CoA Tetra-hydrodipicolinate N-Acyltransferase dapD ; iv N-Ac~I-LL-DAP Aminotransferase EC 2.6.1.17 dupC; v N-ACYLLL-DAP Deacylase EC 3.5.1.18 dupE; vi meso-DAP Epimerase EC 5.1.1.7 dupF; vii meso-DAP Dehydrogenase EC 1.4.1.16 ddh; viii meso-DAP De- carboxylase EC 4.1.1.20 lysA Scheme 1 the industrially important lysine producer Corynebacterium glutamicum.In the last step of the pathway L-lysine (9) is synthesized directly from meso-DAP (8b) by decarboxylation catalysed by the PLP dependent meso-DAP decarb~xylase.~' The situation in plants is less clear. Both L-DHDP synthase and L-DHDP reductase have been obtained from plant extracts as have meso-DAP epimerase and meso-DAP decarboxylase. The presence of enzymes from the middle section of the pathway such as meso-DAP dehydrogenase,28 L-THDP acyl- transferase or LL-N-acyl-DAP deacylase is more contro-~ersial.~~ This has led to the suggestion that direct trans-amination of L-THDP (5) may be occurring to afford LL-DAP @a) though definitive proof is lacking.30 Evidence suggests that lysine biosynthesis in plants may occur largely or entirely within ~hloroplasts.~~ This is in accord with current evidence supporting a bacterial origin for these organelles.32 2 L-Dihydrodipicolinate Synthase EC 4.2.1.52 dapA L-Dihydrodipicolinate (L-DHDP) synthases have been isolated and purified to homogeneity from many source^,^^-^^ and the dapA locus has been cloned from both plants36 and bacteria.".38 The enzyme is a homotetramer in the majority of cases with a subunit M of between 28000 and 35000. The pea (Pisurn sativum) enzyme is reported to be a homotrimer of subunit M Scheme 2 43OO0.39Sequence analysis has revealed 30 YOidentity between the wheat (Triticum aestivum) and E. coli enzymes,*O and 33 O/O identity between E.coli and Brevibacterium lactofermenturn enzyme^.^' Significant homology exists with another pyruvate binding enzyme the B. lactofermenturn enzyme shares 29 Yo identity with E. coli N-acetylneuraminate lyase.*l Three assays have been reported for L-DHDP ~ynthase.~~ Observation of the characteristic absorbance of an adduct between the reaction product and o-aminobenzaldehyde at 540 nm has been used as has a coupled assay utilizing the NAD(P)H dependent L-DHDP reductase (Section 2.1). The most widely used assay relies on rapid spontaneous oxidation at pH 7.4 of the product L-DHUP (4) to dipicolinate (IOa) which can be detected spectrophotometrically at 270 nm (Scheme 2). This procedure is suitable for detailed kinetic analysis because the synthesis of highly pure substrate L-ASA (3) has been achieved,*3 and the product of the reaction unambiguously shown to be dipicolinate (10a).44 Michaelis constants for many of the enzymes have been determined; they range between 0.4 and 3.1 mM for L-ASA (3) and between 0.5 and 11.8 mM for pyruvate (2).Kinetic analysis of the reactions catalysed by the E. ~oli,'~ wheat4s and maize (Zea mays)*6 enzymes suggests that pyruvate (2) binds to the enzyme first with the loss of water followed by binding and reaction of L-ASA (3). The active site of the enzyme has been probed in several ways. Early studies showed that pyruvate (2) forms a reducible imine (NaBH is inhibitory) with the €-amino group of a lysine residue and that for the E.coZi tetramer one equivalent of pyruvate (2) binds per subunit.20 Imine formation with pyruvate (2) has recently been observed directly by electrospray mass spectrometry.14 The enzyme catalyses the reversible exchange of tritium between p-3H pyruvate and water indicative of the formation of an enamine at the active site. Lysine-161 of the E. coli enzyme has been identified as the active site residue through sequencing of tryptic digests of reduced imino protein and in fact this is the only conserved lysine in all known L-DHDP synthase sequence^.^' On the basis of these observations the reaction mechanism has been formulated as in Scheme 3. The E. coli pzyme has been crystallized and a crystal structure at 2.5 A resolution obtained.48 The actiye site lysine is accessible to solvent lying at the bottom of a 10 A deep by 30 A long crevice.Substrates could approach lys-161 through an entrance formed by adjacent aspartate residues Asp-1 87 and Asp-188. These two residues are also conserved between the known L-DHDP synthases ; this may indicate a mechanistic role for these residues possibly in general acid/base catalysis. Numerous studies of substrate analogues and inhibitors have been carried out. The enzyme displays high specificity for both substrates. For the E. coli enzyme analogues of pyruvate (2) such as phosphoenolpyruvate (PEP) phenylpyruvate a-ketobutyrate a-ketoglutarate oxaloacetate and fluoropyru- vate are not recognized. L-ASA (3) analogues such as L-glutamate semialdehyde N-acetyl-L-ASA and succinate semi- aldehyde are also not substrates.D-ASA is not a substrate for NATURAL PRODUCT REPORTS 1996R. J. COX 5 H02C'0 NH;! H02C'NH 5 (2) Enz Enz lr-L-ASA H02C 1 Enz - Enz OH H20 H02C 0C02H (4) L-DHDP Enz Scheme 3 the wheat enzyme. None of these compounds shows any inhibitory activity although fluoropyruvate weakly inhibits the wheat enzyme only (ICso > 10 mM).49 Bromopyruvate alkyl- ates the enzymes and causes inhibition (Ki= 1.6 mM for E. coli 1.8 mM for wheat).49 Alkylation may be at or near the active site for the wheat enzyme since the presence of excess pyruvate (2) affords protecti~n.~~ Alkylation is not at the active site of the E. coli enzyme; studies of inhibition by bromo- pyruvate using electrospray mass spectrometry have shown that each protein subunit is monoalkylated after incubation with four equivalents of this compound.Peaks corresponding to imine formation between lys- 16 1 and bromopyruvate were not detected. Furthermore monoalkylated enzyme subunits retained 72% of their activity; significant loss of activity occurred only on p~lyalkylation.~~ lnvestigations with the E. coli enzyme have revealed that below pH 8 acetopyruvate (1 1) is an effective slow-binding inhibitor (K = 5pM).50 A drop in potency of inhibition at higher pH values however indicates that the protonated form of (1 1) is the active species. Clearly acetopyruvate (1 l) when bound at the enzyme active site could mimic one of the reaction intermediates since it contains structural elements of each substrate.For the plant and some bacterial enzymes the final product of the DAP pathway lysine (9) is regulatory. For the E. coli and wheat enzymes lysine (9) is a non-competitive inhibitor with respect to pyruvate (2) but it inhibits competitively with respect to L,-ASA (3) suggesting that the lysine and L-ASA (3) binding sites overlap. In E. coli and Bacillus sphaericus5' inhibition by lysine (9) is weak (ICso E. coli = 1 mM; K B. sphaericus = 0.6 M) and has not been observed in other bacterial enzymes.s2 The plant enzymes however are charac- terized by potent allosteric inhibition by lysine (ICs, values wheat germ 11 pM ;53 tobacco (Nicotiana sylvestris) 15 pM;54 spinach (Spinacea oleracea) 20 ,lcM;j5 maize 23 pM;46 wheat 5 1 pM4').Lysine analogues show similar patterns of inhibition but are somewhat less effective; for wheat threo-P-hydroxy-L- lysine (THL) (12) is inhibitory (ICso = 141 pM) as is (2-aminoethy1)-L-cysteine (AEC) (13) (ICso = 288 PM).~~ Other plant L-DHDP synthases show similar patterns of behaviour e.g. AEC inhibits the tobacco synthase (ICso = 120PM),~~ and the spinach homologue (ICso= 400 PM).'