Diversity, Structures, and Collagen-Degrading Mechanisms ofBacterial Collagenolytic Proteases

Yu-Zhong Zhang, Li-Yuan Ran, Chun-Yang Li, Xiu-Lan Chen

State Key Laboratory of Microbial Technology and Marine Biotechnology Research Center, Jinan, China

Bacterial collagenolytic proteases are important because of their essential role in global collagen degradation and because oftheir virulence in some human bacterial infections. Bacterial collagenolytic proteases include some metalloproteases of the M9family from Clostridium or Vibrio strains, some serine proteases distributed in the S1, S8, and S53 families, and members of theU32 family. In recent years, there has been remarkable progress in discovering new bacterial collagenolytic proteases and in in-vestigating the collagen-degrading mechanisms of bacterial collagenolytic proteases. This review provides comprehensive in-sight into bacterial collagenolytic proteases, especially focusing on the structures and collagen-degrading mechanisms of repre-sentative bacterial collagenolytic proteases in each family. The roles of bacterial collagenolytic proteases in human diseases andglobal nitrogen cycling, together with the biotechnological and medical applications for these proteases, are also brieflydiscussed.

Collagen is the most abundant and ubiquitous material thatoccurs in the extracellular matrices of animals. Nearly 30% of

the total proteins in mammalian bodies are various types of colla-gens, which are essential structural components of all connectivetissues (1). Collagen serves as an important nitrogen source in theglobal nitrogen cycle, primarily due to its tremendous distributionin the animal kingdom all over the world. Collagens are a largefamily consisting of 28 different members, which share a helicalstructure made up of three polypeptide chains (1). The polypep-tide chains are mostly composed of repeating Gly-X-Y triplets,where X and Y are frequently proline and hydroxyproline, respec-tively. Each polypeptide chain is composed of triple-helical do-mains, which are flanked by nonhelical regions (2). Nonhelicalregions are present in all procollagens (1). Type I collagen has onemajor helical domain, but there are 21 to 26 interruptions in thehelix structure of type IV collagen (3). For type I and III collagens,a large part of each nonhelical region in procollagens, which iscalled propeptide, is proteolytically removed when procollagensare processed into mature molecules, and the remaining parts atthe N and C termini of each mature collagen molecule are calledtelopeptides, which are capable of forming intramolecular andintermolecular cross-links to enhance the tensile strength of col-lagen fibrils (4). In type VI collagen, the C-terminal nonhelicaldomain is kept during posttranslational processing (5).

Collagens can be classified into several subgroups when theyare organized into supramolecular assemblies. Fibril-forming col-lagen, such as type I or III collagen, forms the structural basis ofconnective tissues in all mammals. The molecules of this type ofcollagen axially polymerize to form a microfibril, and then furtheraggregate structures, fibrils and fascicles, are successively formed(6). The individual molecules in fibrillar collagens are staggeredfrom each other by approximately 67 nm, allowing covalent bondswith adjacent molecules that extend beyond their individual ends(7). Type IV collagen, a major constituent of mammalian base-ment membranes, represents another type of supramolecular or-ganization which forms three-dimensional networks (3). Othermajor subgroups include fibril-associated collagens with inter-rupted triple helices, beaded-filament-forming collagens, anchor-ing fibril collagens, and transmembrane collagens (8).

Because of its special structure, collagen is resistant to mostcommon proteases and can be degraded only by a few types ofproteases from mammals or bacteria. These proteases that candegrade one or more types of collagens are regarded as collageno-lytic proteases, and these include mammalian matrix metallopro-teinases (MMPs), mammalian cysteine proteases, and some bac-terial proteases. MMPs have been widely studied for theirimportance in mammal development and for their involvement inhuman diseases, and they have been introduced in other reviews(9–11). Bacterial collagenolytic proteases include some metallo-proteases and serine proteases. The bacterial collagenolytic metal-loproteases reported so far are some members of the M9 family inthe MEROPS database (http://merops.sanger.ac.uk) (12), whichare Clostridium collagenases and Vibrio collagenases. Bacterialcollagenolytic serine proteases are distributed in the S1, S8, andS53 families. In addition, some members of the U32 family,mainly from pathogenic bacteria, are also reported to be collag-enolytic proteases.

