Identification of Metal Ligands in the Clostridium histolyticum ColH Collagenase

Abstract

A Clostridium histolyticum 116-kDa collagenase has an H415EXXH motif but not the third zinc ligand, as found in already characterized zinc metalloproteinases. To identify its catalytic site, we mutated the codons corresponding to the three conserved residues in the motif to other amino acid residues. The mutation affecting His415 or His419 abolished catalytic activity and zinc binding, while that affecting Glu416 did the former but not the latter. These results suggest that the motif forms the catalytic site. We also mutated the codons corresponding to other amino acid residues that are likely zinc ligands. The mutation affecting Glu447 decreased markedly both the enzymatic activity and the zinc content, while that affecting Glu446 or Glu451 had smaller effects on activity and zinc binding. These mutations caused a decrease ink cat but no significant change inKm . These results are consistent with the hypothesis that Glu447 is the third zinc ligand. The spacing of the three zinc ligands is the same in all known clostridial collagenases but not in other known gluzincins, indicating that they form a new gluzincin subfamily. The effects of mutations affecting Glu446 and Glu451 suggest that the two residues are also involved in catalysis, possibly through an interaction with the two zinc-binding histidine residues.

A novel metalloproteinase virulence factor is involved in Bacillus thuringiensis pathogenesis in nematodes and insects

“…ColB could help B. thuringiensis to invade the cuticle barriers of nematode host, similar to other bacterial collagenases involved in the degradation of the extracellular matrices of animal cells (Watanabe, ; Duarte et al ., ; Singh and Bhattacharyya, ). The collagenases in some species, such as C. hisolyticum , C. perfringens , A. veronii and Leptospira interrogans , have been reported to be involved in the infection of hosts as a virulence factor (Jung et al ., ; Duarte et al ., ; Kassegne et al ., ). Sequence alignments in the National Center for Biotechnology Information database show the presence of collagenase genes in more than 300 bacteria species.…”

Discovery of a Potent Inhibitor Class with High Selectivity toward Clostridial Collagenases

“… 34 The peptidase domain harbors the catalytic zinc ion, which is coordinated by the two histidines of the canonical zinc-binding HEXXH motif, and a downstream glutamate. 4 , 34 , 35 , 39 − 41 The glutamate residue in the HEXXH motif acts as the general acid/base, which polarizes the catalytic water essential for catalysis. This polarized water molecule performs the nucleophilic attack, while the zinc ion serves as an oxyanion hole to the carbonyl oxygen of the scissile peptide bond.…”

Collagen V Is a Potential Substrate for Clostridial Collagenase G in Pancreatic Islet Isolation

“…Activator and peptidase domains are involved in the collagenolytic activity [ 3 ] and are commonly contained in ColH and ColG. The active center comprises an HEXXH zinc-binding motif [ 4 ]. Polycystic kidney disease-like domains (PKDs) are located at the middle part of the enzyme, with two PKDs in ColH and one PKD in ColG.…”