Enzymes acting on peptides containing d -amino acid

JOURNAL OF BIOSCIENCEAND BIOENGINEERING Vol. 89, No. 4, 295-306. 2000

REVIEW

Enzymes Acting on Peptides Containing D-Amino Acid YASUHISA Biotechnology

ASANO* AND TINA L. LijBBEHijSEN

Research Center, Toyama Prefectural

Uniyersity,

5180 Kurokawa,

Kosugi,

Toyama 939-0398, Japan

Received 14 January 2OOO/Accepted21 February 2000

Mainly microorganisms but only a few higher organisms are presently known to express enzymes that hydrolyze peptides containing ~-amino acids. These enzymes can be involved in proceedings at the bacterial cell wall, in either assembly or modification, and thus cause resistance to glycopeptide antibiotics, or mediate resistance against blactam antibiotics. In other cases the in vivo function is still unknown. New enzymes screened from nature, such as D-aminopeptidase, o-amino acid amidase, alkaline o-peptidase or o-aminoacylase, offer potential application in the production of o-amino acids, the synthesis of o-amino acid oligomers by promoting the reversed reaction under appropriate conditions, or in the field of semi-synthetic antibiotics. [Key words: D-amino acid, D-aminoacylase, D-aminopeptidase, alkaline D-peptidase, j-lactamase, DD-carboxypeptidase, penicillin-binding protein, vancomycin, Ochrobactrum anthropi, Bacillus cereus, Bacillus sub& Enterociccus] The proteins of living organisms are mostly composed of L-amino acids. D-Amino acids are rarely found except in microorganisms. It has been thought that D-amino acids occur exclusively in bacteria, where they are utilized as building blocks for specialized molecules like antibiotics or cell wall compounds. With recent advances in analytical methodology, however, it has been revealed that more D-amino acids than previously thought are present in nature, implying that several unknown metabolisms of D-amino acids still exist. In this review, the occurrence of D-amino acids in nature is covered, and

tion. D-Ala was found in metabolically stable proteins of the human nervous system at higher concentration in Alzheimer brain than in the respective regions of unaffected brains (5). Other examples of their involvement in aging processes include the racemization of L- to Dasparatyl in erythrocytes (6) and the accumulation of DAsp in aging lens protein (7). Asp-151 in nA-crystallin, the major protein of the mammalian lens, has been reported to be converted into the D-form not by racemization but by stereoconversion (8). The rate is dependent on the formation of succinimide which itself proceeds at a high rate in the presence of a small residue such as Gly, Ala or Ser, at the carboxy-terminus of Asp-151 (9, 10). Another peptide containing D-amino acids was reported, the opioid peptide dermorphin secreted by the frog Phyllomedusa sauvagei (11). D-Amino acids were also found in cephalopods. For a review of D-amino acid-containing peptides from frogs and molluscs see reference (12). D-Amino acids in microorganisms One of the bestknown examples of peptides containing D-amino acids are probably the building blocks of the bacterial cell wall. The cell envelope is stabilized by an exoskeleton consisting of murein (peptidoglycan), a long-chain polymer of alternating N-acetylglucosamine and D-acetyl muramic acid. Adjacent macromolecules are linked by peptide bridges, and the number and composition of their amino acid residues vary according to the bacterial species. Amongst those, D-Glu and D-Ala are found. This structure is present only in bacteria and its uniqueness can be regarded as bacterial “Achilles heel”, offering a target for antimicrobial agents such as /-lactam antibiotics or vancomycin. The former inhibits the enzymes specifically involved in the formation and cleavage of the DD-peptide bonds (13), the latter, which inhibits crosslinking, was considered to be a last resort in fighting the constantly growing number of multi-drug resistant grampositive bacteria. But in the past decade, clinically sig-

recent findings on D-amino acids in mammalian cells are briefly introduced. The main focus is on bacterial

enzymes and their possible application, as few reports on D-amino acids of higher organisms exist at present. OCCURRENCE

OF D-AMINO

ACIDS

D-Amino acids in higher organisms Several D-amino acids were detected in mammalian cells. The synthesis of ~-Asp in pheochrimocytoma cells (PC12) could be demonstrated by time-dependent accumulation in culture (1). Free D-amino acids were found in mammalian tissues, and the presence of D-Ser (2), D-Ala and ~-Asp in the human brain has been reported (comprising up to 30% of the free amino acids) (3). Furthermore, they are prominent in ventricular cerebrospinal fluid (4). A few examples are known where by post-transcriptional modification of amino acids from a peptide chain are partially converted from the L- into the D-form. Due to their possible role in Alzheimer’s disease, aged proteins containing D-amino acids have attracted increasing atten*Corresponding author. Abbreviations: ADEPT, antibody-directed enzyme prodrug therapy; ADP, alkaline D-peptidase; DAP, D-aminopeptidase; DAA, Dstereospecific amino acid arnidase; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; EDTA, ethylenediarrdnetetraacetic acid; HPLC, highpressure liquid chromatography; IS, insertion sequence; MIC, mobile insertion cassette; Lac, lactic acid; PBP, penicillin-binding protein; PCMB, p-chloromercuribenzoic acid; van, vancomycin.

nificant vancomycin resistant strains have appeared, leading to vast research efforts to determine the underlying