In recent years, there has been remarkable progress in identi-fying new bacterial collagenolytic proteases and their degradationmechanisms on collagen. In this paper, a comprehensive intro-duction of various bacterial collagenolytic proteases is presented,with a focus on the structures and collagen-degrading mecha-nisms of representative bacterial collagenolytic proteases. In addi-tion, the roles of bacterial collagenolytic proteases in human dis-eases and global nitrogen cycling, as well as their biotechnologicaland medical applications, are also briefly discussed.

Before further discussing bacterial collagenolytic proteases, it

Accepted manuscript posted online 6 July 2015

Citation Zhang Y-Z, Ran L-Y, Li C-Y, Chen X-L. 2015. Diversity, structures, andcollagen-degrading mechanisms of bacterial collagenolytic proteases. ApplEnviron Microbiol 81:6098 –6107. doi:10.1128/AEM.00883-15.

Editor: F. E. Löffler

Address correspondence to Xiu-Lan Chen, cxl0423@sdu.edu.cn.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.00883-15

MINIREVIEW

6098 aem.asm.org September 2015 Volume 81 Number 18Applied and Environmental Microbiology

on March 16, 2021 by guest

http://aem.asm

.org/D

ownloaded from

is necessary to discuss the relationship between bacterial collag-enolytic proteases and bacterial collagenases to avoid confusion.Bacterial collagenases are usually considered to be enzymes thatcleave helical regions of fibrillar collagen molecules under physi-ological conditions (13). However, this concept of bacterial colla-genase is not always used in such a strict standard in publications.Bacterial proteases which can only hydrolyze denatured collagenor type IV collagen are sometimes called collagenases. Here, wesuggest that the term “bacterial collagenolytic proteases” be usedto refer to all proteases that can degrade at least one type of colla-gen. Bacterial collagenolytic proteases include true collagenasesand other proteases with collagenolytic activity.

CLOSTRIDIUM COLLAGENASES FROM THE M9 FAMILYDiversity. The collagenase secreted by Clostridium histolyticumreported first by MacLennan et al. in 1953 was the first bacterialcollagenase to be discovered and studied (14). Since then, the col-lagenases from Clostridium histolyticum have been studied in de-tail and extensively used in basic science laboratories and for med-ical treatment. These Clostridium collagenases are divided intotwo classes, I and II, with class I being encoded by the gene colGand class II by colH. The C. histolyticum genome contains bothgenes. colG encodes a 1,118-amino-acid precursor, and colH en-codes a 1,021-amino-acid precursor. ColG is secreted as a 1,000-amino-acid protein (Y119 to K1118), and ColH is secreted as a981-amino-acid protein (V41 to R1021) (15–17). In addition tocollagenases ColG and ColH, at least five gelatinases were identi-fied from C. histolyticum (16, 17). The N-terminal amino acidsequences of these gelatinases coincide with that of either ColG orColH, indicating that these gelatinases are produced from a full-length collagenase by the proteolytic removal of C-terminal frag-ments (17). Collagenases from other Clostridium species, such asColA from Clostridium perfringens (18) and ColT from Clostrid-ium tetani (19), have also been reported. All Clostridium collage-nases are members of the M9B subfamily in the MEROPS database(12).

Structure. A Clostridium collagenase molecule is composed oftwo parts: the N-terminal collagenase unit (or module) and theC-terminal recruitment domains (20, 21). The collagenase unitcontains an N-terminal activator domain and a C-terminal pep-tidase domain that contains a conserved zinc-binding motif andfunctions as a catalytic domain. The collagenase unit is able toindependently show collagenolytic activity (20). The recruitmentdomains usually contain one or two collagen-binding domains(CBDs) and one or two polycystic kidney disease (PKD)-like do-mains, but ColT, which has no PKD-like domain, is an exception(18–21). The recruitment domains are not needed for degradationof triple-helical collagen but are most likely needed for larger col-lagen entities such as fibrils (20, 22). Clostridium collagenases usu-ally show differences in their C-terminal recruitment domainsand/or their zinc-binding motifs (Fig. 1).

