The Complete Amino Acid Sequence of the Zn²⁺‐Containing d‐Alanyl‐d‐Alanine‐Cleaving Carboxypeptidase of Streptomyces albus G

Abstract

The 22076-M, Zn2+-containing D-alanyl-u-alanine-cleaving carboxypeptidase of Streptorny~~s ahuls G effectively catalyses the transfer of the N",NL-diacetyl-L-lysyl-u-alanyl electrophilic group of the standard tripeptide substrate N ",N'-diacetyl-L-lysyl-u-alanyl-D-alanine to water. It also performs a weak fl-lactamase activity, hydrolysing penicillin into penicilloate at a very low rate. This protein consists of 212 amino acid residues in a single polypeptide chain. The N terminus is partially blocked as a result of the cyclization of the dipeptide Asn-Gly into anhydroaspartylglycine imide. The protein has been fragmented by cyanogen bromide into five fragments whose sequences have been determined via appropriate subcleavages with various proteases. The ordering of the cyanogen bromide peptide fragments has been carried out (a) by submitting the S-carboxymethylated protein to complete tryptic digestion and labelling the methionine-containing peptides thus obtained with iodo[14C]-acetamide, and (b) by submitting to limited tryptic digestion the S-[2-(4'-pyridyl)ethyl]-cysteine protein whose amino groups have been blocked by reaction with exo-cis-3,6-endoxo-d4-tetrahydrophthalic anhydride prior to digestion. The protein contains six cysteine residues in the form of three disulfide bridges. N o homology is found by comparing this peptidase with other Zn2+-containing enzymes (carboxypeptidase A, thermolysin, carbonic anhydrase B and alcohol dehydrogenase) and several completely or partially sequenced, serine-containing D-alanyl-D-alanine-cleaving peptidases and Zn2 (where ~G l u is a pglutamyl residue and Raa is a diamino acid or an amino acid grouping possessing a free w-amino group) from LAla-DGlu(LRaa-DAla-DAla) pentapeptide units either to water (carboxypeptidation) or to the w-amino group at the L-Raa position of another peptide (transpeptidation). On the basis that the sequence ~R a a -~A l a -~A l a is that part of the carbonyl donor mainly involved in SUbstrdte activity, the tripeptide N",N'-diacetyl-L-lysyl-ualanyl-D-alanine (AczLLys-DAla-DAla) has been used as a substrate analogue to isolate several DD-peptidases exhibiting, with varying efficiencies, carboxypeptidase activity (Acz-~L y s -~A l a -~A l a

Discovery of an Abundant Viral Genus in Polar Regions through the Isolation and Genomic Characterization of a New Virus againstOceanospirillaceae

“…ORF 25 and ORF 26 may be responsible for viral lysis. ORF25 represents a C-terminal domain of zinc D-Ala-D-Ala carboxypeptidases (subfamily M15A, PF08291.14 ), which catalyzes carboxypeptidation but not transpeptidation reactions that are involved in bacterial cell wall metabolism ( 39 – 41 ). d -Ala- d -Ala metallopeptidases have similar active sites and share a core folding motif with lysostaphin-type peptidases.…”