mechanisms. In some species of archaea, high concentra295

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ASANO AND LijBBEHiiSEN

tions of free D-Ser (12-13%), ~-Asp (4-7%) and D-Pro (3-4x) were found (14). The following sources for dipeptides of D-Ala have been reported: rice plants (15), the peptide antibiotic gramidicin S, tyrosidine, and bacitratin (16). Microcystins are a group of cyclic heptapeptide toxins produced by cyanobacteria. Of the 50 variants of microcystin identified to date, the most abundant is microcystin LR, containing D-Ala, D-isoAsn, and DisoGln. In Sphingomonas sp., novel enzymes acting on the hydrolysis of the microcystin LR were discovered. Microcystinase catalyzes the ring opening reaction on the cyclic peptid5 yielding a linear peptide, which is then degraded by Enzyme 2” at the carboxy-terminus site of D-Ala residue to give fragmented peptides. At the present stage, the enzymes have not been characterized fully (17). Opines are categorized as secondary amine dicarboxylic acids (octopine and nopaline families), mannityl opines (agropine) and phosphorylated sugars (agrocinopine). They occur in crown gall tumor tissues in plants induced by an infection of Agrobacterium tumefaciens and muscle tissue of marine invertebrate. The first category, secondary amine dicarboxylic acids, contains D-amino acid structures that have been reviewed recently (18). Involvement of racemases in the formation of D-amino Except for a cofactor-independent serine isomacids erase isolated from the venom of the Agelenopsis aperta spider (19), mostly racemases have been described. Only recently the biosynthesis of n-Ser, which probably acts as a neuromodulator, was discovered in rat brain, and a pyridoxal 5’-phosphate requiring serine racemase could be purified to homogeneity (20, 21). The specific activity of the purified enzyme was as low as 5 pmol L-Ser/mg per h. The enzyme is highly selective for L-Ser, only 1.5% as much activity is displayed toward L-Ala. Another serine racemase was obtained from silkworm (22). In mammals, n-Ser is present at around 0.4 /*mol/g wet weight and the D/L ratio of Ser is as high as 0.4. A D/L ratio of 0.62 was found in cerebral gray matter of the bull (23). However, racemase activity has primarily been reported for a range of bacteria, as unicellular organisms still remain the best investigated source for n-amino acids at the moment. A serine racemase, VanT, involved in vanC phenotype Enterococcus gallinarum BM4174 was described recently. Like the above mentioned serine racemase, it bears a putative pyridoxal attachment site that is also highly conserved in alanine racemase (24). High levels of ~-Asp (more than 40% of total aspartic acid) have been reported in hyperthermophilic archaea. Aspartate racemase activity was exhibited and aspartate racemase homologous genes could be identified by PCR. Additionally, D-enantiomers of Leu, Phe and Lys were detected in Thermococcusstrains and in Desulfurococcus sp. strain SY. Their function is still unknown (25). Poly y-glutamate appears in several Bacilli. It is present in the outer capsule of B. anthracis (26) and as part of the mucous material on fermented soybeans (natto), a typical Japanese food. It consists of 50-80% D-Glu and 20-50% L-Glu (27). The polymer promises broad industrial application as a degradable material in various areas or as a hydrogel (28, 29). A three gene encoded enzyme system in Bacillus subtilis (natto) IF0 3336 enabling the synthesis of poly y-glutamate, has been well characterized, and a glutamate racemase described (27, 30).

PRODUCTION

AND USAGE OF D-AMINO ACIDS

acids are important starting materials for various pharmaceuticals, herbicides and food additives: they act as intermediates for the preparation of p-lactam antibiotics such as semi-synthetic cephalosporins and penicillins (31) or, like D-Ala, play an important role as a synthetic sweetener such as alitame (32, 33). Synthesis of biologically active substances such as peptide hormones or antibiotics incorporating o-amino acids instead of their L-counterparts might lead to metabolically stable and long acting products. Further information about this can be found in (34) and (35). Most L-amino acids are nowadays produced fermentatively, whereas almost all o-amino acids including p-hydroxy-D-phenylglycine are obtained by enzymatic methods. Only for DAla has a fermentative preparation been reported (36). Enzyme catalyzed methods to manufacture D-amino acids have recently been reviewed by Yagasaki and Ozaki (37). New enzymatic methods to synthesize n-amino acids or related peptides are expected to be developed in the near future. The most recent examples include the optical resolution of N-acylamino acids (38), asymmetric hydrolysis of hydantoins (31), the employment of four enzymes, n-amino acid aminotransferase, alanine racemase, alanine dehydrogenase, and formate dehydrogenase with a-keto acid, as a starting material (39, 40), and the optical resolution of racemic amino acid amides (40). Under appropriate conditions, a few of the enzymes described here could also successfully be applied as a catalyst in peptide synthesis. Not only does enzymatic synthesis offer an interesting alternative to chemical synthesis concerning reaction conditions but also, in the case of a o-stereospecific enzyme, will lead to D-oligomers thus elegantly avoiding complicated protection and deprotection steps as might be necessary with the conventional method. This is a major advantage over similar attempts using L-specific proteolytic enzymes like subtilisin (41) or u-chymotrypsin (42) in organic solvents to relax the stereospecificity. D-hIhO

ENZYMES ACTING ACID-CONTAINING

ON D-AMINO PEPTIDES

D-Enzyme By total chemical synthesis, replacing the amino acids with either the corresponding D- or Lforms, the two enantiomers of the HIV-l protease were prepared (43). Reversed-phase HPLC gave identical retention times, with both products having the same amino acid sequence and molecular weight. Only in chiral interaction differences appeared. The enzymatic properties were evaluated with a fluorogenic assay. The enzymes displayed reciprocal chiral substrate specificity for the substrate, a hexapeptide analog of the natural GAG cleavage site. Thus, the o-enzyme cleaved merely the Damino acid peptide, and the L-form was active on the Lsubstrate only. Similarly, enantiomers of the pseudoinhibitor MVTlOl were effective only against the corresponding enzyme enantiomer whereas an achiral inhibitor, Evans Blue, inhibited of both enzymes. In vivo, protease inhibitors prevent the final processing of important HIV proteins and thus at least temporarily block the development of AIDS. Further data about enzyme activity for other substrates, K,, Ki or V,,,, values have not been presented.

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ENZYMES ACTING ON PEPTIDES CONTAINING

D-Aminoacylase The enzyme specifically deacetylates N-acetyl-D-amino acids while leaving the corresponding L-amino acid derivates in their acetylated form and therefore is useful for the resolution of racemic N-acyl-DL-amino acids. In industry, the usage of acylase has primarily been aimed at obtaining L-amino acids, the first commercial process producing L-Met was developed by Tanabe Seiyaku Company in Japan using an immobilized aminoacylase from Aspergillus oryzae as the catalyst (38). Only a few D-aminoacylases have been reported in bacteria such as Pseudomonas, Streptomyces, and Alcaligenes. Little is known about their physiological function. The first enzyme was described by Kameda et al. The resolution of N-benzoyl-DL-Phe with a partially purified enzyme from Pseudomonaswas achieved although the activity was rather poor, properties of the enzyme were not given (44, 45). Partially purified D-aminoacylase from Streptomyces tuirus produced good results in the resolution of N-acetyl-DLPhe-Gly. From a 50mM racemic mixture, the D-form could be obtained at 99.9% optical purity after incubation with 26.5 units of the enzyme for 6 h (46). Details about the enzyme were not available. D-Aminoacylase has also been reported in various strains of Alcaligenes: A. denitr@cans DA181 (47), A. faecalis (48) and A. xylosoxydans subsp. xylosoxydans A-6 (49). The latter acts on N-acetyl-derivates of D-Leu, D-Phe, D-NorLeu, D-Met and D-Val (K, 9.8 mM) but little of the native enzyme was produced. Thus the encoding gene was cloned, expressed (50) and overproduced in Escherichia coli (5 l), this finally allowed its purification. Table 1 summarizes some of the general properties of the three D-aminoacylases from Alcaligenes sp. A novel enzyme, N-acylamino acid racemase which catalyzes the interconversion of the enantiomers of Nacylamino acid, was found in Amycolatopsis sp.TS-l-60 (52). It can be assumed that the optical resolution process will be much improved to directly yield D-amino acid by the use of these enzymes. Dn-Hydrolyzing enzymes involved in cell wall synthesis A number of cytoplasmic membrane enzymes are involved in the construction of the bacterial cell wall. They are the targets of penicillin, and accordingly are called penicillin-binding proteins (PBP). High- M, PBPs (PBP-la, -lb, -2, -3) are bifunctional and combine transglycosylase and DD-transpeptidase activity. They catalyze the elongation of the glycan chain and are responsible for the peptidoglycan cross-linking (53). The latter mechanism causes the rupture of the DAla-D-Ala bond in the pentapeptide units. Whereas