Although the overall structure of Clostridium collagenases isstill not completely known, the crystal structures of the collage-nase unit of ColG (20), the peptidase domains of ColH and ColT(21), the PKD-like domains of ColG and ColH (22, 23), and theCBDs of ColG and ColH (24, 25) have been solved. The N-termi-nal activator domain and the C-terminal peptidase domain in thecollagenase unit of ColG form a saddle-shaped architecture. Theactivator domain, the left saddle flap, comprises an array of 12parallel �-helices, and the peptidase domain, the right saddle flap,

adopts a thermolysin-like peptidase fold (Fig. 2) (20). In additionto an active zinc ion, a calcium ion is also seen in the active site ofClostridium collagenases. Both ions are required for the full enzy-matic activity (21). The three PKD-like domains from ColG andColH are structurally similar, adopting a V-set Ig-like fold. EachPKD-like domain contains a calcium ion, which is likely involvedin interdomain alignment and aids the domain stability. However,some structural differences between the ColG and ColH PKD-likedomains have also been found. There are surface aromatic resi-dues in the ColH PKD-like domains but not in the ColG PKD-likedomain (23). The CBDs from ColG and ColH adopt a �-sheetsandwich fold, in which a collagen-binding cleft is seen. Two cal-cium ions are coordinated in a CBD molecule, and these are crit-ical for maintaining the function and stability of the CBDs (24,25). It has not yet been determined whether the CBD can functionas a helicase to unwind the collagen triple helix.

Collagenolytic mechanism. The class I and II collagenases are

FIG 1 Schematic diagrams of the domain organization of typical collageno-lytic proteases from bacteria. The domain structures of mature clostridial col-lagenases ColG (BAA77453), ColH (BAA34542), and ColT (AAO37456) weredrawn with reference to a paper by Eckhard et al. (21), and the others werededuced from the sequences of mature ColA (BAA02941) secreted by Clostrid-ium perfringens, VMC (AAC23708) from Vibrio mimicus, VAC (CAA44501)from Vibrio alginolyticus, VHC (BAK39964) from Grimontia (Vibrio) hollisae,SOT (BAI44325) from Streptomyces omiyaensis, MO-1 (BAF30978) from Geo-bacillus collagenovorans MO-1, MCP-01 (ABD14413) from Pseudoalteromonassp. SM9913, myroicolsin (AEC33275) from Myroides profundi D25, kumamo-lisin-As (BAC41257) from Alicyclobacillus sendaiensis, and PrtC (AAA25650)from Porphyromonas gingivalis. Both Clostridium and Vibrio collagenases aregluzincins with Glu as the third proteinaceous zinc ligand.

Minireview

September 2015 Volume 81 Number 18 aem.asm.org 6099Applied and Environmental Microbiology

on March 16, 2021 by guest

http://aem.asm

.org/D

ownloaded from

true endopeptidases. ColG is suggested to have two states for col-lagen hydrolysis. The activator domain and the catalytic domainin the collagenase module remain mostly closed during collagencleavage but relax to the open ground state once the collagen isdegraded (20). Unlike MMPs, which cleave native collagens intocharacteristic three-quarter and one-quarter fragments, the colla-genases from Clostridium histolyticum hydrolyze native collagensinto a mixture of small peptides (26, 27). The class I and II colla-genases initially attack distinct hyperreactive sites at Y-Gly bondsin the repeating Gly-X-Y collagen sequence (27). The amino acidpreference at P1 to P3 and P1= to P3= of both classes of collagenaseshas been determined with synthetic peptides (28, 29), and this has

been comprehensively described by Van Wart (30). In recentyears, new materials and techniques, such as immobilized posi-tional peptide libraries and proteomic identification of proteasecleavage sites, have been used to study the substrate specificity ofClostridium collagenases, and new profiles of the prime andnonprime cleavage site specificities and their molecular bases havebeen illustrated (31, 32). The two classes of collagenases bind todifferent portions of the collagen and have different specificitiesfor cutting native collagens, showing synergy in collagen degrada-tion (33, 34).

