Streptomyces AlbusGD‐Ala‐D‐Ala Carboxypeptidase

Abstract

The zinc D ‐Ala‐ D ‐Ala carboxypeptidase hydrolyzes the C‐terminal peptide bond of peptides of general structure R‐ D ‐Ala‐ D ‐Xaa. The lytic activity of the enzyme and its extracellular location suggest that it might be used by Streptomyces for fighting competitors in its ecological niche since the enzyme does not hydrolyze the Streptomyces peptidoglycan. The sequence of the 213‐residue mature protein is related to the VanX, VanY, and VanXY proteins of vancomycin‐resistant enterococci. The molecule is an ellipsoid consisting of two globular domains in which the N‐terminal and the C‐terminal ends are pulled apart. The small N‐terminal domain, from residue Asp1 to Pro83, has an all‐α‐helices structure and exhibits a typical three‐helix bundle fold, characteristic of the peptidoglycan binding domain. The largest C‐terminal domain belongs to the α + β type secondary structure, and is related to the N‐terminal domain of the murine sonic hedgehog, a signaling protein involved in natal morphogenesis and formation of embryonic patterning centers, and to the VanX D ‐Ala‐ D ‐Ala dipeptidase of Enterococcus faecium , a protein involved in the vancomycin resistance systems in streptococci. The mixed five‐stranded β‐sheet forms the core of the C‐terminal domain and the lining of one side of the catalytic cavity. One zinc ion occupies a roughly central position in the active site and is coordinated by the His154 NE2, the Asp161 OD1, and the His197 ND1. Classical β‐lactam compounds, which readily inactivate active site serine DD ‐peptidases, are very sluggish inactivators of the zinc D ‐Ala‐ D ‐Ala carboxypeptidase. The best competitive inhibitors are thiol derivatives such as 2‐ and 3‐mercaptopropionate ( K i = 50 and 5 nM) that might behave as bidentate ligands of the Zn 2+ ion.

Structure to function prediction of hypothetical protein KPN_00953 (Ycbk) from Klebsiella pneumoniae MGH 78578 highlights possible role in cell wall metabolism

“…This resulted in the identification of one potential template, termed 1LBU with sequence identity of only 23%. 1LBU is a crystal structure of muramoyl-pentapeptide carboxypeptidase, a Zn 2+ D-Ala-D-Ala carboxypeptidase from Streptomyces albus [20] which contain the particular Peptidase_M15_3 superfamily domain. 1LBU was also ranked top by Phyre2 search within CombFunc server as having the highest structural similarity KPN_0053.…”

“…The intactness of both the secondary structure topology and the three Zn 2+ ligand-binding residues of the built model suggest that KPN_00953 may function as a cell wall (peptidoglycan)-hydrolyzing enzyme, similar to a few characterized Zn D-Ala-D-Ala metallopeptidases such as muramoyl-pentapeptide carboxypeptidase from S. albus [20] and VanX from Enterococcus faecalis [19]. Closer inspection on the sequence of KPN_00953 in comparison to the abovementioned proteins revealed that it does not contain the characteristic H-x-H motif which is predominantly found in nearly all D-Ala-D-Ala metallopeptidases, except VanX [40].…”

“…Template identification based on this domain has led to the building of a 3D model of KPN_00953 via homology modeling using the crystal structure of muramoyl-pentapeptide carboxypeptidase (PDB id: 1LBU), a Zn D-Ala-D-Ala metallopeptidase from S. albus [20] as the template. The built model has been verified to be acceptable and topologically conserved with other available structures related to peptidases such as the L-alanoyl-D-glutamate endopeptidase domain of Listeria bacteriophage endolysin Ply500 (PDB id: 2VO9) [35].…”

“…Muramoyl-pentapeptide carboxypeptidase, which is used as a template in the homology modeling of KPN_00953, is a specific Zn D-Ala-D-Ala carboxypeptidase [20]. It was reported that this particular enzyme from Streptomyces hydrolyzes the C-terminal peptide bond of peptides of general structure R-D-Ala-D-Xaa.…”

Functional and structural characterization of a novel putative cysteine protease cell wall-modifying multi-domain enzyme selected from a microbial metagenome

“…Biosynthesis of PG is carried out by a variety of conserved enzymes, such as racemases (which generate D-amino acids), glycosyl transferases (which form the hexose polymers), and peptyltransferases and transpeptidases (which form interpeptide linkages)1415. The cell wall must go through reorganization during vegetative growth, development and cell division, which requires enzymes that hydrolyze various linkages in PG131617. These enzymes include peptidases that cleave the cross-linking peptides, and glycosidases, such as lysozymes, that degrade the polysaccharide backbone18.…”