D-AMINO ACID

297

high-M, PBPs are essential, the loss of the low- M, PBPs interestingly can be tolerated, the bacteria will grow normally (54-57). So while their in uivo function remains unknown, their in vitro traits have been researched. PBP-5, and -6 and the recently discovered DacD (=PBP 6b) (54) are DD-carboxypeptidases (58). PBP-4, -7, and -8 are DD-endopeptidases (murein meso-diaminopimelateD-alanine DD-endopeptidase activity) with PBP 4 also displaying a weak DD-carboxypeptidase activity. PBPs have been reviewed in detail in (53), and (59, 60) summarize the multi-modular PBPs. Do-Carboxypeptidase This enzyme catalyzes a step in the cell wall biosynthesis by hydrolyzing the D-AlaD-Ala bonds using a reactive site serine residue (53, 58). Acting on pentapeptide muropeptides, it thus controls the extent of crosslinking between adjacent peptidoglycan macromolecules (59). Inhibition of these enzymes by ,l-lactam antibiotics leads to stagnation of cell growth. A rare example of a cytoplasma-located enzyme is Dn-carboxypeptidase from Streptomyces R61. Nonetheless it serves as a model for the normally membrane-bound low-M, DD-peptidases and has been reviewed in detail (58). Many mutational and kinetical data are available (61-63). The refined crystallographic structure of the enzyme has been solved at 1.6A resolution (64). A further example for a DD-carboxypeptidase is VanY, an enzyme involved in vancomycin resistance. It will be described in one of the following sections. Only recently a penicillin resistant DD-carboxypeptidase has been described in a gram-negative bacterium, Myxococcus xanthus, for the first time (65). It did not reveal significant similarity to DD-carboxypeptidases PBP5 and PBP6. Data about substrate specificity is not available at present. Application to peptide synthesis Utilizing muramoylpentapeptide carboxypeptidase (EC 3.4.17.8) isolated from Bacillus subtilis, a tripeptide containing two D-Ala residues could be synthesized (66). To the substrate Na,NE-diacetyl+Lys-D-Ala-D-Lac, D-Ala-OCH3 was added as the nucleophile. The resulting products were DLac and Na,NE-diacetyl+Lys-D-Ala and the tripeptide ND:,Nt-diacetyl-L-Lys-(D-Ala)2-OCH3. The stereospecificity of the reaction could be demonstrated by addition of L-Ala-OCH3 as the nucleophile. In this case no product formation could be observed. A tripeptide yield of approximately 40% was obtainable after 10 h incubation. ,!SLactamase (EC 3.5.2.6) ,&Lactamases cause the hydrolysis of the amide bond in the ,3-lactam ring of penicillins and cephalosporins and accordingly are responsible for ,3-lactam resistance in many bacteria.

TABLE 1. Properties of D-aminoacylases from Alcaligenes A. denitrificans DA18ls (47)

‘w PI Optimum pH Specific activity (U/mg)

58000 4.4 7.5 2380 (N-acetyl-D-Met, 37°C)

A. faecalis DA1 (48)

55000 5.4 1.5 580 (N-acetyl-D-Met, 37°C)

A. xylosoxydans subsp. xylosoxydans A6 (49)

52000 7.0 990

Inhibition Yes Yes Zn2+ (1 mM) Yes Yes EDTA (1 mM) Activation No No Co2+ (1 mM) The enzymes from Akaligenes sp. displayed only negligible activity toward N-acetyk-amino acids, whereas those from Streptomyces and Pseudomonas acted with less strict stereospecificity.

298

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AND LiiBBEHiiSEN

Based upon amino acid sequence, they are separated into four major classes:class A serine penicillases, class B Zn2+-dependentp-lactamases, class C serine cephalosporinases, and class D oxacillin-hydrolyzing serine enzymes. As a large number of detailed reviews are already available, we merely refer to several publications. Serine b-lactamase is described in (53), and mechanisms of action and inhibition are covered in (67). (68) deals with the variety and distribution of ,f3-lactamase among clinical isolates, and (69-71) with characterization and classification; according to amino acid sequencea phylogenetic tree was proposed (72). As referred to later, alkaline Dpeptidaseshowed a distinct /3-lactamaseactivity. D-Aminopeptidase (EC 3.4.11.19) The screening for a new enzyme catalyzing the synthesis of D-Ala-Nalkyl amides, similar to the unique structure of alitame (L-Asp-D-Ala thiethane amide), an artificial sweetener developed by Pfizer Inc. (32, 33), led to the discovery of D-aminopeptidase (DAP). It was isolated from Ochrobactrum anthropi sp. SCRC Cl-38. DAP is expressedconstitutively. It is an intracellular enzyme acting on low molecular weight D-amino acid amide, ester, and oligopeptides containing D-Ala or D-Ser (73-77). The V,,/K, values decreasedmarkedly as the substituents become larger, while they change little with increasing peptide: (D-Ala)4 was the best substrate. Aminopeptidases release the amino-terminal residue from peptide substrates.The stereochemistryof the second amino acid from the amino-terminus is of no importance. At present only enzymesfrom 0. anthropi are known to act on peptides containing amino-terminal D-residues. Their primary structure displays a strong similarity to DD-carboxypeptidase and p-lactamase (78) and thus DAP can be considered a penicillin-recognizing enzyme (79). Significant inhibition was caused by ,!3-lactamcompounds although they are not suitable as a substrate. With a relative molecular weight of 122,000, DAP is composed of two identical subunits (M,=59,000). The active center bears the essential amino acid sequenceSXXK, which is a conserved motif in penicillin-recognizing enzymes. By generatingseveral mutant enzymesat the active center, it was shown that Ser 62 is responsible for the nucleophilic attack on the carbon of the peptide bond to be hydrolysed in a substrate or the p-lactam carbonyl carbon in an antibiotic molecule. Taking into account the enzyme’s high stereospecificity and also its rather narrow substrate specificity, accepting merely low molecular weight amino acid derivatives, severalconceivablephysiological roles could be suggested(73). It may hydrolyze peptidoglycan fragments composed of D-Ala and Gly (80), the dipeptide D-Ala-D-Ala, which is a product of the D-alanyl-D-alanine ligase (81), or degrade naturally occurring D-amino acid derivatives as synthesizedin the rice plant (15). From 0. anthropi LMG7991, two further D-aminopeptidases (A and B) could be isolated (82), both of which were active on D-Ala-p-nitroanilide. D-kninopeptidase B was purified to 90% homogeneity and its amino-terminal sequenceexhibited 60% homology with the DAP enzyme from 0. anthropi SCRC Cl-38 (74). The second enzyme, D-aminopeptidase A, hydrolyzes p-nitroanilide derivatives of Gly and D- and L-Ala with a preference for the D-substrate whereas the L-configuration is utilized more efficiently in the case of free amino-terminals. Examinations with tripeptides demonstrated the release of the amino-terminal residue first, thus the enzyme has