VIBRIO COLLAGENASES FROM THE M9 FAMILYDiversity and structure. The extracellular proteases from Vibriospp. have been widely studied because of their important roles invirulence. Vibrio extracellular proteases are divided into threeclasses based on their structure and function (35, 36). The class Iproteases, which are members of the M4 family, are not discussedin this article, because they have no collagenolytic activity. Theproteases in class II and class III all have collagenolytic activity andare all members of subfamily M9A in the MEROPS database (12,36). However, the enzymes in these two classes have significantdifferences in structure and function (Fig. 1 and Table 1). Notably,the class II proteases have a zinc-binding motif (HEYTH), containno C-terminal domain, and cannot hydrolyze casein (37, 38),while the class III proteases have a zinc-binding motif (HEYVH),contain a PKD-like domain and a prepetidase C-terminal (PPC)domain at their carboxyl terminus, and can hydrolyze casein (36,39, 40). However, the collagenase from Grimontia hollisae 1706B,which was first named Vibrio hollisae 1706B in 1982 (41) andreclassified in 2003 (42), is an exception among the class III pro-teases (43). Although it has the same zinc-binding motif as theother class III members, this enzyme does not have a C-terminalPKD-like domain and cannot hydrolyze casein. It has been sug-gested that it represents a new group of Vibrio collagenases (43).Thus far, no crystal structure of a Vibrio collagenase or its domainhas been solved.

Collagenolytic mechanism. Vibrio collagenases cleave triple-helical collagen in a manner similar to MMPs in the first step ofdegradation. They attack at a point three-quarters of the way fromthe N terminus by hydrolyzing the preferential peptide bond,XaaOGly. Vibrio collagenase cleaves native collagen at a muchhigher rate than vertebrate MMP1 (44, 45). Vibrio collagenasehydrolyzes the Pz (4-phenylazobenzyloxycarbonyl) peptide (Pz-Pro-Leu-Gly-Pro-D-Arg) by cleaving the same peptide bond(LeuOGly) as Clostridium collagenase (44, 45). Because the crys-tal structure of Vibrio collagenase is not solved, it is still unclearhow Vibrio collagenase recognizes and degrades collagen. Al-though the class II proteases do not contain a C-terminal exten-sion, it is reported that the FAXWXXT motif in the carboxyl ter-minus of Vibrio mimicus collagenase is involved in binding tocollagen (46). In contrast, neither the PKD-like domain nor thePPC domain of the class III proteases is yet demonstrated to func-tion as a collagen-binding domain.

BACTERIAL SERINE COLLAGENOLYTIC PROTEASES FROMTHE S1 FAMILY

Of the reported collagenolytic serine proteases in the S1 family,most are from invertebrate animals. The protease isolated from afiddler crab (Uca pugilator) was the first serine protease found tobe capable of cleaving native type I triple-helical collagen. The

FIG 2 Structures of ColG (M9), ColH (M9), MCP-01 (S8), and kumamoli-sin-As (S53). For ColG, the activator domain and the peptidase domain of thecollagenase unit (PDB accession number 2Y50) (20) are colored in purple andgray, respectively, and the PKD (PDB accession number 4AQO) (21) and theCBD (PDB accession number 1NQD) (24) are colored in yellow and orange,respectively. For ColH, the peptidase domain (PDB accession number 4AR1)(21), the PKD (PDB accession number 4U7K) (23), and the CBD (PDB acces-sion number 3JQW) (25) are colored in gray, yellow, and orange, respectively.The catalytic domains of both MCP-01 (PDB accession number 3VV3) (65)and kumamolisin-As (PDB accession number 1SN7) (72) are shown in gray.The catalytic triads of serine collagenolytic proteases MCP-01 (Asp49, His104,and Ser269) and kumamolisin-As (Ser278, Glu78, and Asp82) are shown instick representation. The Zn2� in ColG and ColH is shown in orange, andCa2� is colored in green for all enzymes.