~ The pea enzyme is inhibited by ~-a-(2-aminoethoxyvinyl)glycine (AVG) (14) (ICso = 155pM). Interestingly activators that are analogues of DAP (though not DAP itself) have also been discovered for the pea enzyme. The phosphono-DAP analogue (15) gave 13 YO (1 1) (12) THL (13) AEC (14) AVG (15) rate enhancement at 1.2 mM while P-hydroxy-DAP isomer (57b) gave 38 % rate enhancement at 1.2 mM.39 Heterologous expression of the unregulated L-DHDP synthases from Coryne-bacterium glutamicum in canola and soybean5' and from E.coli in tobacco chloroplast^^^ leads to increased levels of L-lysine synthesis in these plants clearly indicating how feedback control of the native plant enzymes limits overall lysine production. The observation that the E. coli enzyme is inhibited by dipicolinate (10a) (ICso = 1.2 mM)47 has encouraged a sys-tematic investigation of numerous heterocycles as potential inhibitor^.^^ Substituted pyridines and piperidines (Figure 1) were found to be moderate inhibitors (ICsovalues < 1 mM). The best inhibitors are compounds such as the ditetrazole (16) diimidate (17) the dinitrile (18) and the N-oxide (19).Compounds (1 8) and (19) inhibit noncompetitively against either substrate. Esters inhibit more strongly than their acid congeners and a planar disposition of the substituents is preferred. These relationships are clearly shown for compounds (10a,b) and (20a-d). L-DHDP synthases isolated from Bacillus spp. show some- what different properties towards inhibitors. Dipicolinate (10a) is a constituent of spores from these species and is synthesized from L-DHDP (4) during sp~rulation.'~ Dipicolinate (10a) is not an inhibitor of these enzymes although a-,€-diketopimelate (2 1) is a good competitive inhibitor [Ki = 220 pM versus L-ASA (3) and Ki = 156 pM against pyruvate (2)].35 N MeOAOMe I 'N N.N NH NH (16) ICm= 0.25 mM (17) IC = 0.2 mM NCOCN 0-(18) (19) ICW= 0.3 mM ICgj = 0.2 mM K = 1.25 mM vs L-ASA(3) & = 0.29 mM vs L-ASA(3) K,= 0.35 mM vs pyruvate (2) 4 = 0.06 mM yspyruvate (2) RO2C 0C02R (loa) R = H; ICw = 1.2 mM (lob) R = Me; ICs0= 0.7 mM RO,C*"+ 0C02R R02C QC02R H H (20a) R = H; no inhibition (2Oc) R = H; no inhibition (20b) R = Me; ICW= 5.2 mM pod) R = Me; ICs0 = 0.7 mM Figure 1 Heterocyclic inhibitors of L-DHDP synthase H* L-DHDP reductase Scheme 4 2.1 L-Dihydrodipicolinate Reductase EC 1.3.1.26 dapB L-DHDP reductases have been purified from E.~oli,~~ maize60 and Bacillus ~pp.,~' and activity detected in Chlamydomonas corn soybean and tobacco.29 All of the enzymes studied so far catalyse the irreversible reduction of L-DHDP (4) utilizing either NADPH or NADH as an electron source.Two assays have been described utilizing synthetic L-DHDP (4) (this material may be generated chemically or enzymatically and is reported to be stable if stored at pH > 10).59.60For crude samples and as an aid to screening restoration of DAP biosynthesis to a lysate of an E. coli dapB mutant (deficient in L-DHDP reductase) has been observed. For quantitative activity measurements oxidation of NADPH may be mon- itored at 340 nm (Scheme 4). The E. coli enzyme is a homotetramer of approximate M 110000 to 120000. The E. coli dapB gene has been sequenced by two groups the predicted subunit being a polypeptide of 273 amino acids.62 Nucleotide sequencing of the chromosomal gene predicted a M of 28 798.62 The gene has been cloned directly by PCR and the product overexpressed in E.coV3 The PCR product sequence predicts M 28 757 which matched the M of the cloned and overexpressed gene product of 28 758 & 8 measured by electrospray mass spe~trometry.~~ The maize enzyme which resembles the E. coli enzyme in its behaviour towards substrates and inhibitors is smaller having a M of approximately 80 OO0.60 The availability of overexpressed protein in E. coli has facilitated mechanistic and crystallographic sudie~.~~ The cofactor donates its 4-p0-R hydride to the substrate at its y-position. Michaelis constants have been determined for the substrate L-DHDP (4) (K = 50 f12 pM) and the cofactor NADPH (K = 8.0f2.5 pM).Kinetic analyses suggest that the enzyme binds the cofactor and the substrate sequentially before reaction and release of product and oxidized cofactor. Dipicolinate (10a) is a linear competitive inhibitor with respect to the substrate (Ki = 26 & 6 pM) but inhibits uncompetitively against the cofactor (Ki = 330 & 50 pM). Binding by (10a) at the L-DHDP binding site indicates that the substrate (4) is probably bound in its cyclic state. Product inhibition by NADP' was also observed being competitive with respect to NADPH (Ki = 190& 35 pM) and noncompetitive versus sub- strate (Ki = 700f200 pM). Much weaker inhibition has been observed for other substrate analogues such as iso-phthalic acid (22) (ICs0-2 mM) and compounds with only one carboxylate such as pipecolic (23) and picolinic (24) acids are very poor inhibitors (IC5,,> 20 mM).Piperidine dicarboxylic acids (20a,c) are not inhibitory. The E. coli enzyme is unusual in that either NADPH or NADH are accepted as cofactor. In early work it was noted that with NADH as cofactor the Vm, was approximately half of the value observed when NADPH was Values for V/K7however show that NADH is the better substrate by a factor of two because of its four-fold lower Km.63The conclusions from the kinetic studiesoare supported by the X-ray crystal structure obtained at 2.2 A resolution. The subunit consists of a cofactor binding domain and a substrate binding domain.NADPH is bound in the crystal and the proposed binding site of the substrate would juxtapose the substrate and cofactor in the correct orientation for reaction.64 The maize enzyme is similar to the E. coli enzyme in its NATURAL PRODUCT REPORTS 1996 0 Ho2c-Co2H 0 H02C C02H H 0C02H C02H -N N-response to inhibitors and its kinetic characteristics. This situation contrasts with enzymes isolated from Bacillus spp. where dipicolinate (IOa) is an important constituent of spores and is produced by oxidation of L-DHDP (4). The Bacillus reductases are large (approximately 150000 Da) and differ in their response to dipicolinate (10a).61 This compound is a good inhibitor (Ki = 85 pM for B. cereus) but it inhibits non-competitively versus L-DHDP (4) suggesting a regulatory role.Kinetic analyses and the observation of product inhibition at high NAD(P)+ concentrations indicate a sequential ordered mechanism analogous to that determined for the E. coli enzyme. A third class of L-DHDP reductase has been isolated from sporulating B. s~bti1i.s.~~ This enzyme is a homotetramer of subunit M -18500 which again may use either NADPH or NADH. It differs markedly from the other L-DHDP reductases in containing flavin mononucleotide (FMN). Dipicolinate (1Oa) is again inhibitory (IC50 -0.4mM); it inhibits non/ uncompetitively with respect to L-DHDP (4) but competitively against NAD(P)H.66 Mechanistic studies have revealed that a ternary complex is not formed; rather NAD(P)H binds first and reduces FMN before the loss of NAD(P)+ followed by the binding and reduction of L-DHDP (4).Inhibition by di-picolinate (10a) and o-phenanthroline (25) (1C5, -70 pM) is positively cooperative. The enzyme reduces various synthetic dyes and therefore may not be a dedicated L-DHDP reductase. 2.2 Succinyl CoA :L-TetrahydrodipicolinateN-Succinyl-transferase dapD The succinyltransferase has been purified to homogeneity from E. ~oli.