J. BIOSCI. BIOENG.,

an L-aminopeptidaseactivity with a hydrolysis rate for r,-Ala-Gly-Gly of 1.25/Imol/min per mg enzyme compared to only 0.04 for the o-analog. However, on ester and amide substrates, a higher rate of hydrolysis could be observed for the D-substrateswith 0.23 pmol/min per mg enzyme for D-Ala-NH* and 0.09 for L-Ala-NH2 or when comparing L-Ala-OCH3 and D-Ala-OCH3 with rates of 0.09 and 1.2 respectively (83), thus D-aminopeptidase A possessesa D-amidase/D-esterase activity as well. Crystallization and preliminary X-ray analysis of the enzyme have been performed (84). Properties of DAP and D-aminopeptidaseA are compared in Table 2. Application to synthesis of o-amino acids and their derivatives The DAP gene was cloned into E. coli and an expressionplasmid was constructed (85). The enzyme comprised about 30% of the extractable cellular protein. Whole transformant cells or cell-free extract were used as a catalyst to synthesizeD-amino acids. Starting from 5.0 M racemic alanine amide HCl, 220 g/l (2.5 M) of D-Ala was obtained after 4.5 h with whole cells. Usage of cell-free extracts led to the synthesisof D2-aminobutyric acid, D-Met, D-NorVal and D-NorLeu from their corresponding amides with a yield of 100, 97, 86 and 100x, respectively. The enzyme-catalyzedsynthesis of D-Ala oligomers was also examined (86). In nonaqueous media, best results were achieved with watersaturated toluene, the urethane-pre-polymer PU-6 immobilized enzyme synthesized(D-Ala)a and (D-Ala)3 from D-Ala-OCH3 HCI with a yield of 56% and 6%, respectively. Products of higher molecular weight could not be obtained. K,, of the reaction (19,500minr) was found to be several ten thousand times that of known oligomerization procedures for amino acid esters. Additionally, the immobilized enzyme proved to be a catalyst for the stereospecific synthesis of D-Ala-N-alkyl amide from an amine and either D-amino acid amide or Damino acid methylester. With DL-Ala-OCH3 HCl as the acyl donor, D-Ala-3-aminopentanecould be obtained in 45.2% yield with an optical purity of more than 99% e.e. (87). Mutants of DAP with increasedthermal stability were generated(88). As shown in Fig. 1, when the intermediate D-amino acyl-enzyme complex is hydrolyzed, a D-amino acid is formed. When the complex is deacylated by a nucleophile with a higher nucleophilicity than water, a peptide bond with the D-amino acid will be formed. D-Ala-Nalkyl amide can be synthesizedby reacting amine to racemic alanine amide or ester. Such an enzyme would also be useful for preparing D-amino acid by an asymmetric hydrolysis from racemic amino acid amides prepared by a hydration of amino nitrile, which is synthesized from an aldehyde and cyanide by the Strecker method. By this route, n-amino acids may be prepared in three steps, as demonstratedin Fig.2, less than the Damino acylaseroute, which requires four steps. Application to prodrugs In a study aimed to improve the effectiveness of the anticancer drug methotrexate, DAP was consideredto be a potential candidate for antibody-directed enzyme prodrug therapy (ADEPT). As most drugs are not selective for tumor tissue only and also harm normal tissue, studies are underway to develop inactive prodrugs that will be activated at the tumor site. With the ADEPT strategy, an antibody conjugated enzymecan be located on the tumor surface (89). Alkaline D-peptidase (D-stereospecific peptide hydrolase, EC 3.4.11.~) A screeningby Asano and cowor-

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D-AMINO ACID

299

TABLE 2. Properties of DAP and DAP-A from 0. anthropi E.C. Nr Strain M Nr of subunits IV, of subunits Optimum PH Temp. (“C) PI Apparent K,,, (mM)

n-Aminopeptidase (DAP) (73) 3.4.11.19 0. anthropi SCRC Cl-38 122000 2 59ooo

26565. 13737

8.5 45 4.2

5.0

Rate of hydrolysis (,umol/min mg enzyme) Inhibition

Not inhibited by

Requirements for the substrate N terminus

0. anthropi LMG 7991

45000

2 (a?)

0.51 0.65 D-Ala-D-Ala 10.2 D-Ala-D-&a-D-Ala 0.57 D-Ala-D-Ala-D-Ma-D-Ala 0.32 D-Ala amide 600 n-Ala-Gly 1000 D-Ala-D-Ma-D-Ma 866 20-50x: Ca2+, NiZ+, Cd2+, Cu2 +, Zn2+(l mM) 5,5’-dithiobis(2-nitrobenzoic acid) (1 mM); Nethylmaleimide (10 mM) 25-100x: Ag+, Hg2+ (O.O2mM), PCMB (0.074 mM) Li+, Na+, Ba2+, Bar+, Fe2+, MgZ+, Sn2+, Al”, Fe3+, Pb3+, EDTA, 8-oxyquionoline, a,a’-dipyridyl, o-phenanthroline, sodium azide (1 mM); KCN (0.2 mM); monoiodoacetate, phenyhnethylsulfonyl fluoride (1 mM); pepstatin A, leupeptin (20 ,nM) n-configuration preferred. Slow rates with some Lderivatives D-Ala-pNA