Minireview

6100 aem.asm.org September 2015 Volume 81 Number 18Applied and Environmental Microbiology

on March 16, 2021 by guest

http://aem.asm

.org/D

ownloaded from

crystal structure of this protease was solved in 1997, and structuralanalysis showed that it has no collagen-binding domain and thatthe substrate-binding pocket is enlarged relative to that of trypsin,owing to the insertion of several amino acids (47). Its cleavagepattern on collagen is similar to that of MMPs (47, 48). Fewerreports on the S1 collagenolytic serine proteases from bacteriaexist. Uesugi et al. (50) purified an S1 collagenolytic protease, SOT(Streptomyces omiyaensis trypsin), from Streptomyces omiyaensis,and it has 77% identity with the trypsin SGT (Streptomyces griseustrypsin) from S. griseus in the primary structure (49, 50). SOT andSGT show similar pH and temperature optima, thermostabilities,and substrate preferences. Both SOT and SGT greatly hydrolyzetype I collagen, showing much higher activities than commercialC. histolyticum collagenase type I (Sigma). SOT also has a highactivity towards type IV collagen, but the ability of SGT to hydro-lyze type IV collagen is poor. A comparison of the substrate spec-ificities of the constructed chimeras of SOT and SGT indicates thatthe N-terminal domains of these enzymes are associated with their

specificity toward structural protein substrates (50). Because SOTand SGT can degrade type I collagen at 37°C, these S1 collageno-lytic proteases can be regarded as bacterial collagenases.

BACTERIAL SERINE COLLAGENOLYTIC PROTEASES FROMTHE S8 FAMILYDiversity. The S8 family, also known as the subtilisin or subtilasefamily, is the second-largest family of serine proteases. Proteasesin this family are all characterized by an Asp/His/Ser catalytic triadand an alpha/beta-fold catalytic center containing a seven-stranded parallel �-sheet (51). While most proteases in the S8family have no collagenolytic activity, some S8 proteases havebeen reported in recent years to be collagenolytic proteases. Thethermostable protease from Geobacillus collagenovorans MO-1was the first reported S8 collagenolytic protease. This enzyme hasa C-terminal collagen-binding domain, and its cleavage sites oncollagen are varied but specific (52, 53). Several more S8 proteaseswith collagenolytic activity have been reported from environmen-

TABLE 1 Characteristics of representative collagenolytic proteases

Family Class Name OriginMol mass(kDa)

Motif/catalytictriad

GenBankaccessionno. Substrates

M9B I ColG Clostridium histolyticum 116 HEYTH BAA77453 Type I, II, and III collagens(15, 28)

I ColA Clostridium perfringens type CNCIB 10662

116 HEFTH BAA02941 Type I collagen, Pz peptide,azocoll (18)

II ColH Clostridium histolyticum 98 HEYTH BAA34542 Type I, II, and III collagens(16)

M9A II VMC Vibrio mimicus ATCC 33653 62 HEYTH AAC23708 Type I, II, and III collagens,gelatin, Cbz-GPLGP, Cbz-GPGGPA (37)

II PrtVp Vibrio parahaemolyticus 93 62 HEYTH CAA86734 Type I, II, III, and IV collagens,FALGPA (38)

III VAC Vibrio alginolyticus 82 HEYVH CAA44501 Gelatin, casein, collagen,synthetic substrate (39)

III VPPC Vibrio parahaemolyticus 04 90 HEYVH AAG59883 Type I collagen, gelatin, casein,Cbz-GPGGPA (36)

III VPM Vibrio parahaemolyticusFYZ8621.4

90 HEYVH ABF19104 Type I, II, III, and IV collagens,gelatin, casein, Cbz-GPGGPA (40)

S1 SOT Streptomyces omiyaensis 23 His75, Asp120,Ser210

BAI44325 Type I and IV collagens (50)

SGT Streptomyces griseus ATCC10137

23 His73, Asp118,Ser208

AAA26820 Type I collagen (50)

S8 Geobacillus collagenovoransMO-1

105 Asp196, His260,Ser590

BAF30978 Type I and IV collagens (52)

MCP-01 Pseudoalteromonas sp.SM9913

66 Asp49, His104,Ser269

ABD14413 Type I, II, and IV collagens,gelatin, casein, Pz peptide(63)

Myroicolsin Myroides profundi D25 56 Asp129, His174,Ser378

AEC33275 Type I collagen, gelatin (66)

S53 Kumamolisin-As Alicyclobacillus sendaiensisNTAP-1

37 Glu78, Asp82,Ser278

BAC41257 Gelatin, relaxed collagen (69)

U32 PrtC Porphyromonas gingivalisATCC 53977

70 (dimer) AAA25650 Reconstituted type I collagen,heat-denatured type Icollagen, azocoll (78)

Minireview

September 2015 Volume 81 Number 18 aem.asm.org 6101Applied and Environmental Microbiology

on March 16, 2021 by guest

http://aem.asm

.org/D

ownloaded from

tal bacteria and archaea (54–59) and from human pathogens (60–62). Recently, two S8 proteases from deep-sea bacteria, deseasinMCP-01 from Pseudoalteromonas sp. strain SM9913 and myroi-colsin from Myroides profundi D25, were characterized as collag-enolytic proteases, and their actions toward collagen degradationwere also revealed (63–66).

