Kumamolisin-As (previously called ScpA) is the first known example of a collagenase from the sedolisin family (MEROPS S53). This enzyme is active at low pH and in elevated temperatures. In this study that used x-ray crystallographic and biochemical methods, we investigated the structural basis of the preference of this enzyme for collagen and the importance of a glutamate residue in the unique catalytic triad (Ser 278 -Glu 78 -Asp 82 ) for enzymatic activity. Crystal structures of the uninhibited enzyme and its complex with a covalently bound inhibitor, N-acetyl-isoleucyl-prolyl-phenylalaninal, showed the occurrence of a narrow S2 pocket and a groove that encompasses the active site and is rich in negative charges. Limited endoproteolysis studies of bovine type-I collagen as well as kinetic studies using peptide libraries randomized at P1 and P1, showed very strong preference for arginine at the P1 position, which correlated very well with the presence of a negatively charged residue in the S1 pocket of the enzyme. All of these features, together with those predicted through comparisons with fiddler crab collagenase, a serine peptidase, rationalize the enzyme's preference for collagen. A comparison of the Arrhenius plots of the activities of kumamolisin-As with either collagen or peptides as substrates suggests that collagen should be relaxed before proteolysis can occur. The E78H mutant, in which the catalytic triad was engineered to resemble that of subtilisin, showed only 0.01% activity of the wild-type enzyme, and its structure revealed that Ser 278 , His 78 , and Asp 82 do not interact with each other; thus, the canonical catalytic triad is disrupted.A novel peptidase, initially named ScpA (1) and now called kumamolisin-As (2), was recently identified by us in the culture filtrate of a thermoacidophilic soil bacterium Alicyclobacillus sendaiensis strain NTAP-1 (1). Specificity analyses using macromolecular substrates including globular and other fibrillar proteins showed that kumamolisin-As is highly specific for collagen (3, 4) and thus could be considered as a collagenase, although with some unusual properties. Most noticeably, this enzyme exhibits the maximum activity at acidic pH ϳ4.0. This is in striking contrast to all known collagenases, which are either zinc-dependent metallopeptidases (5) or chymotrypsinlike serine proteinases (6, 7), with an optimum pH for activity at neutral to alkaline regions.A primary structure analysis of this novel "acid collagenase" revealed that it is a member of the sedolisin family, a recently established class of serine peptidases with a unique catalytic triad, Ser-Glu-Asp, in place of the Ser-His-Asp triad of classical serine peptidases (2, 4). Moreover, the enzyme was found to be very similar in its primary structure to kumamolisin, a well characterized member of the family (8 -10), exhibiting 92.7% identity with its mature form. This high level of identity led to the change of the name from the initially used ScpA (1) to kumamolisin-As (2). Kumamolisin-As was the fir...

Enzymatic degradation of collagen produces peptides, the collagen peptides, which show a variety of bioactivities of industrial interest. Alicyclobacillus sendaiensis strain NTAP-1, a slightly thermophilic, acidophilic bacterium, extracellularly produces a novel thermostable collagenolytic activity, which exhibits its optimum at the acidic region (pH 3.9) and is potentially applicable to the efficient production of such peptides. Here, we describe the purification to homogeneity, characterization, gene cloning, and heterologous expression of this enzyme, which we call ScpA. Purified ScpA is a monomeric, pepstatin-insensitive carboxyl proteinase with a molecular mass of 37 kDa which exhibited the highest reactivity toward collagen (type I, from a bovine Achilles tendon) among the macromolecular substrates examined. On the basis of the sequences of the peptides obtained by digestion of collagen with ScpA, the following synthetic peptides were designed as substrates for ScpA and kinetically analyzed: Phe-Gly-Pro-Ala*Gly-Pro-Ile-Gly (k cat , 5.41 s ؊1 ; K m , 32 M) and Met-Gly-ProArg*Gly-Phe-Pro-Gly-Ser (k cat , 351 s ؊1 ; K m , 214 M), where the asterisks denote the scissile bonds. The cloned scpA gene encoded a protein of 553 amino acids with a calculated molecular mass of 57,167 