D-Ala amide

kers for n-peptide-degradingm icroorganismswith synthetic (D-Phe)das a substrateled to discovery the strain Bacillus cereus DFCB. From the culture broth, an extracellular n-stereospecificendopeptidase,alkaline D-peptidase (ADP), was isolated and purified to homogeneity (90-92). The enzyme was strictly stereospecifictowards oligopeptidescomposedof D-Phe such as (D-Phe),, (relative activity: 100x, K,,, value: 0.398mM), (D-Phe)3(90%, O .l27mM), (D-Phe)6(1.8%) forming (D-Phe)z and (DPhe). Its m o d e of action was examinedand it was shown that it acts as a n-stereospecificdipeptidyl endopeptidase,hydrolyzing the secondbond from the a m ino-terminus first. Maximal activity was displayed at about pH 10.3, accordingly the enzymewas named alkaline Dpeptidase.A weak p-lactamaseactivity towards a m p icillin (8.9%, 73.1mM) and penicillin G (9.7%, 48.9mM) could be detected, but neither DD-carboxypeptidase nor DAP activities were found. The iV& of the enzymewas

-

DAP-A (83)

o-Ala-pNA r.-Ala-pNA

0.54 0.36

Gly-D-Ala Gly-L-Ala o-Ala-Gly-Gly

0.02 0.09 0.04

Antipain, aprotinin, bestatin, chymostatin, fransepoxysuccinyl-L-leucylamido-(4-gua.nido)butane(E64), EDTA, leupeptin, pefabloc SC, o-phenanthroline Free a-amino group L-configuration required for optimal activity Basic amino acids (Arg, Lys) > Phe > aliphatic amino acids (Leu, Gly, Ala) > hydroxylated amino acid CW Peptides with His or Trp residue are not hydrolyzed

37,952. It is composedof a single peptide chain. After cloning and sequencing,similarities in primary structure to severalother enzymeswere found, including nn-carboxypeptidasefrom Streptomyces R61 (35.0% identical over 346 a m ino acids), PBPs and class C P-lactamases. The typical sequenceSXXK is conservedin all of those enzymes,therefore ADP can be categorizedas one of the “Penicillin-recognizing enzymes” (79). Recently, Komeda et al. sequencedthe adp gene and found that three similar genesare tandemly located in the B. cereus genome (Komeda, H. et ai., Abstr. Annu. Meet. Sot. Biosci. Bioeng., Japan, p. 247, 1998) A study by M a h illon et al. on naturally occurring mobile insertion cassettes(MIC) from B. cereus showedthe existenceof two left IS231 ends flanking the adp gene. The ADP activity was not only present in the original isolate, but detectedin all, but one B. cereusstrain, test-

Amino Acid RJH-COX Hx-g3

I?A

R%.

COOH

RCHO -g?tiE‘i. R

H~.kd+@md~;$

RCH-COOH

COO”

;&,

RCH-CONH-R’

FIG. 1. Formation of acyl-enzyme complex.

D-Amlno

FIG. 2. Synthesis of n-amino acid from aldehyde.

Acid

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ed. Effective tram-mobilization is causedby IS231 transposase(93). Application to D-Phe oligopeptide synthesis Kinetically controlled peptide synthesis could be performed with ADP (94). By placing the PCR-amplified adp under the promoter of pKK223-3, the expression plasmid pKADP was constructed and then transformed into E. coli JM109. ADP was purified to a specific activity of 2.0 units/mg. In an alkaline aqueous medium @ H 11.5) in the presenceof DMSO and MgS04, the enzyme was able to polymerize D-PheOCH3 HCl. The formation of (D-Phe)z,(D-Pheb and (D-Phe)4could be confirmed and quantified by HPLC. Maximum yields of (D-Phe)zand (D-Phe)3 of 12.5% and 6.5%, respectively, were obtained at a substrate concentration of 50mM. At higher concentrations, the yield decreased,probably caused by substrate inhibition of ADP. Studies on the time course of oligomerization lead to the production of (D-Phe)z and (D-Phe),, in 25.4% and 8.6% yield, respectively, when 50 m M of the substrate was incubated for 8 h with ADP (2.0U/ml and 0.4 U/ml, respectively) in 1 O O m M triethylamine-HCI (pH 11.5). ~-Amino acid amidase Increasingattention has been paid to D-amino acid amidasesas they can be used as a catalyst in the stereospecificproduction of D-amino acids by hydrolyzing D-amino acid amides. Ozaki et al. (95) of Kyowa Hakko Kogyo screenedfor D-alanine amide amidase producers to prepare D-Ala from Dr.-Ala-NH2 and isolated Arthrobacter sp. NJ-26. The strain possessedan amide hydrolase with a high D-stereospecificitytowards alanine amide and also towards Gly-NH2. A low activity for L-Ala-NH2 could be detected, with a relative velocity of only 0.67% (K,, 26.1 mM) of that toward the Damide (K,,,, 4.19mM). Reactivity for other amide substrates was very weak. After purification near to homogeneity (about 260-fold), the relative molecular weight was calculated to be approximately 67,000 and 51,000 by gel filtration and SDS-PAGE, respectively. The enzyme showed maximal activity at 45°C and pH 7.5. Neither a requirement for metal ions nor for cofactors could be detected. Activity was effectively induced by Ala-NH2 (D-, L-, DL-), whereasthe hydrolyzed products, D-, L-, and DL-Ala or other amide substrates could not cause induction. There was no evidence for hydrolyzing activity on dipeptides or amino acid esters. Substrate inhibition was not observedat up to 3.4M of DL-Ala-NH2. Product inhibition of the D-amidase activity was not observed at all. After optimization of the culture conditions, log/l of wet cells in a total volume of 2 1 kinetically resolved 210 g/l (2.4 M) of DL-Ala-NH2 to give 105g/l of D-Ala with an optical purity of more than 99% e.e. A D-stereospecificamino acid amidase (DAA) accepting a broader range of substrates was isolated from 0. anthropi SV3 (96, 97). This enzyme catalyzes the stereospecifichydrolysis of o-amino acid amides yielding D-amino acids and ammonia. Amongst its major substrates were D-Phe-NH2 (relative activity: lOO%), D-TyrNH2 (98%), D-Trp-NH2 (79%) D-Leu-NH2 (37%) and DAla-NH2 (23%). K, values for the first three substrates were calculated to be 0.44, 0.50, and 0.48, respectively. Boc-D-Ala-NH2 was inactive as a substrate, indicating that the enzyme prefers D-amino acid amides with a free n-amino group. Its A4, was estimated to be about 38,000 by gel filtration on HPLC. Maximal activity was displayed at pH 7.5 to 8.0. Inhibitory effectswere investigat-