~~ While chemical syntheses of the substrate L-THDP (5) have recently been initial investigations of this enzyme had to await the development of a reliable enzymatic synthesis utilizing meso-DAP dehydrogenase (Section 2.6). Existence of the acetyl transferase variant in Bacillus spp. has been inferred from the observation of N-acetyl-LL-DAP (7b) accumulation in a B.megaterium mutant lacking DAP-deacylase activity.69 The standard assay utilizes radio-labelled L-THDP (5) and an in situ generation of succinyl CoA. Aliquots of the reaction mixture are acidified to pH 2 and the radioactivity determined of fractions eluted from a cation exchange column. The eluted fraction consists of the product N-succinyl-a-amino-e-ketopimelate (6a); protonated L-THDP (5) remains bound to the resin. The reaction may be more conveniently followed by observation of the release of free thiol from succinyl CoA utilizing Ellman's reagent 5,5'-dithiobis (2-nitrobenzoic acid) monitored at 21 5 nm.70 The E. coli enzyme is a homodimer of subunit M 31 000 determined by gel electrophoresis; this is in agreement with the value of 30040 calculated from the dapD nucleotide sequence.71 The apparent K, for L-THDP (5) is 22 pM for succinyl CoA 15 pM with k,, 43.3 s-l.The reaction is reversible under standard conditions the initial forward reaction proceeds some 380 fold faster than the reverse. The acyclic compound L-a-aminopimelate (L-cz-AP) (26) has been used as an alternative substrate for the enzyme [K = 1 mM k,,,/K = 1.3YOthat of L-THDP (5)].72 The enantiomer D-a-aminopimelate (27) is a competitive inhibitor [Ki = 0.76 mM against L-THDP (5) 0.31 mM against L-a-AP (26)] NATURAL PRODUCT REPORTS 1996R.J. COX (5) L-THDP (35) succinylCoA Ho2CmC02H AB XY (28a) B = OH; X = NH,; A = Y = H (28b) A=OH; X=NH2; B=Y=H (28~) B=OH; Y=NHz; A=X=H (28d) A=OH; Y=NH2; B=X=H A (29) (30) DHT which appears to bind to the enzyme more tightly.These observations led to an investigation of binding by a number of acyclic substrate analogues. It was found that the enzyme was specific for diacids having a carbon chain length of seven. The presence of an a-hydroxy moiety was preferred slightly over a-amino for binding. Thus LL-a-amino-e-hydroxypimelate(28a) was succinylated at 43% of the rate of L-~-AP (26) but the statistical mixture of isomers (28a-d) was succinylated 21 YO faster than L-a-AP. Since the D-a-amino configuration (28c,d) is not succinylated and the LL isomer (28a) is succinylated slowly these results suggest that the a-L-amino-e-D-hydroxy- pimelate (28b) isomer is a very good substrate but further measurements were not made.The conformationally restricted (ring-like) compound (2E,SE)-y-ketohepta-2,5-dienedioic acid (29) is an inhibitor [y= 0.53 mM against L-~-AP (26)]. Cyclic L-THDP analogues have also been tested against the succinyltransferase. One substrate was found 3,4-dihydro-2H-1,4-thiazine-3,5-dicarboxylicacid (DHT) (30) [K",pp = 2 mM k,,,/K",PP = 0.5 % that of L-THDP (5)]. Other cyclic compounds are generally poor inhibitors (Figure 2). It is noteworthy however that unsaturated compounds bearing trans carboxyl groups are better inhibitors than those with carboxylates disposed cis. One compound m-a-hydroxytetrahydropyran-a,€-dicarboxylate (HTHP) (34) is a very potent competitive inhibitor [Ki = 58 nM against L-u-AP (26)].These observations have been used to propose a stereo-chemical model for the mechanism of the succinyltransferase (Scheme 5).72 The model proposes that the enzyme binds L-THDP (5) in its cyclic form then catalyses the addition of water to the re face of the imine. The trans-piperidine dicarboxylate intermediate (35) then reacts with succinyl CoA H02C 0k02H HO2C (20a) X = NH; K = 2.0 mM (20b) X = NH; K,= 63 mM (31a) X = 0; K = 0.68 mM (31 b) X = 0; 6= 3.9 mM (32a) X=CH2; K,=144mM (32b) X=CH2; &=265mM (33a) X=S; K,= 1.1 mM (33b) X = S; K,= 4.4 mM H02C 0C02H (loa) X=N; $=12.8mM (22) X= CH; K,=8.5mM Figure 2 Inhibitors of succinyl CoA :L-tetrahydrodipicolinatesuccinyl-transferase H02CI {I (27) O-a-AP II H20 (34) DL-HTHP (28b) Stereochemical model for the mechanism of succinyl CoA L-tetrahydrodipicolinate succinyltransferase Scheme 5 and the ring is opened.A feature of this model is that the acyclic substrates and inhibitors must bind in a ring-like manner in which the carboxyl groups are disposed in the same trans conformation as L-THDP (5). This conformation forces the disposition of the a and e substituents as shown in Scheme 5 and accounts for both the apparently good substrate activities of (28b) and the inhbition by D-~-AP (27) and DL-HTHP (34) since these compounds can mimic the conformation of the intermediate (35). 2.3 N-Succinyl-LL-DAPAminotransferase EC 2.6.1.17 dapC The aminotransferase has been purified to homogeneity from E.coli and its activity detected in both Gram-negative and Gram-positive bacteria.73 The E. coli enzyme is a homodimer of subunit M 39900.74 The enzyme is PLP dependent and requires L-glutamate (36) as the amine donor. Early studies utilized substrate (6a) isolated from an E. coli mutant blocked in deacylase a~tivity.'~ The assay consisted of measuring LL-DAP (8a) production by a reaction mixture containing an excess of DAP desuccinylase (Section 2.4). A more convenient continuous spectrometric assay has been described in which the forward reaction is coupled to glutamate dehydrogenase (EC 1.4.1.4) in the presence of NADPH and ammonium ions (Scheme 6).74 Under physiological conditions the reaction is freely reversible and the reverse reaction may be monitored by observing the disappearance of N-succinyl-LL-DAP (7a).The Michaelis constants for the natural substrates have been measured for L-glutamate (36) K = 1.21 mM; for L-N-succinyl-a-amino-e-ketopimelate(6a) K = 0.18 mM k,, = 86 s-l. Kinetic analyses suggest a sequential reaction mechanism in which L-glutamate (36) reacts with the pyridoxal phosphate form of the enzyme donating its amino group via aldimine quinonoid and ketimine intermediates (Scheme 7). The sub- strate (6a) then reacts in the reverse direction regenerating the PLP form of the enzyme with transfer of the amino group onto NATURAL PRODUCT REPORTS 1996 Ho2Cmco2H Ho2CYco2H 0 0 HNPCo2H NH2 0 7-7- (74 HOpCwC02H I NH2 0 Ho2cY-co2H (36) + H20 u NADP’CI-ii NADPH + NH&I Assay for N-succinyl-LL-DAP aminotransferase.Enzymes i N-succinyl-LL-DAP aminotransferase ; ii glutamate dehydrogenase EC 1.4.1.4 Scheme 6 H02CVR NH2 H02CvR Enz-NH2 0$4H f H Michaelis complex external aldimine Enz-NH3+ &H H02C I( H02C 0 H Enz-NH2 (NH2 Enz-NH2 (YH quinonoid intermediate m*-+0 H H Michaelis complex ketimine Scheme 7 the product (7a). These properties are typical of PLP-dependent CBZ (38) (kcat/Pzp= 23.5 YO) and dihydrocinnamoyl (39) aminotransferases and are consistent with those of the ‘model’ (kCa,/K”,PP = 7.7 %) are also acceptable. Interestingly the sub- system of aspartate amino transferase (EC 2.6.1.1).76 stitution of succinyl by acetyl (40) the presumed natural Early studies showed the E.coli enzyme to be specific for the substrate for the Bacillus spp. aminotransferase is less favoured natural substrates L-glutamate (36) and L-N-succinyl-a-amino- (k,,,/Pzp = 4.5 YO).The amide funtionality may even be s-ketopimelate (6a). The enzyme tolerates modifications of the deleted :the racemic substrate analogue (41) showed detectable acyl side chain with the substitution of succinyl by cinnamoyl activity (k,,,/K”,pp = 0.7 YO).Other modifications are not toler- (37) being particularly favoured (kca,/KzP= 70 YOof the value ated removal of a carboxyl results in compounds such as for the natural substrate). Other aromatic substituents such as L-e-N-CBZ lysine (42) and N-succinyl-s-amino-a-ketohexanoic HO2CW.