, a Gram-positive spore-forming anaerobic bacterium that causes gas gangrene (clostridial myonecrosis) with 15–30% mortality (Burke & Opeskin, 1999
; Sasaki et al.
) was first described by Weinberg & Seguin (1916
) and was given its scientific name in 1923 (Bergey et al.
). While the histotoxicity of clostridia is primarily caused by specific toxins (Hatheway, 1990
), infection is triggered by the production of various proteases including collagenases that assist in the infiltration of the host by degrading various types of collagen and gelatin (Brüggemann & Gottschalk, 2004
Collagen is widely distributed throughout the mammalian body and is a major constituent of skin, ligaments, tendons and cartilage as well as the organic component of bones, teeth and the cornea. Furthermore, collagen is an integral component of the connective tissue that surrounds most organs. Indeed, it constitutes approximately 30% of the total protein in mammals. Consequently, any process that results in degradation or loss of integrity of this protein is likely to have significant implications for health (Harrington, 1996
; Awad et al.
). Furthermore, a diverse spectrum of biotechnological applications of bacterial collagenases exists that includes their use in meat tenderizing, as additives to laundry detergents, for wound healing, for islet-cell isolation and in the treatment of sciatica in herniated intervertebral discs or of retained placenta (Chu, 1987
; Sank et al.
; Hesse et al.
; Fecteau et al.
; Haffner et al.
; Yeh et al.
; Watanabe, 2004
The versatility of bacterial collagenases is related to their broad substrate specificity, which has been investigated in great detail for the enzymes from C. histolyticum
. These enzymes have been shown to display a broad substrate specificity that contrasts with the strict specificity of mammalian collagenases (Bond & van Wart, 1984
; Schlage, 1988
; Ravanti & Kähäri, 2000
; Matsushita et al.
; Toyoshima et al.
; Hu et al.
). Clostridial collagenases are large multi-modular proteins that consist of a signal peptide, a putative prodomain, a catalytic domain, one or two polycystic kidney disease-like (PKD-like) domains of unknown function and up to three collagen-binding domains (CBDs; Matsushita et al.
; Rawlings et al.
). With the crystal structure determination of the calcium-binding domain, a major breakthrough in the understanding of the Ca2+
-dependence of collagen binding and presentation has been achieved (Wilson et al.
Collagenase G (ColG) from C. histolyticum
contains 1118 amino acids and possesses a single PKD-like domain and a duplicated CBD domain in addition to the catalytic domain. Comprising approximately 670 amino acids, the latter contains the consensus motif HEXX
H that harbours the catalytic Zn2+
ion. In contrast to the zinc ligation in metzincins such as MMPs, bacterial collagenases are believed to employ glutamate as a third zinc ligand (Jung et al.
), thus belonging to the subfamily of gluzincins, albeit with a different spacing pattern to that in gluzincins of known three-dimensional structure (Kyrieleis et al.
In this communication, we report the cloning, recombinant expression, crystallization and preliminary X-ray diffraction experiments of the 77 kDa catalytic domain.