J. BIOSCI. BIOENG.. ed, but neither chelating nor sulfhydryl reagentshad any effect on the enzyme activity. Serine protease inhibitors on the other hand, such as phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate, led to 80-95% inhibition when incubated with the enzyme. In a recent study, the DAA encoding gene has been cloned and sequenced revealing active site motifs typical for PBP and p-lactamasessuch as SXXK, YXN and H(K)XG (97). The perfect conservation of these residues suggeststhat DAA can be classified as one of the penicillin-recognizing enzymes (79). The SXXK motif was also found in DAP (78) and interestingly, in a new carboxypeptidasefrom Brevibacterium linens IF012171 (98), screened as a catalyst for regioselectivecleavageof a diol diacrylate. Despite further homology in some motifs, the enzyme was neither active towards substrates for ,&lactamases and DD-peptidaSeS nor inhibited by them. Table 3 shows some of the generalproperties, relative activities, and inhibitors of the two amidasesdescribedhere. Non-stereospecificdipeptidase (peptidyl-D-amino acid hydrolase, EC 3.4.13.17) From the digestive juice of the cephalopod Loligo vulgaris Lam., an enzyme hydrolyzing peptidyl D-amino acids was purified (99). It was only active against small peptides, acting as a carboxypeptidase. Polypeptides or amino acid derivates, amidesor esterswere not hydrolyzed. The native enzyme (A4,=140,000) consists of two subunits of h4,=104,000 and M ,=36,000, respectively.Maximum activity was displayed at pH 8.0. Neither inhibition nor increaseof enzyme activity was observedin the presenceof metal ions Co2+, Mn2+, Mg2+ or Zn2+ at a concentration of 1 m M , or EDTA at 50 m M . &-values for Gly-D-Ala, Gly-LAla, L-Ala-D-Ala, L-Ala-L-Ala, D-Leu-D-Leuand r.-Leu-LLeu have been reported as 5.2, 7.7, 2.5, 2.8, 5.4, and 8.6 m M , respectively. Membrane dipeptidase (renal membrane peptidase, EC 3.4.13.19) The enzymewas first purified to homogeneity from hog kidney in 1965 (100). Gly-Gly was hydrolyzed most effectively with a specific activity of 116pmol/min per mg. D-Leu-Gly was not attacked, GlyD-Leu was hydrolyzed (39 pmol/min per mg). Dipeptides bearing a carboxy-group at the amino-terminus were not accepted as a substrate thus indicating the requirement for a specific optical configuration at the amino-terminus. Cleavageof tripeptides, such as L-Leu-Gly-Gly, LAla-Gly-Gly or L-Ser-Gly-Gly, was not promoted by the enzyme. Activity was only displayed in the presenceof the cofactor zinc, with one atom of zinc per mole of enzyme. A D-amino acid peptide-hydrolyzing enzyme from pig kidney cortex was purified 816fold (101). With a relative molecular weight of 99,000 (two identical subunits, M ,=48,000) and maximum activity at pH7.8, this enzyme showeda high specificitytowards a variety of dipeptides bearing a D-amino acid at the carboxy-terminus. However, peptides with an amino-terminal D-residue were poor substrates.The K, values of the former indicate that a carboxy-terminal D-amino acid containing dipeptide binds to the enzyme as well as to a dipeptide with an L-residue at the same position. K, values for Gly-D-Ser, Gly-L-Serare 75, 80 m M ; r,-Leu-D-Leu,t,-LeuL-Leu 0.15, 0.16 m M ; Gly-D-ASP,Gly-~-Asp 17, 14mM. Interestingly the K, value for Gly-D-Ala (2.7 mM) is about 30 times lower than that for Gly-L-Ala (77 mM). But generally, the V,, values were lower for the D- than for the L-amino acid dipeptides (V,, values in the above

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TABLE 3. Properties of two D-amino acid amidases Organism

Arthrobacter sp. NJ-26 (95)

0. anthropi SV3 (96, 97)

M

67000 (gel filtration) 51000 (SDS) 1

40082

Nr of subunits Optimum PH 7.5 Temperature (“C) 45 5.2 PI Relative activity (%) and apparent Km (mM) (if reported) for D-Phe amide 0.64% D-Tyr amide Not tested D-Leu amide 0.37% D-Ala amide 100 % L-Ala amide 0.67% Gly amide 97.1 % Inhibition EDTA (10 mM) Zn2+, Cu2+, PCMB (1 mM)

1 9 45 5.3 100% 0.44mM 98% 0.5 m M 37%

23%

Not accepted as a substrate No Yes

Serine protease inhibitor Specific activity of final preparation

1384 units/mg (D-Ala amide, 37°C)

mentioned order in units/mg: 1600, 1800; 21, 170; 75, 160; 660, 1600). Only a few tripeptides were hydrolyzed, therefore the enzymewas classifiedas a dipeptidase. Significant activity was displayed for D-Ala-Gly-Gly and LAla-Gly-Gly at 5.7% and 5.6% of the rate of Gly-D-Ala hydrolysis, respectively,at a concentration of 10m M . As Gly but neither D- nor L-Ala were liberated from the tripeptides, it was suggestedthat the enzyme acts as a carboxypeptidase.Compared with peptidyl-D-amino acid hydrolase from the cephalopodLoligo vulgaris Lam (99) or leucine-aminopeptidase (EC 3.4.11.l), a MrP or Mg2+ requiring enzyme from swine kidney, active not only on leucine peptides but also on several D-amino acid containing amides or dipeptides (102), membrane dipeptidase has been reported to be respectively more than lo-fold, and lOOO-foldmore effective toward Gly-DAla under similar conditions at 37°C. Dn-Dipeptidase involved in vancomycin resistance

Vancomycin The glycopeptide antibiotic vancomycin acts against gram-positive bacteria and thus is a powerful tool in the treatment of infections caused by strains such as Staphylococcusaureus or Enterococcous faecalis. Interestingly, it binds to the D-Ala-D-Ala moiety of the peptidoglycan precursors thus blocking the subsequenttranspeptidation step, whereas usually antibiotics would inhibit one of the enzymesinvolved. The crosslinking betweenadjacent peptidoglycanstrandsis inhibited and as a result the bacterial cell wall is weak-