-H02C y-yCO;HB H02c-3 0 HN HNK 0 HN 0 0 0 H02c~C02 HNK Ho2CY-YC0,H H Ho2cp oa 0 HNY n 0 NATURAL PRODUCT REPORTS 1996-R.J. COX 35 ""'Y-l Ho2cYC02H H02CY-Yco2H 0 HNl-Co2H HNPCo2H HNK-Co2H OH 0 0 0 (43) (44) (45) Ho2CmCo2H HNPco2H HNR HNNH 0 0 (Ma) R' = succinyl; R2 = H (47) (48) (46b) R' = H; R2 = succinyl c."c02H NH2 NH2 H02C wCo2H (84 HNPco2H CO H Enz-NH2 0$H NH2 0 H02C-Enz-N9 f (74 H02C I H H Michaelis complex stable hydrazone Scheme 8 PEP ~COSCOA H02C Assay for N-succinyl-LL-DAP desuccinylase. Enzymes i N-succinyl- acid (43) which are neither substrates nor inhibitors. The c-LL-DAP desuccinylase EC 3.5.1.18; ii porcine succinate thiokinase keto/amino substituent is also of key importance compounds EC 6.2.1.4; iii pyruvate kinase EC 2.7.1.40 ;iv lactate dehydrogenase lacking it such as DL-N-succinyl-a-aminopimelate (44) and L-EC 1.1.1.27 N-succinyl-a-amino-ehydroxypimelate(49 showed no de-Scheme 9 tectable inhibition.Both a-carbons must possess L-configu- ration since a mixture of N-succinyl-meso-DAP isomers (46a,b) was inactive with respect to the reverse reaction. The enzyme like other PLP-dependent aminotransferases is in good agreement with a calculated subunit M of 41129 inhibited by hydroxylamine hydrazine and their substituted determined from the nucleotide sequence of the E. coli dapE analogues. These compounds react with pyridoxal phosphate locus.s4 The dapE locus from Corynebacterium glutamicum has or hydra~one'~ also been ~equenced;~~ inferred amino acid sequence at the active site forming a stable nitr~ne~~ the which cannot react further and which is also resistant to indicates a subunit M of 39942.hydrolysis (Scheme 8). This fact was exploited in the design of The E. coli enzyme requires a metal ion ideally cobalt(I1) (K specific potent inhibitors of the enzyme. Substituted hydrazino = 4.0 pM) but zinc (K = 1.2 pM) is also effective though 2.2 substrate analogues have been synthesized with the motifs fold less so. Early studies showed that iron(m) nickel@) and required for good recognition specifically a DAP skeleton with manganese@) ions can substitute with similar efficacies to zinc. a succinyl(47) or CBZ (48) substituted amine. These compounds The enzyme is functionally similar to other carboxypeptidases,s6 are potent tight slow-binding inhibitors of the enzyme [q and shows 38.7 % sequence similarity with the cobalt(I1)- values:'' 22 nM for (47); 54 nM for (48)].74 Substrate binding dependent acetylornithine deacetylase EC 3.5.1.16 from E.coli by the model PLP enzyme aspartate aminotransferase is and 3 1.5% sequence similarity with Pseudomonas sp. G2- accompanied by a conformational closure of the active site carb~xypeptidase.~~ The deduced amino acid sequence of the which promotes fast reaction and product release.s0 In the case C. glutamicum desuccinylase is 23% homologous with the E. of inhibition of N-succinyl-LL-DAP aminotransferase by these coli enzyme.85 substituted hydrazines a similar but prolonged closure of the Assays have been developed to detect either succinate or LL-active site may be occurring.This fact is reflected in the long DAP (8a) liberation. A spectrometric method has been off-rate half times79 for these two inhibitors 78 and 45 min described for the quantification of succinate production respectively. (Scheme 9). This coupled system relies on the reaction of released succinate with coenzyme A catalysed by porcine succinate thiokinase (EC 6.2.1.4). Inosine 5'-triphosphate (ITP) 2.4 N-Succinyl-LL-DAP Desuccinylase EC 3.5.1.18 dapE is concomitantly converted into inosine 5'-diphosphate (IDP) The product of the aminotransferase N-succinyl-LL-DAP (7a) which in turn reacts with phosphoenoylpyruvate (PEP) to form in the case of E.coli N-acetyl-LL-DAP (7b) in the case of many pyruvate (2) catalysed by pyruvate kinase (EC 2.7.1.40). Bacillus sp~.,~ is subject to deacylatiom81 The E. coli de-Pyruvate (2) is then reduced by lactate dehydrogenase (EC succinylase has been purified to homogeneity,s2 and DAP 1.1.1.27) and the consequent decrease in NADPH concentration deacylase activity detected in numerous bacterial species.83 The is observed at 340 nm. E. coli enzyme is dimeric at low concentrations (< 1 mg ml-l) Michaelis constants for the natural substrate (7a) have been but forms a tetramer at higher concentrations; the subunit M determined (K = 410 pM k,, = 266 s-l) and systematic has been determined to be approximately 40000. This figure is studies of potential substrate analogues carried out.The E. coli NATURAL PRODUCT REPORTS 1996 mco2H Ho2c-Y-Yo2H Ho2cm HNPCo2H HNPCo2H0 NH2 0 NH2 NH2 HNPC02H 0 (49) (50) (51) Ho2cY-Yco2H YNHHNT-Co2H' 0 0 (9) desuccinylase hydrolysed a statistical mixture of N-succinyl- DD-DAP (8c) is not a substrate. The stereochemistry of the non- DAP stereoisomers (49) rapidly releasing 25 % of the succinate reacting a-carbon therefore controls the substrate specificity of and LL-DAP (8a). A further 25 YOof the succinate was released the enzyme since catalysis at the reacting a-carbon is fully slowly concomitantly with meso-DAP (8b) but no further reversible (Scheme 10). The Michaelis constants for LL-DAP hydrolysis was observed even with prolonged incubation. (8a) (K = 160 pM k,, = 84s-l k,,,/K,, = 525000 M-ls-l) These observations were rationalized by assuming initial rapid and meso-DAP (8b) (K = 360pM k,, = 67 s-l k,,,/K,, = hydrolysis of the LL-N-succinyl-DAP isomer (7a) followed by 186000 M-ls-l) have been determined and the equilibrium slow hydrolysis of the N-succinyl-meso-DAP isomer with the L-constant was shown by HPLC analysis of products to be 2.0 amide (approximately 30% of the rate of the LL isomer at (reflecting the statistical distribution of LL and meso isomers).10 mM). The 50% of isomers bearing D-configured a-amido Early investigations showed that the enzyme does not require carbons were presumably not substrates for the reaction. Both PLP for activity and that it is not inhibited by hydrazine or carboxyls are required for substrate activity L-e-N-succinyl-hydroxylamine.Furthermore a reducible imine is not an lysine (50) was not a substrate while L-a-N-succinyllysine (5 1) intermediate since sodium borohydride is not inhibitory as it is was a very poor substrate (0.016 % of the rate of the natural with L-DHDP synthase (Section 2.0). Nicotinamide and flavin substrate at 10 mM). A mixture of isomers of N-succinyl-a- cofactors are unnecessary and metals appear to play no part in aminopimelate (44)was also a very poor substrate (0.036 YOthe the reaction. Tritium at the substrate a-position is exchanged rate of the natural substrate at 10 mM) with a K of 16 mM. rapidly with solvent. Dithiothreitol must be present for the The L-N-acetyl substrate analogue (7b) (the substrate for the enzyme to remain active and time dependent inhibition by Bacillus spp.deacylases) showed no substrate activity; neither iodoacetamide was observed. One equivalent of iodoacetamide did L-N-succinyl-a-amino-e-ketopimelate (6a) or N-acety1-N'- is bound per enzyme and activity is protected by the presence succinyl-DAP (52). of substrate. These observations suggested that meso-DAP epimerase operates via base catalysed a-proton abstraction. A thorough investigation of the exchange of tritium between 2.5 meso-DAP Epimerase EC 5.1.1.7 dapF the substrate a-carbon and the solvent has been carried The epimerase has been detected in both Gram-positive and Each turnover is accompanied by the loss of an a-proton to 89 Gram-negative bacteriaa8 as well as in plant species.29+ solvent and a solvent derived proton is preferentially delivered Purification of the enzyme has proven problematical in part to the substrate.It was found that the kinetic isotope effects for because of the presence of a free thiol residue necessary for tritium exchange differed for LL and meso substrates. The activity. The Bacillus megaterium enzyme has been partially results are consistent with a mechanism in which two bases purified (20 fold),g0 but the E. coli enzyme has been purified to must be at work (Scheme 11); a single base mechanism The involving deprotonation from one face followed by repro- > 85 Ohpurity and has thus attracted the most attenti~n.