Synthesis of d-phenylalanine oligopeptides catalyzed by alkaline d-peptidase from Bacillus cereus DF4-B

doi:10.1016/S1381-1177(98)00136-2

Journal of Molecular Catalysis B: Enzymatic

Volume 6, Issue 3, 11 March 1999, Pages 379-386

https://doi.org/10.1016/S1381-1177(98)00136-2 Get rights and content

Abstract

Synthesis of d-phenylalanine oligopeptides from d-phenylalanine methylester has been demonstrated by use of alkaline d-peptidase (ADP) from Bacillus cereus. An expression plasmid pKADP was constructed by placing the PCR-amplified ADP gene (adp) under the tac promoter of pKK223-3. Oligomerization of d-phenylalanine methylester by use of the purified ADP from the transformant Escherichia coli was investigated under several conditions. d-Phenylalanine dimer, (d-Phe)₂, and trimer, (d-Phe)₃, were produced in 25.4% and 8.6% yield, respectively, when 50 mM of the substrate was incubated for 8 h with ADP (2.0 U/ml and 0.4 U/ml, respectively) in 100 mM triethylamine–HCl (pH 11.5). Addition of dimethylsulfoxide to the reaction mixture resulted in the production of tetramer, (d-Phe)₄ in 6.7% yield with the decrease of the (d-Phe)₂ and (d-Phe)₃ production. This is the first study on the synthesis of d-phenylalanine oligomers by use of a d-stereospecific endopeptidase.

Introduction

Enzyme-catalyzed peptide bond formation represents a promising alternative to chemical synthesis procedures 1, 2, 3. Several attempts have been reported on the in vitro synthesis of the d-amino acid containing peptides and amides 4, 5, 6, 7, 8, 9, 10. Naturally, however, these enzymatic reactions have an inevitable drawback as they are not stereospecific for substrates with D-configurations. A d-stereospecific peptidase, d-aminopeptidase was isolated from Ochrobactrum anthropi and characterized 11, 12. The enzyme can be applied for kinetic resolution of racemic amino acid amides to yield d-amino acids 11, 13, synthesis of N-alkylamides from dl-amino acid esters and amines [14], and synthesis of d-alanine oligopeptides from d-alanine methylester [15]. We also characterized a novel extracellular d-stereospecific endopeptidase `Alkaline d-peptidase (ADP)' from a bacterium Bacillus cereus strain DF4-B, isolated with a synthetic substrate (d-Phe)₄ [16]. The enzyme (Mr: 37,952, monomer) acts as a dipeptidyl endopeptidase strictly d-stereospecific toward oligopeptides such as (d-Phe)₃ and (d-Phe)₄. The ADP gene (adp) was isolated and its deduced amino acid sequence revealed that the ADP could be classified as a member of penicillin-recognizing enzymes.

Since the ADP was found to be a serine peptidase [16], we attempted to use it for kinetically controlled peptide synthesis. In this study, we describe for the first time the synthesis of d-phenylalanine oligomers by use of the ADP as catalyst in an alkaline aqueous medium.

Section snippets

Materials

d-Phenylalanine methylester as a substrate and authentic (d-Phe)₂, (d-Phe)₃, and (d-Phe)₄ were chemically synthesized from d-phenylalanine by the procedure as described previously [16]. $^{1} H$ -NMR spectrum was recorded in CD₃OD using a JEOL JNM-EX400 spectrometer (Tokyo, Japan), with tetramethylsilane as the internal standard. Mass spectrum was recorded on a JEOL JMS-AX500 mass spectrometer (Tokyo, Japan) under fast atom bombardment (FAB) conditions. DEAE-Toyopearl 650 M and Butyl-Toyopearl 650 M

Enzymatic oligomerization in aqueous solution

We have previously described the characterization of the alkaline d-peptidase (ADP) purified from B. cereus DF4-B [16]. The enzyme not only hydrolyzed (d-Phe)₃ and (d-Phe)₄ to form (d-Phe)₂ and d-phenylalanine, but also acted on d-phenylalanine methylester and d-phenylalanine amide to form (d-Phe)₂. This finding gave us the opportunity to further investigate the synthesis of d-phenylalanine oligopeptides from d-phenylalanine methylester by use of the ADP.