EDTA (50 mM), cu,a’-dipyridyl (5 mM) No Semicarbazide, KCN, NaNs (5 mM) No Zn2+ (1 mM) Yes 5,5’-Dithiobis(2-nitrobenzoic acid), PCMB (0.2 mM) No N-Ethylmaeleimide (5 mM) No Phenyhnethane sulfonyl fluoride (0.1 mM) Yes Diisopropyl fluorophosphate (0.1 mM) Yes 367 units/mg (D-Phe amide, 30°C)

ened. Vancomycin had turned into a last resort in eliminating multi-drug resistant strains such as MRSA, but in recent years an increasing number of clinically resistant bacteria have appeared.As a number of enzymesare involved, their basic roles are describedin Table 4. Further description can be found in the following paragraph. Five types Enzyme system in gram-positive bacteria of resistanceagainst vancomycin are known. Transposon Tn1546 (or closely related forms) mediatesVanA phenotype, high-level resistance to vancomycin and teicoplanin. Generally, the transposon is carried by a conjugative plasmid (103). For resistanceagainst vancomycin, the expression of five genes has been found necessary. Van B type strains have resistance to vancomycin but remain susceptibleto teicoplanin (104). The responsible van genes are usually part of conjugative chromosomal elements (90-250 kb) (105). The vancomycin inducible vanS/vanR, in VanB-type strains vanSB/vanRB,respectively (106), encoded two-component regulatory system (107), activates transcription of the HAXY operon. vanH codes for a dehydrogenasereducing pyruvate to D-lactate (108), which is used by the ligase VanA for synthesizing a D-Ala-D-LaCdepsipeptide(109). Accordingly resistant strains incorporate peptidoglycan terminating in the depsipeptide rather than D-Ala-D-Ala into their cell wall, resulting in a 103 to 104-fold lower affinity for the glycopeptide (108) and cell wall synthesisis able to proceed in the presenceof vancomycin. Finally, the vanX

TABLE 4. Enzymes involved in vancomycin resistance VanR Vans VanH Van4 VanX VanY

Transcriptional activator Response regulator Stimulates VanR-dependent transcription Histidine kinase (transmembrane) Reduction of pyruvate to D-Lac which is necessaryfor VanA action D-Specific cu-ketoreductase Production of depsipeptide D-Ala-D-Lac that competes with the normal D-Ala-D-Ala D-Ala-D-Lac ligase Hydrolysis of dipeptide remainders, thus enabling the accumulation of the depsipeptide D-Ala-D-Ala hydrolyzing D,D-peptidase (Zn’+reqUiring; cytoplasma located) By removal of D-Ala it converts pentapeptide to tetrapeptide DD-Carboxypeptidase (Zn2+ requiring) Production of the depsipeptide D-Ala-D-Ser D-Ala-D-Ser IigaSe (present in VanC type enterococci only) Racemization of L- to DL-serine Racemase(Membrane bound)

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gene product is required (110). It is a D-Ala-D-Ala hydrolyzing DD-peptidaselocated in the cytoplasm. As the enzymeexclusivelycleavesdipeptideswith free aminoand carboxy-termini, neither D-Ala-D-Lac, pentadepsipeptide, nor pentapeptide are suitable substrates. The catalytic hydrolysis of D-Ala-D-Lac was investigated and found to be lower than 0.005 ssl (vs. 4.7 s-i for D-AlaD-Ala) (111). Thus D-Ala-D-Ala is cleaved almost exclusively. In an examination of substrate specificity of VanX from Enterococcusfaecium expressedin E. coli, only D-Ala-Gly was accepted as further substrate, with 26% of the rate of hydrolysis of D-Ala-D-Ala (110). As a consequenceof VanX activity, pentapeptide production decreaseswhereas the synthesis of the pentadepsipeptide is favored. Several divalent metal cations are able to activate the enzyme, and it was found, that VanX is Zn2+-dependent(111). Kinetic data on VanX is given in Table 5. It has to be pointed out that in the presenceof divalent metal ions, much higher K,, values than given in the table were achieved for D-Ala-D-Ala as the substrate,the maximum being 788+79 at 1 m M Ni*+. A recent mutagenesisstudy on VanX from E. faecium identified putative enzyme ligands for zinc coordination, residuesHisl16, Asp123 and His184 (112). Not only to gain insight into the catalytic mechanism but also with drug design in m ind, inhibition studies on VanX from E. faecium were performed. Phosphinate analogs of D-Ala-D-Ala, m imicking a proposed tetrahedral intermediate during hydrolysis were studied. 3((1-Aminoethyl)phosphoryl)-2-methyl-propionicacid and two of its derivates proved to be slow binding inhibitors with a Ki of 1.5 ,uM (0.73 ,uM and 1.53,uM) (113). A further enzyme, the DD-carboxypeptidaseVanY, contributes to vancomycin resistancebut is not necessarilyrequired. High presenceof D-Ala results in increasedsynthesis of D-Ala-D-Ala. Dipeptides that escape VanX hydrolysis will therefore compete with D-Ala-D-Lat. If a significant amount is incorporated into peptidoglycan precursors, vancomycin susceptibility will be restored. VanY works in serieswith VanX: by removal of D-Ala it converts pentapeptide to tetrapeptide, thus destroying the target for vancomycin (114). In contrast to bacterial PBP DD-carboxypeptidases,VanY is penicillin-insensitive and lacks the classicalSXXK active site motif of PBPs (115). Like VanX, the enzyme is Zn2+-dependent.Other than the closely related VanA and VanB phenotypes, the intrinsically resistant VanC-type enterococci (E. gallinarum and E. casse&¶avus)possesa secondligase VanC that synthesizes D-Ala-D-Ser (116, 117), and via the membranebound VanT, the racemization of L- to DL-Ser is mediated (24). VanD-type-resistancehas been described for E. faecium B M 4339 (118), and recently, E. faecalis BM4405 was reported to be of a new type, VanE (119). Occurrence of Do-dipeptidases in gram-negative bacteria A periplasmic D-Ala-D-Ala dipeptidase (PcgL) was also identified in the gram-negativeSalmonella enterica (120). Sequenceanalysis revealed only low level identity between PcgL and VanX but typical conserved motifs including the zinc coordination residues (112) were found. Furthermore, PcgL exhibits a substrate specificity similar to that of VanX, with neither tripeptides nor N-blocked dipeptides hydrolyzed. But as gramnegative bacteria have a protective outer membrane,they are not susceptibleto vancomycin, the enzymes’physiological role must be other than hydrolyzing D-Ala-DAla. Examination of the G+C content implies that the

TABLE 5. Substrate specificity andkineticdataof VanXawith dipeptides anddepsipeptide asa substrate; datafrom (111)

