Two new aminopeptidases from Ochrobactrum anthropi active on D-alanyl-p-nitroanilide

Abstract

Abstract.Two new enzymes which hydrolyse D-alanyl-on the tripeptide L-Ala-Gly-Gly but it was not possible p-nitroanilide have been detected in Ochrobactrum an-to be certain that the same protein was responsible for thropi LMG7991 extracts. The first enzyme, DmpB, was both p-nitroanilide and peptide hydrolysing activities. purified to homogeneity and found to be homologous to The gene encoding the DmpA protein was cloned and sequenced. The deduced protein sequence exhibits varythe Dap protein produced by O. anthropi SCRC C1-38 (ATCC49237). The second enzyme, DmpA, exhibits a ing degrees of similarity with those corresponding to similar substrate profile when tested on p-nitroanilide several open reading frames found in the genomes of other prokaryotic organisms, including Mycobacteria. derivatives of glycine and L/D-alanine, but the amounts produced by the Ochrobactrum strain were not sufficient None of these gene products has been isolated or characterised, but a tentative relationship can be proposed with to allow complete purification. Interestingly, the DmpA preparation also exhibited an L-aminopeptidase activity the NylC amidase from Fla6obacterium sp. K172.Key words. Peptidase; stereospecificity; amidohydrolase.Aminopeptidases release the amino-terminal residue from peptide substrates. Most are L-aminopeptidases and exhibit a wide variety of molecular masses, quaternary structures, catalytic residues and specificity profiles [1 -10]. So far, the only aminopeptidase active on peptides containing N-terminal D-residues is that from Ochrobactrum anthropi SCRC C1-38. It has been characterized and named D-aminopeptidase (Dap) (E.C 3.4.11.19) [10,11]. Its sequence exhibits 25% identity with that of the Streptomyces R61 DD-carboxypeptidase and the residues most important for the catalytic mechanism of i-lactamases and DD-carboxypeptidases [11][12][13][14][15] appeared to be conserved. Here we report the isolation and purification of a similar D-aminopeptidase from O. anthropi LMG7991. Moreover, the same strain produces a second enzyme which hydrolyses D-alanyl-pnitroanilide but not its N-acetylated derivative, and thus represents a new potential member of the Daminopeptidase family. The study of D-aminopeptidases will contribute general information on this poorly characterized enzyme group and will help to elucidate the possible evolutionary relationship between D-aminopep-* Corresponding author.

l-Selective Amidase with Extremely Broad Substrate Specificity fromOchrobactrum anthropiNCIMB 40321

“…Among these are a number of amide bond-hydrolyzing enzymes, i.e., the D-aminopeptidase from O. anthropi SCRC C1-38 (4), which is identical to DmpB from Ochrobactrum anthropi LMG7991 (22), the L-aminopeptidase (DmpA) from strain LMG7991 (22), and the D-amino acid amidase from O. anthropi SV3 (3). Comparison of the nucleotide sequences of these biocatalysts with the lamA gene sequence clearly shows that the L-amidase from strain NCIMB 40321, described in this paper, is not related to any of these amide hydrolases described before.…”

Hydrolysis of Amides

“…The first enzyme (DmpB) was purified to 90% homogeneity from the wild-type strain, and its NH 2 -terminal amino acid sequence was determined. This exhibited nearly 60% identity with the N-terminus of DAP from O. anthropi SCRC C1-38, suggesting similar properties, which was supported by the cross reactivity of DmpB with rabbit anti-DAP antibodies [278]. The second enzyme, DmpA [279], hydrolyzed the p-nitroanilide, amide, and ester derivatives of glycine and D-alanine more efficiently than that of L-alanine [280].…”

“…About a decade after the first D-selective amino acid amide hydrolyzing enzymes were described by Asano [263,264], a search for novel D-alanine-p-nitroanilide hydrolyzing enzymes led to the isolation of two other intracellular D-aminopeptidases from O. anthropi LMG7991 [278]. The first enzyme (DmpB) was purified to 90% homogeneity from the wild-type strain, and its NH 2 -terminal amino acid sequence was determined.…”

“…The O. anthropi dmpA gene was cloned and sequenced and actively expressed in E. coli under the control of the vector borne lac promoter [278]. It encodes a 375-residue inactive polypeptide that partly accumulated in E. coli in inclusion bodies.…”

Simple method for transformation of Ochrobactrum anthropi

“…In recent decades, O. anthropi has become a source of useful enzymes including D-aminopeptidase (Asano et al 1989b), L-aminopeptidase (DmpA) (Fanuel et al 1999), D-amino acid amidase (Asano et al 1989a) and the L-amidase (Sonke et al 2005). Ochrobactrum strains are of particular interest for bioremediation (Lebuhn et al 2006).…”