ADP was purified from E. coli

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Alkaline d-Peptidase
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Aminolytic reaction catalyzed by d-stereospecific amidohydrolases from Streptomyces spp
2011, Biochimie
Citation Excerpt :
Among them, peptidases such as d,d-carboxypeptidase from Streptomyces sp. R61 [30], d-aminopeptidase from Ochrobactrum anthropi [8], and d-peptidase from Bacillus cereus [9] have the function of aminolysis reaction. The enzymes have catalytic serine residues and belong to the clan SE, S12 peptidase family.
From investigation of 2000 soil isolates, we identified two serine-type amidohydrolases that can hydrolyze d-aminoacyl derivatives from the culture supernatant of Streptomyces species 82F2 and 83D12. The enzymes, redesignated as 82F2-DAP and 83D12-DAP, were purified for homogeneity and characterized. Each enzyme had molecular mass of approximately 40 kDa, and each showed moderate stability with respect to temperature and pH. Among hydrolytic activities toward d-aminoacyl-pNAs, the enzymes showed strict specificity toward d-Phe-pNA, but showed broad specificity toward d-aminoacyl esters. The specific activity for d-Phe-pNA hydrolysis of 82F2-DAP was ten-fold higher than that of 83D12-DAP. As a second function, each enzyme showed peptide bond formation activity by its function of aminolysis reaction. Based on results of d-Phe–d-Phe synthesis under various conditions, we propose a reaction mechanism for d-Phe–d-Phe production. Furthermore, the enzymes exhibited peptide elongation activity, producing oligo homopeptide in a one-pot reaction. We cloned the genes encoding each enzyme, which revealed that the primary structure of each enzyme showed 30–60% identity with those of peptidases belonging to the clan SE, S12 peptidase family categorized as serine peptidase with d-stereospecificity.
Crystal Structure and Functional Characterization of a D-Stereospecific Amino Acid Amidase from Ochrobactrum anthropi SV3, a New Member of the Penicillin-recognizing Proteins
2007, Journal of Molecular Biology
d-Amino acid amidase (DAA) from Ochrobactrum anthropi SV3, which catalyzes the stereospecific hydrolysis of d-amino acid amides to yield the d-amino acid and ammonia, has attracted increasing attention as a catalyst for the stereospecific production of d-amino acids. In order to clarify the structure-function relationships of DAA, the crystal structures of native DAA, and of the d-phenylalanine/DAA complex, were determined at 2.1 and at 2.4 Å resolution, respectively. Both crystals contain six subunits (A–F) in the asymmetric unit. The fold of DAA is similar to that of the penicillin-recognizing proteins, especially d-alanyl-d-alanine-carboxypeptidase from Streptomyces R61, and class C β-lactamase from Entereobacter cloacae strain GC1. The catalytic residues of DAA and the nucleophilic water molecule for deacylation were assigned based on these structures. DAA has a flexible Ω-loop, similar to class C β-lactamase. DAA forms a pseudo acyl-enzyme intermediate between Ser60 O^γ and the carbonyl moiety of d-phenylalanine in subunits A, B, C, D, and E, but not in subunit F. The difference between subunit F and the other subunits (A, B, C, D and E) might be attributed to the order/disorder structure of the Ω-loop: the structure of this loop cannot assigned in subunit F. Deacylation of subunit F may be facilitated by the relative movement of deprotonated His307 toward Tyr149. His307 N^ε2 extracts the proton from Tyr149 O^η, then Tyr149 O^η attacks a nucleophilic water molecule as a general base. Gln214 on the Ω-loop is essential for forming a network of water molecules that contains the nucleophilic water needed for deacylation. Although peptidase activity is found in almost all penicillin-recognizing proteins, DAA lacks peptidase activity. The lack of transpeptidase and carboxypeptidase activities may be attributed to steric hindrance of the substrate-binding pocket by a loop comprised of residues 278–290 and the Ω-loop.
D-amino acid dipeptide production utilizing D-alanine-D-alanine ligases with novel substrate specificity
2005, Journal of Bioscience and Bioengineering
