Characteristics of a New Enantioselective Thermostable Dipeptidase from Brevibacillus borstelensis BCS-1 and Its Application to Synthesis of a d -Amino-Acid-Containing Dipeptide

Abstract

Many biologically active peptides, including antibiotics (6), peptide sweeteners (13), enkephalins (19), and prodrugs used in chemotherapy, contain D-amino acid residues. Although the kinetically controlled enzymatic synthesis of peptides containing D-amino acids has been investigated with several proteases, including ␣-chymotrypsin and subtilisin (14,23,24), the Lspecificity of these enzymes makes it impossible to stereospecifically synthesize peptides that contain D-amino acids. D-stereospecific peptidases are recognized as suitable enzyme catalysts for the synthesis of peptides containing D-amino acids, due to the ability of these enzymes to use racemates as acyl group donors and acceptors, as well as the minimal protection and deprotection step of the protective group. Despite the potential of D-stereospecific peptidases, there have been relatively few reports on the production of peptides containing D-amino acids, except for the carboxypeptidase-catalyzed synthesis of a tripeptide containing D-Ala-D-Ala (11) and the D-aminopeptidase-catalyzed synthesis of a D-Ala oligomer (14) in organic solvents. The major problem with these systems is the difficulty in screening and cloning the enzymes, inasmuch as certain dipeptidases act on dipeptides containing D-amino acids.The VanX protein, which is involved in vancomycin resistance, has been reported to hydrolyze the peptide bond of D-Ala-D-Ala, although it does not hydrolyze N-or C-terminalprotected dipeptides, such as N-acetyl-D-Ala-D-Ala and D-Ala-D-Ala-o-methyl ester (25), making it inappropriate for the synthesis of D-amino-acid-containing peptides. A dipeptidase from Acinetobacter calcoaceticus (ACDP) has also been found to hydrolyze dipeptides with a D-amino acid at the Cterminus, but it does not hydrolyze C-terminal modified dipeptides (5). To overcome these problems, we recently developed a simple and rapid screening method for microorganisms producing D-stereospecific peptidases (17), and we used this method to identify a thermophile that produces a D-stereospecific peptidase.In this paper we report on the cloning, expression, and characterization of a thermostable D-stereospecific dipeptidase (BDP) from Brevibacillus borstelensis BCS-1. We also tested the industrial applicability of this enzyme in the production of the D-amino-acid-containing dipeptide Z-D-Asp-D-AlaOBzl as a model system for the synthesis of the dipeptide sweetener alitame (D-Asp-D-AlaNH 2 ) in an organic solvent system. MATERIALS AND METHODSMaterials. Pepstatin, EDTA, and antipain were purchased from Roche Molecular Biochemicals (Mannheim, Germany), and ninhydrin, bestatin, iodoacetic acid, and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma (St. Louis, Mo.). The various di-and tripeptides, L-AlaNH 2 , L-AlaNH 2 , and L-AlaOMe, were purchased from Bachem (Bubendorf, Switzerland); 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC) and N-hydroxybenzotriazole hydrate (HOBt) were purchased from Aldrich (St. Louis, Mo.); and Z-L-AspOH, Z-L-AlaOH, and L-AlaOBzl-p-tosylate wer...

Structure, Mechanism, and Substrate Profile for Sco3058: The Closest Bacterial Homologue to Human Renal Dipeptidase,

“…The renal dipeptidase-like enzyme BCS-1 from Brevibacillus borstelensis has a tryptophan in place of the residue equivalent to His-152. BCS-1 is reported to have respectable activity (k cat /K m = 2 Â 10 5 M -1 s -1 ), which supports the conclusion that either His-152 does not play a crucial role in the chemical mechanism or the mechanism for substrate hydrolysis of enzymes such as BCS-1 varies from that of hRDP (11).…”

“…With rat renal dipeptidase, L -Ala-Gly is hydrolyzed faster than L -Ala- L -Ala and Gly- D -Ala is hydrolyzed faster than Gly- L -Ala (3). Mammalian renal dipeptidases and some of their microbial homologues have been assayed with various substrates, but a complete substrate specificity profile for these types of enzymes has not been determined (2–4, 6, 10, 11, 12). …”

“…With rat renal dipeptidase, l -Ala-Gly is hydrolyzed faster than l -Ala- l -Ala and Gly- d -Ala is hydrolyzed faster than Gly- l -Ala . Mammalian renal dipeptidases and some of their microbial homologues have been assayed with various substrates, but a complete substrate specificity profile for these types of enzymes has not been determined − ,,− . 1 Abbreviations: hRDP, human renal dipeptidase; AHS, amidohydrolase superfamily; IPTG, isopropyl β- d -thiogalactopyranoside; PEG, polyethylene glycol; PDB, Protein Data Bank; SDS−PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; rmsd, root-mean-square deviation. …”

Dipeptide Synthesis by an Aminopeptidase from Streptomyces septatus TH-2 and Its Application to Synthesis of Biologically Active Peptides

“…In addition, some amino acids, such as tyrosine (Tyr), which is considered as an essential amino acid for some premature infants, have a low solubility. Because these immiscible amino acids become soluble by ligation to another amino acid, alanyl-tyrosine (AlaTyr) is a potential source of tyrosine (10, 11).Several metallopeptidases, such as thermolysin, synthesize peptides in organic solvents (6,20,24). Thermolysin prefers chemically N-protected peptides as substrates for hydrolysis, and therefore N-protected amino acids are used as acyl donors in dipeptide synthesis by thermolysin.…”

“…Several metallopeptidases, such as thermolysin, synthesize peptides in organic solvents (6,20,24). Thermolysin prefers chemically N-protected peptides as substrates for hydrolysis, and therefore N-protected amino acids are used as acyl donors in dipeptide synthesis by thermolysin.…”