D-da-D-Ala

D-Ala-D-Serb D-Ser-D-Ala

1 kO.1 2.8kO.8 1.7kO.l

12.3kO.4 4.7kO.5 1.3io.3

242

D-Ala-D-LaC

4.7 kO.2 1.8 kO.2 0.35t0.01

Notdetectable<0.005

a VanXfromE. faecium BM4147 was overexpressedin E. coli. bV m8xand KC, were obtained by using Dr.-Ala-Dr.-Seras a substrate assuming that 25% of the racemic mixture is the D,D-peptide and the other three isomers have no inhibitory effect. Activity was also displayed on D-Ma-D-Phe, D-Ala-Gly, DL-AlaDL-Val, DL-Ala-DL-Am, but no further data was reported.

pcgL gene has been incorporated into the genome by horizontal gene transfer. Nutrient acquisition, virulence and resistance to a toxic compound were suggestedas potential functions. Further VanX homologs have been identified in Streptomyces toyocaensis, E. coli and Synechocystis sp. strain PCC6803. By searchingfor the key residuesof the zincbinding motif in bacterial genomedatabases(121), VanX type open reading frames were found to display 64%, 27% and 16% similarity, respectively. Despite low overall sequencesimilarity, the dipeptidase activity and the conserved active site residues imply the usage of a similiar proteolytic mechanism. The catalytic efficiencies of VanX homologs from different strains were compared (122). For D-Ala-D-Ala, the following K, values were reported: EntVanX (E. faecafis), 80,~M; StoVanX (S. toyocaensis),4 ,uM; and DdpX (E. colz), 14,000,uM. CONCLUDING

REMARKS

As shown in this review, knowledge on the enzymes acting on D-amino acid containing peptides appears to be very much restricted compared with that acting on peptides composed of L-amino acids: there are only around ten well studied D-stereospecificenzymesand the enzymological properties have only been investigated with simple D-amino acid derivatives or oligopeptides. Their properties are summarized as follows. The stereospecificity is rather strict with the bacterial enzymes, while those of non-stereospecificdipeptidase and membrane dipeptidase of animal origin are loose: in some case racemic peptides are utilized. On the active sites of the enzymes, D-aminoacylase, class B /?-lactamase, and DD-dipeptidase involved in vancomycin resistanceare Zn*+- dependent. The class A, C, and D f3-lactamases,DD-hydrolyzingenzymesand PBP involved in cell wall synthesis,and newly found DAP, ADP, and DAA from 0. anthropi are serine enzymes,which are all structurally related and can be categorizedas Penicillinrecognizing enzymes, although DAA is not sensitive to penicillin. On the stereochemistryand mode of action of the enzymes, most of them appear to recognize the Dconfiguration of one of the amino acid and some act on the acyl group at the amino group (D-aminoacylase),or hydrolyze the carboxy terminus amide bond (DD-carboxypeptidase, DAP, ADP and DAA). The configuration of the amino acid at the C-termini of the peptides varies: DD-carboxypeptidaseand DD-dipeptidaseinvolved in vancomycin resistancerequire DD stereochemistryon the peptides, while DAP and ADP utilize peptides containing either D- or L-amino acids, with preference to D-amino

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acids. ADP is an endopeptidase acting on substrates whose N-terminal groups are masked, and this feature resemblesthat of ,&lactamasewhich hydrolyzes the /I-lactam ring. As D-amino acid-hydrolyzing enzymes are attractive biocatalysts, the developmentof methods for their application as well as the discovery of new enzymes is of great interest. Asano et al. synthesized(D-ASP)* and (DGlu)s having amide bonds between the 1-carboxylic acid and the 2-amino group in order to screen for new m icrobial degradersof unnatural n-amino acid peptides (123). M icroorganisms from soil acclimated to a medium containing the oligopeptides were successfully isolated. The octamers were consumed by the m icroorganisms. We expect that new enzymesand related cryptic biochemistry will be exploited in this manner. With further advancesin the developmentof analytical methodology (1, 29, which provides new or more sensitiveways of investigating an activity, more enzymeswill be found. A new method useful for the search of D-amino acid-containing peptides has been described recently. Based on the Edman degradation principle, the use of the fluorogenic Edman reagent 7-[(iVJV-dimethylamino)sulfonyi]-2,1,3benzoxadiazol4-yl isothiocyanate and combination of reversed-phaseand chiral stationary-phaseHPLC allows D/L configuration identification of peptides (124, 125). Another reason for the lim ited number of studies of Damino acid containing peptides seemsto be the lack of suitable commercially available substrates. Thus, prior to screening for degrading m icroorganisms and characterizing the enzymes, lengthy synthesis has to be often undertaken. But once at hand, even the usage of rather simple n-amino acid derivates resulted in the discovery of new m icrobial enzymes. ACKNOWLEDGMENTS This work was supported in part by Grants-in Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan. REFERENCES 1. Long, Z., Homma, H., Lee, J. A., Fukushiia, T., Santa, T., Iwatsubo, T., Yamada, R., and Imai, K.: Biosynthesis of Daspartate in mammalian cells. FEBS Lett., 434, 231-235 (1998). 2. Kumashiro, S., Hashimoto, A., and Nishikawa, T.: Free D-serine in post-mortem brains and spinal cords of individuals with and without neuropsychiatric diseases. Brain Res., 681, 117125 (1995). 3. Fisher, G. H., D’Aniello, A., Vetere, A., Padula, L., Cosano, G.P., and Man, E. H.: Free D-aspartate in normal and Alzheimer brain. Brain Res. Bull., 26, 983-985 (1991). 4. Fisher, G. H., Lorenzo, N., Abe, H., Fujita, E., Frey, W. H., Emory, C., Di Fiore, M. M., and D’AnieBo, A.: Free D- and L-amino acids in ventricular cerebrospinal fluid from Alzheimer and normal subjects. Amino Acids, I5, 263-269 (1998). 5. D’AnieBo. A.. Vetere. A.. Fisher. G. H.. Cusano. G.. Chavez. M., and Fetrucelli, i.: Presence of r&tnine in proteins of normal and Alzheimer human brain. Brain Res., 592, 44-48 (1992). 6. McFadden, P. N. and Clarke, S.: Methylation at D-aspartyl residues in erythrocytes: possible step in the repair of aged membrane proteins. Proc. Natl. Acad. Sci. USA, 79, 24% 2464 (1982). I. Masters, P. M., Bada, J. L., and Zigler, J. S, Jr.: Aspartic acid racemization in heavy molecular weight crystallins and water-insoluble protein from normal human lenses and cataracts. Proc.

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