D-Alanine-D-alanine ligase (Ddl) is an important enzyme in the synthesis of bacterial peptidoglycan. The genes encoding Ddls from Escherichia coli K12 (EcDdlB), Oceanobacillus iheyensis JCM 11309 (OiDdl), Synechocystis sp. PCC 6803 (SsDdl) and Thermotoga maritima ATCC 43589 (TmDdl), the genomic DNA sequences of which have been determined, were cloned and the substrate specificities of these recombinant Ddls were investigated. Although OiDdl had a high substrate specificity for D-alanine; EcDdlB, SsDdl and TmDdl showed broad substrate specificities for D-serine, D-threonine, D-cysteine and glycine, in addition to D-alanine. Four D-amino acid dipeptides were produced using EcDdlB, and D-amino acid homo-dipeptides were successfully produced at high yields except for D-threonyl-D-threonine.
Genes for an alkaline D-stereospecific endopeptidase and its homolog are located in tandem on Bacillus cereus genome
2003, FEMS Microbiology Letters
Alkaline D-peptidase (Adp) from Bacillus cereus DF4-B is a D-stereospecific endopeptidase acting on oligopeptides composed of D-phenylalanine and the primary structure deduced from its gene, adp, shows a similarity with D-stereospecific hydrolases from Ochrobactrum anthropi strains. We have isolated DNA fragments covering the flanking region of adp from DF4-B genome and found an additional gene, adp2, located upstream of adp. The deduced amino acid sequence of Adp2 showed 96% and 85% identity with those of Adp from B. cereus strains AH559 and DF4-B, respectively. The recombinant Adp2 expressed in Escherichia coli was purified to homogeneity and characterized. It had hydrolyzing activity toward (D-Phe)₃, (D-Phe)₄, and (D-Phe)₆ but did not act on (L-Phe)₄, D-Phe-NH₂, and L-Phe-NH₂, some characteristics that are closely related to those of Adp from strain DF4-B. These results indicate that highly homologous genes encoding D-stereospecific endopeptidases are arranged in a tandem manner on the genomic DNA of B. cereus DF4-B.
Enhancement of the thermostability and catalytic activity of D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3 by directed evolution
2003, Journal of Molecular Catalysis B: Enzymatic
d-Amino-acid amidases, which catalyze the stereospecific hydrolysis of d-amino-acid amide to yield d-amino acid and ammonia, have attracted increasing attention as catalysts for stereospecific production of d-amino acids. We screened for the enzyme variants with improved thermostability generated by a directed evolution method with the goal of the application of evolved enzyme to the production of d-amino acids. Random mutagenesis by error-prone PCR and a filter-based screening was repeated twice, and as a result the most thermostable mutant BFB40 was obtained. Gene analysis of the BFB40 mutant indicated that the mutant enzyme had K278 M and E303 V mutations. To compare the enzyme characteristics with the wild-type enzyme, the mutant enzyme, BFB40, was purified from the Escherichia coli (E. coli) transformant. Both the thermostability and apparent optimum temperature of the BFB40 were shifted upward by 5 °C compared with those of the wild-type enzyme. The apparent K_m value for d-phenylalaninamide of BFB40 enzyme was almost the same with that of the wild-type enzyme, whereas V_max value was enhanced about three-fold. Almost complete hydrolysis of d-phenylalaninamide was achieved in 2 h from 1.0 M of racemic phenylalaninamide–HCl using the cells of E. coli transformant expressing BFB40 enzyme, the conversion of which was 1.7-fold higher than the case using cells expressing wild-type enzyme after the same reaction time.

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¹: Dedicated to Professor Hideaki Yamada in honor of his 70th birthday.

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Synthesis of d-phenylalanine oligopeptides catalyzed by alkaline d-peptidase from Bacillus cereus DF4-B1

Abstract

Introduction

Section snippets

Materials

Enzymatic oligomerization in aqueous solution

Enzyme Microb. Technol.

FEBS Lett.

J. Biol. Chem.

Tetrahedron

J. Biol. Chem.

Anal. Biochem.

J. Mol. Cata. B

Angew. Chem. Int. Ed. Engl.

J. Org. Chem.

Synthesis of d-phenylalanine oligopeptides catalyzed by alkaline d-peptidase from Bacillus cereus DF4-B¹