~' E. coli dapF locus has been cloned,92 sequencedg3 and tonation from the opposite face is not supported exper- The epimerase is a monomeric polypeptide of imentally.meso-DAP epimerase resembles the well studied overexpre~sed.~~ M 34000 as determined by gel filtration; this figure has been enzyme proline racemase in this respect.95 However in the case refined to 30265 from the dapF nucleotide sequence. The of proline racemase where both bases are thiols the kinetic enzyme has been assayed by monitoring the release of 3H from isotope effects for each substrate enantiomer are nearly a-tritiated DAP or via the NADP' dependent oxidation of identical. This suggests that the bases involved in meso-DAP meso-DAP (8b) to L-THDP (5) by meso-DAP dehydrogenase epimerase are likely to be different. It has been argued on (section 2.6). purely kinetic grounds that one of the meso-DAP epimerase The enzyme interconverts LL-DAP (8a) and meso-DAP (8b); bases is in fact a thiol.This is consistent with the observation H02C w C 0 2 H I I H02c-To2H =Ho2Cmco2H =I-NHP NH2 NH2 NH2 NH2 NH2 DD-DAP (8a) LL-DAP (8b) meso-DAP (8~) Scheme 10 CO2H * C02H H-B+ -=B-H ,I~H -B H< B d NH2 d NH2 Scheme 11 NATURAL PRODUCT REPORTS 1996-R. J. COX H02C& C02H Ho2cnH:2H HN (53) azi-DAP NH2 AB NH2 (56a)B=NH2; Y=F; A=X=H (56b) A=NH2; Y=F B=X=H (56c)B=NH2; X=F A=Y=H (56d)A=NH2; X=F; B=Y=H Scheme 12 Ho2cY-Yco2H NH2 NH2 NH2 -H02C QCO, (5) L-THDP (54) (55) Scheme 13 that the irreversible inhibitor azi-DAP (53) (Ki2 25 pM) specifically covalently labels Cys-73 of meso-DAP epimerase (Scheme 12).96 Other epimerases such as mandelate racemase (EC 5.1 .XQg7 operating by 'two-base ' mechanisms have been intensively studied because of the apparent paradox of relatively poor active-site bases being able to abstract very weakly acidic protons from the substrate a-carbon.Recently the concept of short strong hydrogen bondsgs between an active site residue and the substrate carboxylate in the transition state (or similar intermediate) has been validated e~perimentally.~~ These 'low barrier' hydrogen bonds can stabilize the transition state significantly thus influencing the pK of the a-proton and it may be that a similar process allows meso-DAP epimerase to exchange substrate a-protons as observed. The proposed 'two-base' mechanism could proceed in a stepwise or concerted fashion but in either case negative charge should be concentrated at the a-carbon during reaction.This feature has directed the design of possible meso-DAP epimerase inhibitors towards compounds unstable to elimination (i.e. /3 or N-substituted) or towards compounds that are planar at the a-carbon which could mimic the putative planar transition state (Scheme 11). Thus a mixture of /3-chloro-DAP (54) stereo- isomers potently inhibits the epimerase (Ki = 200 nM).loo Inhibition is reversible and competitive with substrate. At low inhibitor concentrations however a time dependent decrease of inhibition was observed indicative of inhibitor turnover. The product of this reaction was reduced by meso-DAP dehydrogenase in the presence of NADPH; it was thus identified as L-THDP (5).It is likely therefore that the epimerase catalyses elimination of HCl from P-chloro-DAP forming the intermediate (55) (Scheme 13) which is planar at the reacting a-carbon and which is therefore a mimic of the postulated transition state (Scheme 11). This enamine then slowly isomerizes to L-THDP(5),which is not inhibitory. Since meso- DAP dehydrogenase is specific for L-THDP (5) (Section 2.6) the substrate for the epimerase must have possessed L-configuration at the distal (non reacting) end. This is consistent with the known substrate specificity of the epimerase. The synthesis in stereochemically pure form of the four stereoisomers of P-fluoro-DAP (56a-d) possessing e-L con-F F H H H H Stereochemical rationalization of reactions of P-fluoro-DAP stereo-isomers (56a-d) catalysed by rneso-DAP epimerase.Scheme 14 figuration has been achieved.lol All of these compounds are good inhibitors of the epimerase [IC, values (56a) 10pM; (56b) 25pM; (56c) 4pM; (56d) 8pM].As expected the elimination of HF from each isomer is catalysed by the epimerase and the eventual product is L-THDP (5). The characteristics of these elimination reactions vary however. The rates of HF elimination and epimerization at the reacting a-carbon have been independently determined.l0' For one pair of isomers (56c,d) the elimination is slow but epimerization is fast and the two isomers are in rapid equilibrium. For the other pair (56a,b) only fast elimination is observed.These results suggest the stereochemical course shown in Scheme 14 in which the position of the charged groups and the bulky substituent R are fixed in the enzyme active site [the same conclusions may be drawn from a scheme in which the a+ bond of (56a-d) has been rotated by 180" yielding the intermediate (E)-(55)]. Fast elimination is expected when H and F are fixed either syn or NATURAL PRODUCT REPORTS 1996 OH H02C~0-~C02HH02CwC02H H02C*C02H N-0 NH2 N-0 NH NH2 NH2 (574 N-0 N-0 Ho2CY-YCo2H NH2 HN. NH2 (64) (65) (59) Ho2cmH:H NH2 NH Ho2CmP(oH)2 (67) (684 B=X=NH2; A=Y=H AB X H02cmco2H Y (60) (61a) B =X= NH2; A=Y=H (61b) B=Y=NH,; A=X=H (61~)A =X = NH2; B=Y=H (61d) A = Y = NH2; B=X=H anti-coplanar in the enzyme active site as in stereoisomers (56a,b).lo2 When H and F are fixed gauche as in stereoisomers (56c,d) epimerization occurs rapidly and HF elimination is slow.P-Hydroxy DAP (57a,b) isomers have also been synthe- Sized.lOO,101,103 These compounds are poor inhibitors of the epimerase (IC50= 2.5 and 4 mM respectively) since H,O is not eliminated; (57b) is not epimerized at all and (57a) is epimerized at the a-centre distal from the hydroxy group. A mixture of stereoisomers of N-hydroxy-DAP (58) reversibly and com-petitively inhibited the epimerase (K,= 56 pM).lo4 N-Amino- DAP (59) was a much poorer inhibitor (K,= 2.9 mM). It has been suggested that elimination of water from (58) would lead to the planar imine (60) which could either bind to the epimerase tightly per se or reversibly react with one of the active site bases.Another strategy for the design of possible inhibitors of DAP-epimerase relies on the perturbation of the a-proton pK by the substitution of carboxylate by phosphonate. The doubly charged phosphonate could also mimic a possible aci-carbanion transition state as is observed in the potent slow-binding inhibition of alanine racemase (EC 5.1.1.I) by phosphono lo5 It analogues of ala~~ine.~~' was thought that a similar approach could yield useful meso-DAP epimerase inhibitors.lo6 Four stereochemically pure phosphono-DAP isomers (6 1 a-d) have been synthesized.lo7 These compounds are relatively poor competitive inhibitors of meso-DAP epimerase (K,= 3.9-7.2 mM) however and none of them are substrates.Heterocyclic compounds (62-66) in which a planar configu- ration about the a-carbon is rigidly held have also been synthesized as potential inhibitors of the epimerase.los Similar compounds can inhibit other 'two-base' epimerases such as proline racema~e.'