Da. Heterologous expression of the scpA gene in the Escherichia coli cells yielded a mature 37-kDa species after a two-step proteolytic cleavage of the precursor protein. Sequencing of the scpA gene revealed that ScpA was a collagenolytic member of the serine-carboxyl proteinase family (the S53 family according to the MEROPS database), which is a recently identified proteinase family on the basis of crystallography results. Unexpectedly, ScpA was highly similar to a member of this family, kumamolysin, whose specificity toward macromolecular substrates has not been defined.Collagen is an insoluble structural protein that accounts for approximately 30% of the total weight of animal proteins and is produced in large quantities as a by-product in livestock industries. It has recently been shown that the enzymatic degradation of collagen as well as that of gelatin, a denatured form of collagen, allows efficient utilization of these structural proteins. This degradation produces peptides, the collagen peptides, which have been shown to have several biological activities of industrial and medical interest (27), leading to the establishment of a wide variety of applications, e.g., an immunotherapeutic agent (7, 9), a moisturizer for cosmetics, a preservative (2), and seasoning and dietary materials (4).Generally, the enzymatic degradation of collagen is not affected by ordinary digestive proteinases but requires the collagen-specific, Zn 2ϩ -dependent metalloproteinases (collagenases). Although microbial collagenases have been found in a wide variety of mesophilic bacterial strains (3), the industrialscale application of known bacterial collagenases for collagen peptide production has been hampered because of their insufficient stability. In 2000...

The sedolisin family of proteolytic enzymes (now identified in the MEROPS database [1] as S53) was initially known as pepstatin-insensitive acid peptidases [2,3]. However, recent crystallographic and modeling studies revealed that the sedolisins (sedolisin, kumamolisin, kumamolisin-As, and CLN2) have an overall fold that is very similar to that of subtilisin [4][5][6][7][8]. The active sites of these enzymes contain a unique catalytic triad, Ser-Glu-Asp, in place of the canonical Ser-His-Asp triad of the classical serine peptidases. In the latter case, the Ser and His residues act as nucleophilic and general acid ⁄ base catalysts, respectively [9,10]. The Asp residue of the catalytic triad of sedolisins, although conserved in its nature, originates from a different part Kumamolisin-As is an acid collagenase with a subtilisin-like fold. Its active site contains a unique catalytic triad, Ser278-Glu78-Asp82, and a putative transition-state stabilizing residue, Asp164. In this study, the mutants D164N and E78H ⁄ D164N were engineered in order to replace parts of the catalytic machinery of kumamolisin-As with the residues found in the equivalent positions in subtilisin. Unlike the wild-type and D164N proenzymes, which undergo instantaneous processing to produce their 37-kDa mature forms, the expressed E78H ⁄ D164N proenzyme exists as an equilibrated mixture of the nicked and intact forms of the precursor. X-ray crystallographic structures of the mature forms of the two mutants showed that, in each of them, the catalytic Ser278 makes direct hydrogen bonds with the side chain of Asn164. In addition, His78 of the double mutant is distant from Ser278 and Asp82, and the catalytic triad no longer exists. Consistent with these structural alterations around the active site, these mutants showed only low catalytic activity (relative k cat at pH 4.0 1.3% for D164N and 0.0001% for E78H ⁄ D164N). pH-dependent kinetic studies showed that the single D164N substitution did not significantly alter the logk cat vs. pH and log(k cat ⁄ K m ) vs. pH profiles of the enzyme. In contrast, the double mutation resulted in a dramatic switch of the logk cat vs. pH profile to one that was consistent with catalysis by means of the Ser278-His78 dyad and Asn164, which may also account for the observed ligation ⁄ cleavage equilibrium of the precursor of E78H ⁄ D164N. These results corroborate the mechanistic importance of the glutamate-mediated catalytic triad and oxyanion-stabilizing aspartic acid residue for low-pH peptidase activity of the enzyme.Abbreviations IQF, internally quenched fluorogenic.