~~ None of these compounds showed sig- nificant inhibition perhaps because of the steric bulk of the ring or possibly because of the inability of the ring substituents to take up the correct conformation to mimic the postulated transition state geometry. Several other miscellaneous compounds have been tested. The epimerase has been suggested to be the target of the antibacterial compound y-methylene DAP (67),"O although this material is only a moderate inhibitor (K,=0.95 mM).lo4 The sulfur containing DAP analogue meso-lanthionine (68b) is also a moderate mixed competitive inhibitor (K,=0.18 mM).Its LL isomer (68a) inhibits competitively (K,=0.42 mM) while the DD analogue (68c) shows no inhibition. Oxidation of the sulfur to a sulfoxide (69) or sulfone (70) led to progressively poorer inhibitors (Ki = 11 and 21 mM respectively for meso isomers).lo4 (68b) A=X=NH2; B=Y=H (6&) A=Y=NH2; B=X=H 2.6 meso-DAP Dehydrogenase EC 1.4.1.16 ddh The biosynthesis of meso-DAP (8b) via acylated intermediates and meso-DAP epimerase as described above is bypassed in some organisms. In this variation of the pathway the direct reductive amination of L-THDP (5) to meso-DAP (8b) is catalysed by the NADPH dependent meso-DAP dehydro- genase.This enzyme occurs in Bacillus sphaericuslll (which lacks meso-DAP epimerase and enzymes of the acylated pathway)l12 and some other Gram-negative and Gram-positive bacteria.'l37 114 An early report suggested the presence of meso-DAP dehydrogenase in plants,28 although more recent work seems to have refuted this finding.29 The enzyme has been purified to homogeneity from Corynebacterium gl~tarnicum'~~ and Brevibacterium spp.'15 but the most highly studied enzyme is that from B. sphaericus,l16 which has a high dehydrogenase activity.l13 The reaction is reversible maximal velocity in the forward direction of reductive amination is displayed at pH values of 7.5-8.5.Maximal reverse velocity occurs at pH values between 9.0 and 10.5,"' although under forcing conditions (i.e. using NADP' with meso-DAP in the absence of NH,) the reverse reaction may be studied at pH 7.8. The reaction is conveniently assayed by observation of cofactor consumption in either direction or via a sensitive coupled reaction with formazan in the reverse direction.lls The bacterial enzymes are homodimers of subunit M approximately 39000 and two active sites are present per dimer. The ddh locus from C. glutamicum has been cloned and sequenced,ll9 and the inferred peptide has a M of 35 099.lZ0 Michaelis parameters have been determined for the natural substrates and cofactors for reactions catalysed by the B. sphearicus C.glutamicum and Brevibacterium spp. enzymes K values for L-THDP (5) -0.2 mM; NADPH -0.2 mM; NH = 12-62 mM; meso-DAP (8b) =2.5-6 mM; NADP' = 0.83 pM for B. sphaericus -0.1 mM for the others. Detailed kinetic analyses of reactions catalysed by the B. sphaericus enzyme have revealed that the reaction is sequentially ~rdered,"~ like the classical glutamate dehydrogenase mech- anism. In the forward reaction NADPH binds first followed by L-THDP (5) and then ammonia. After reaction meso-DAP is released followed by NADP'. The reverse sequence is observed for the oxidative deamination of meso-DAP. The reaction is unusual in that oxidation takes place at a D centre; meso-DAP is transformed into L-THDP (5) in the reverse reaction. Since neither DD-(8c) nor LL-DAP (8a) are substrates for this reaction it is evident that L stereochemistry is required at the non reacting a-carbon as in the case of meso- DAP epimerase (Section 2.5).The forward reaction is likely to NATURAL PRODUCT REPORTS 1996-R. J. COX n NH3 IFNADPH H02C-CO2H Scheme 15 proceed via ring opening of L-THDP (5) by ammonia forming a planar imine intermediate (60) (Scheme 15). Imine reduction then forms the wcentre of meso-DAP. The 4-pro-S hydrogen of NADPH is transferred stereospecifically in the rate-determining step. Like meso-DAP epimerase substrate specificity is tight ; DAP analogues lacking carboxylate or amine groups or shortened by a methylene group are not substrates. meso-Lanthionine (68b) is a poor substrate ( VmaX/Km = 0.2O/O of the value for meso-DAP) and oxidation of the central sulfur lowered substrate activity further.lo4 This result was surprising in view of the fact that lanthionine (68) is only 5 O/O longer than DAP and that at least one of the stereoisomers of the bulkier DAP analogue y-methylene-DAP (67) is a somewhat better substrate (y,ldx/Km = 0.4% of the value for meso-DAP).The observed results may be due to the decreased pK,s of the protonated carboxylate and imine functionalities of the lanthionine imino intermediate. Oxidation of sulfur could reduce these pK,s further explaining the extremely poor activities of the sulfone and sulfoxide.lo4 Two compounds N- hydroxy-DAP (58) and N-amino DAP (59) were substrates; mixtures of stereoisomers turned over at 22 and 4% of the rate for meso-DAP respectively.Fluoro-DAP stereoisomer (56b) an analogue of the natural substrate meso-DAP (Sb) showed neither substrate nor inhibitor activity against the B. sphaericus dehydrogenase.lO' Phosphono-DAP analogues (6 la4) also show no substrate activities although binding evidently occurs since these compounds are weakly inhibitory. It has been suggested that inhibition may occur because of the reduced propensity of the amine adjacent to the phosphono group to undergo oxidation or to cyclize onto the a-imino carbon at the reacting end. The two nzeso isomers bind more tightly than either LL (61a) (K = 12 mM) or DD (61d) (K,= 26 mM) analogues. The best of them (61c) (K,= 4.3 mM) would place the L-phosphono group at the non reacting end if specificity is assumed to be similar to DAP analogues.The other meso analogue (61b) (K,= 7.4 mM) would place the phosphono group at the reacting end. DAP isomers themselves are competitive inhibitors of the forward reaction with K values of 3.1 4.0 and 4.2 mM for LL (8a) DD (8c) and meso-DAP (8b) respectively."' The isoxazoline (62) shows potent inhibitory activity against meso-DAP dehydrogenase from B. sphaericus. At pH 7.8 this compound is a potent inhibitor of both the forward [K,= 4.2 pM versus L-THDP (5)] and reverse reactions [K,= 23 pM with respect to nzeso-DAP (8b)] although inhibition falls off with rising pH (ICs0= 4.1 mM at pH 10.5). Initially it was thought that this compound might be a good mimic of the postulated imine intermediate (60) with the L-amine filling the non-reacting site and the planar oxime bound at the reacting end.However kinetic analysis showed that for the reverse reaction (62) inhibited noncompetitively versus meso-DAP and uncompetitively against NADP' (K = 9.2 pM). In the forward direction (62) is a competitive inhibitor against L-THDP (5). Hence the isoxazoline inhibitor competes only for the L-THDP (5) binding site and does not occupy the meso-DAP binding site. These results imply separate binding sites for the two substrates. The L-THDP (5) binding site may contain ionizable residues since both inhibition by (62) and substrate activity of L-THDP (5) fall off dramatically at high pH.Inhibition by (62) was not merely due to to the isoxazoline moiety. Similar compounds with alternative side chains were very poor inhibitors. In particular the isoxazoline (63) differing only in ring junction stereochemistry showed a poor 13 YO inhibition at 1 mM. The distal amino acid moiety is clearly required because removal of the side chain (64) or replacement by carboxyl (65) led to even poorer inhibitors of the dehydrogenase (7.5 and .c 2 % inhibition respectively at 7.75 mM). The imidazole analogue (66) was also a very poor inhibitor (< 7% inhibition at 1 mM). The B. sphaericus enzyme contains one sulfhydryl group per subunit and is susceptible to inhibition by charged or bulky sulfhydryl reagents such as Hg2+ or Ellman's reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB).Enzyme inhibited by formation of a disulfide with DTNB could be reactivated by treatment with cyanide forming an enzyme-cyanide adduct. It was concluded therefore that the sulfhydryl residue was not required for enzymic activity.12' 2.7 meso-DAP Decarboxylase EC 4.1.1.20 ZysA Decarboxylation of meso-DAP (8b) is catalysed in a PLP- dependent reaction to produce L-lysine (9) as the ultimate product of the pathway. meso-DAP decarboxylase is extremely widespread in both plants and bacteria; it has been purified from a number of sources including E. coli,122, 123 Bacillus sphaericus,lZ4 duckweed (Lemna perp~silla)~~~ and wheat (Triti- cunz vulgaris).126 The B. sphaericus enzyme is a homodimer of approx M 80000,whereas the E.colienzyme is a homotetramer with a subunit M of 46099 determined by genetic analysis.12' The lysA loci from a range of other bacterial species including 129 B. methan~licus,~~~ B. subtilis,128. Mycobacterium tubercu- Ios~s,~~~ and Pseudomonas aeru- Corynebacterium gl~tamicuml~~ gino~al~~ have also been cloned and sequenced. The genes encode similarly sized proteins (M = 4500W9000) with significant sequence ~imi1arities.l~~ The reaction has been assayed in several ways. Early studies relied on manometric measurement of CO evolution or colorimetric determination of lysine (9) by its reaction with ninhydrin. Neither method is reliable enough for kinetic measurements however and more recent studies have measured the release of 14C02from radio-labelled DAP,lS5 or determined lysine (9) formation using the enzyme L-lysine-a-ketoglutarate e-aminotransferase (EC 2.1.6.36) in non-continuous Assays have also been developed for the simultaneous determination of epimerase and decarboxylase activity.13' The decarboxylase is specific for meso-DAP (K -1.7 mM for most bacterial enzymes 0.16-0.35 mM for plant homologues).A mixture of lanthionine isomers (68a-c) was turned over by the B. sphaericus enzyme at approximately 5% of the rate for meso-DAP but although many other amino acids and their analogues have been examined no other substrates have been found. Since the enzymes studied to date accept neither LL (8a) nor DD-DAP (8c) as substrates or inhibitors it may again be concluded that L configuration is required at the non reacting a-carbon.The enzymes are unique among PLP-dependent amino acid decarboxylases in catalysing reaction at a D centre. This fact is reflected by protein sequence studies which suggest that DAP- decarboxylases are closely related as a group but seem to be unrelated to most other PLP-dependent enzymes.1"". 1'34,13*. 139 The stereochemical course of the reaction catalysed by the B. ~phaericus'~~ and wheat enzymes has been studied in detail. In both cases decarboxylation occurs with inversion of stereo- chemistry again contrary to the state of affairs observed for other PLP-dependent decarboxylases where reaction is ac- NATURAL PRODUCT REPORTS 1996 external aldimine Reactions catalysed by PLP-dependent L-amino acid decarboxylases (path Scheme companied by retention of stereochemistry.This apparent dichotomy in the stereochemical preference and course of reaction has been accounted for in two ways. A 'swinging door' mechanism has been proposed in which a rotation of the substrate-PLP complex occurring after loss of CO would allow protonation from the same direction as CO loss. This model would involve a drastic conformational change of the enzyme bound substrate however. An alternative hypothesis would link the mechanism of meso-DAP decarboxylase with other PLP-dependent decarboxylases if a common quinonoid intermediate (71) was formed in the enzyme active site (Scheme 16). Protonation of this intermediate from the re face at the a-carbon in each case would result in the observed stereochemical outcome (i.e.inversion at D centres by DAP decarboxylases and retention at L-centres in other PLP-dependent decarboxyl- ases). The design of effective inhibitors for DAP-decarboxylase has been hampered by the apparently very tight substrate specificity. Neither LL nor DD-DAP inhibit the reaction,24 but L-lysine (9) and its analogues; such as AEC (13) are generally weak competitive inhibitors of many of the enzymes with IC, values > 20 mM.141* 142 meso-Lanthionine (68b) is a somewhat better inhibitor of the enzymes from B. sphaericus and wheat germ with IC,,s of 10 mM and 14 mM respectively. The lanthionine sulfoxides (69) are better still with IC, values of -1 mM.Further oxidation of the sulfur does not increase potency the sulfone analogues (70) showed poorer inhibition (IC50 -10 mM).143 As expected the enzyme is inhibited by 'carbonyl' reagents such as hydroxylamine and isonicotinic acid hydrazide (72) (isonia~id),~~. 144 but detailed investigations have not been carried out. It would be expected that DAP analogues of these compounds could be potent and specific inhibitors of meso-DAP decarboxylase in analogy to the inhibition by similar compounds of the PLP-dependent N-swccinyl-LL-DAP amino- transferase in time-dependent reactions (Section 2.3). Mixtures of isomers of N-amino-DAP (59) and N-hydroxy-DAP (58) however were found merely to be effective competitive inhibitors of the enzymes from B.sphaericus (Ki = 100 and 84 pM respectively) and wheat germ (Ki = 910 and 710 pM respectively). a-Difluoromethyl amino acids are also known inhibitors of PLP-dependent enzymes. These compounds can undergo a series of elimination reactions catalysed in the active site resulting in irreversible enzyme ina~tivati0n.l~~ The a-difluoro- methyl DAP analogue (73) was however merely a weak competitive inhibitor (1C5 -10 mM) of the decarboxylases from wheat germ and B. sphaericus. a-Methyl-DAP isomers H H quinonoid intermediate (71) NH2 L) and meso-DAP decarboxylases (path D) 16 H "yN *NH2 (72) (73) Ho2CmCo2H Ho2cY-YCo2H Me NH2 NH2 NH2 NH2 (74) (75) (76) (77) (74) were also poor inhibitors."O These findings underline the tight substrate specificity of meso-DAP decarboxylase since many other PLP-dependent decarboxylases can accept suitable a-methyl or a-difluoromethyl substrate analogues into their active Unsaturated substrate analogues could also be expected to be good inhibitors and a number of them have been tested against the decarboxylase from E.coli. The best of these compounds the mixture of stereoisomers (75) was a moderate competitive inhibitor (Ki = 180 pM) but structural modification for example y-methylation (76) led to much poorer inhibitors. The individual isomers of y-methylene-DAP (67) are also uniformly poor inhibitors (-30 % inhibition at 10 mM).l1° 3 Antibiotic Properties of DAP Pathway Inhibitors Naturally occurring DAP analogues with biological activities would appear to be very rare possibly reflecting the critical importance of the products of the DAP pathway to bacterial growth and development.An alanyl dipeptide (77) of a-hydroxymethyl-DAP which shows antibiotic activity against E. coli has been isolated from Micromonospora chalcea how-ever.147 The activity against E. coli on minimal media is synergistically enhanced by a number of peptidoglycan bio- synthesis inhibitors such as penicillin-G and chloro-D-alanine NATURAL PRODUCT REPORTS 1996-R. J. COX H02C0C02H HO2C4$CO2H OH OH 0 suggesting that inhibition of DAP biosynthesis may be occurring. These effects are not evident on nutrient agar which may contain DAP or lysine. Efficient transport of potential DAP biosynthesis inhibitors through the cell membrane is clearly desirable.DAP itself would appear to be transported via the cystine uptake mechani~m,'~~ at least in E. coli 14g and Salmonella typhi- murium.150 DAP analogues may also be transported as di- or tri-peptides which are taken up by efficient cellular transport systems. This strategy has been demonstrated to be effective for other peptidoglycan biosynthesis inhibitors such as the peptidic antibiotic alaphosphin which is cleaved in vivo to release the alanine racemase (EC 5.1.1.1) inhibitor pho~phonoa1anine.l~~ In cell-free protein extracts of E. coli the succinyltransferase inhibitor L-OI-AP (26) blocks DAP biosynthesis and causes a build-up of the precursor L-THDP (5).L-OI-AP(26) itself showed no antibacterial activity however perhaps because of poor transport into the cell. When L-OI-AP was included in alanyl dipeptides good antibacterial activity was observed. The peptide (78) was the most potent with MICs of 1-16 pg ml-l against a range of Gram-negative bacteria. High internal cellular concentrations of up to 30mM L-OI-AP (26) were measured when bacterial cells had been treated with these alanyl dipeptides ; this is a concentration significantly higher than the K, of L-OI-AP (1 mM). The dipeptides caused depression of DAP biosynthesis in 'resting' E. coli cells at 2.4 mM and caused lysis of growing Enterobacter cloacae at 0.2 mM. These effects were reversed in the presence of LL-DAP @a) showing that DAP biosynthesis was indeed the likely target of action.These encouraging results suggest that even modest enzyme inhibitors (in fact L-OI-AP is a poor substrate of the succinyl- transferase) can be effective antibiotics if properly delivered. It would be expected that alanyl peptides of more potent enzyme inhibitors could be extremely effective antibiotics. This line of reasoning has recently encouraged the synthesis of a depsi-peptide analogue (79) of enantiomerically pure L-HTHP (80) [Ki of racemate 58 nM against L-OI-AP (26)] along with other potential transition state inhibitors (81) and (82) of the s~ccinyltransferase.~~~ The strategy has been adopted for other potential DAP biosynthesis inhibitors such as phosphono- DAP analogues. These generally show little or no antibacterial activity although the meso-compound (61b) inhibits the growth of Salmonella tryphimurium at 1 pg ml-l.This organism has a low intracellular pool of DAP,150 and growth inhibition is reversed by LL-DAP @a) meso-DAP (8b) and L-cysteine suggesting phosphono-DAP uptake is via the usual cystine mechanism.lo7 The tripeptide (83) is a more effective growth inhibitor and is active against a wider range of bacteria (e.g. MICs of 4-32 pg ml-' versus E. coli and Citrobacter freundii). Other compounds targeted against DAP biosynthesis have been tested. N-Amino (59) and N-hydroxy-DAP (58) good inhibitors of meso-DAP epimerase and poor substrates of meso-DAP dehydrogenase inhibit the growth of Bacillus megaterium at 20 pg m1-l' while y-methylene-DAP (67) (not a particularly good inhibitor of any of the later enzymes) inhibits the growth of E.coli and Pseudomonas aeruginosa at 4 pg ml-l. The heterocyclic dehydrogenase inhibitor (62) does not inhibit growth of Proteus vulgaris or Corynebacterium glutamicum which do not require the dehydrogenase for DAP biosynthesis but it inhibits growth of Bacillus sphaericus which does. Inhibitors of meso-DAP epimerase such as P-chloro-DAP (54) and P-fluoro-DAP (56) stereoisomers are inactive or weak growth inhibitors of E. coli although where activity is seen it is reversed in the presence of DAP.lol Other similar compounds can actually substitute for DAP in mutants auxotrophic for DAP. Compounds such as /I-hydroxy-DAP (57) a very weak inhibitor of the epimerase can be incorporated into the peptidoglycan of E.coli in the absence of DAP. It is thought that the promiscuity of the DAP-condensing enzyme acting during the synthesis of muramyl peptides is responsible. Lanthionine (68) y-methyl-DAP (84) and cystathionine (85) may also be incorp~rated.'~~~~~~ LL-DAP (8a) can also be incorporated into the peptidoglycan of E. coli instead of meso- DAP (8b) in dapF mutants lacking meso-DAP-epimerase.15* Indeed the engineered increase in biosynthesis of alternative substrates for the DAP-condensing enzyme can overcome the absence of DAP in bacterial strains in which dapA (coding for L-DHDP synthase) has been deleted although L-lysine must still be supplied to allow protein ~ynthesis.'~~ 4 Overview Investigations into a wide range of bacteria have revealed two main routes between L-THDP (5) and meso-DAP (8b) directly via the dehydrogenase or indirectly via acylated intermediates.The succinylated and acetylated pathways may be quite distinct since acetylated intermediates are very poor substrates for at least two of the enzymes from the succinyl-blocked pathway. Further work is required on the enzymes from the acetylated pathway chiefly occurring in Bacillus spp. Some enzymes from plants are still 'missing' in particular those linking L-THDP (5) with LL-DAP (8b). The plant enzymes studied to date are remarkably similar in catalytic mechanism to their bacterial homologues although in most cases plant enzymes tend to be more highly regu1ated.l' Understanding of the underlying regulation processes has led to expression of genes for unregulated lysine biosynthetic enzymes in transgenic organ- isms.This has allowed the production of plant and bacterial strains with enhanced lysine production. The enzymes of the DAP pathway to L-lysine have provided many challenges to chemists and biochemists. At the same time novel features of enzyme mechanism and structure have been revealed. For example the stereochemical course of meso- DAP dehydrogenase is unique amongst dehydrogenases. Other enzymes are also very unusual meso-DAP decarboxylases are not related to most other PLP dependent decarboxylases from a structural or stereochemical viewpoint. meso-DAP epimerase a member of the small class of 'two-base' amino acid racemases is another fascinating enzyme and many mechanistic questions have yet to be answered.These three functionally unrelated enzymes are of particular interest for the way the stereo- 42 chemistry of the non-reacting substrate a-carbon controls the substrate specificity and for the apparent very tight dis-crimination for the DAP skeleton. Until recently attention has focused mainly on those enzymes of the pathway where substrates or proteins have been available. The lack of readily available substrates has stimulated efforts towards synthesis of the three optically pure isomers of DAP (8a-c).15s-159 Synthetic effort in this area has been considerable and has resulted in the generation of a library of DAP analogues.Development of specific inhibitors for lysine biosynthetic enzymes has also relied heavily on novel syntheses. In many cases the elucidation of mechanism and the de- velopment of substrates and inhibitors has occurred syner- gistically. Molecular biological techniques are beginning to provide DAP pathway enzymes for studies requiring consistent sources of protein such as crystallography. These techniques will also allow site-directed mutagenesis to facilitate further mechanistic studies. Coupled with the bank of synthetic substrates and inhibitors these methods will lead inevitably to a better understanding of the structures and functions of these enzymes. This will further aid the development of potential inhibitors especially for those enzymes towards the end of the pathway where substrate specificity appears to be very tight.Better candidates for inhibition may ultimately be enzymes from the middle of the pathway such as the succinyltransferase and aminotransferase. Substrate specificity of these enzymes is less rigorous and very potent (nM range) inhibitors have been developed. Additionally since some compounds with DAP skeletons can be incorporated into peptidoglycan it would be beneficial to block DAP biosynthesis at an early stage. In conclusion while potent broad spectrum antimicrobial compounds have not yet emerged from the study of DAP pathway inhibition the scene is set for rapid development. 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