Structure and collagenolytic mechanism. Deseasin MCP-01contains an S8 catalytic domain, a linker, a proprotein convertasedomain (P domain), and a PKD-like domain (Fig. 1) (67). ItsC-terminal PKD-like domain is responsible for collagen binding,and Try36 in the PKD-like domain plays a key role in collagenbinding (63). The PKD-like domain of MCP-01 can swell collagenbut cannot unwind the collagen triple helix (64). The structure ofthe catalytic domain of MCP-01 has been solved (65), and thisshowed that it adopts a fold similar to subtilisin Carlsberg, theprotype of the S8 family, which has no collagenolytic activity. Thecatalytic triad of MCP-01 is composed of Asp49, His104, andSer269 (Fig. 2 and Table 1). Structural and mutational analysesindicated that compared to subtilisin Carlsberg, MCP-1 has anenlarged substrate-binding pocket, which is necessary for collagenrecognition. The substrate-binding pocket is composed of threeloops, and the acidic and aromatic residues on these loops form anegatively charged, hydrophobic environment for collagen bind-ing. The catalytic domain of MCP-01 alone can degrade type Icollagen, but with a lower efficiency than the intact MCP-01, sug-gesting that the PKD domain in MCP-01 is helpful for collagendegradation (65).

MCP-01 cleaves type I, II, and IV collagens, gelatin, and fishcollagen (63). Atomic force microscopy observation and bio-chemical analysis of the action of MCP-01 on bovine type I colla-gen fibers confirmed that both MCP-01 and its catalytic domainprogressively release single fibrils from collagen fibers and releasecollagen monomers from fibrils mainly by hydrolyzing proteogly-cans and telopeptides in the collagen fibers (65). The catalyticdomain can further degrade type I triple-helical collagen andcleave collagen chains with multiple sites (63, 65). MCP-01 dis-played a nonstrict preference for peptide bonds with Pro or basicresidues at the P1 site and/or Gly at the P1= site in collagen. His211in the S1 pocket is the key residue responsible for the preference ofMCP-01 for the P1 basic residues (65).

The S8 protease myroicolsin from Myroides profundi D25 alsohas a C-terminal domain, a �-jelly roll domain (Fig. 1) (66),which, however, has no collagen-binding ability. Myroicolsin hadbroad specificity toward several types of collagens, including types

I, II, and IV collagens, gelatin, and fish collagen. Scanning electronmicroscope and atomic force microscope observations combinedwith biochemical analyses confirmed that myroicolsin exerts asimilar action on collagen fiber disassembly as MCP-01. Myroi-colsin showed different cleavage patterns on native collagen anddenatured collagen. On native collagen, the P1 position is oftenoccupied by Gly, Arg, Pro, or Phe, and the P1= position is occupiedby Gly. On denatured collagen, the P1 position is always a basicresidue (Lys and Arg), and the P1= position is Gly. Ran et al. pro-posed a collagen degradation model of myroicolsin based on theirresults (Fig. 3) (66). Myroicolsin could firstly break the interfibril-lar proteoglycan bridges, leading to disassembly of the tight struc-ture of collagen fiber and exposure of collagen fibrils. Then, telo-peptides at the molecular ends are hydrolyzed by myroicolsin,which leads to the breaking of cross-links between collagen mono-mers and the release of monomers. Thus, myroicolsin gains accessto the attack sites on collagen monomers. Finally, the fibrillarstructure of collagen is completely destroyed, and collagen mono-mers are degraded into peptides and free amino acids (66).

Bacterial collagenolytic proteases in the S8 family, includingMCP-01, myroicolsin, and the thermostable protease from Geo-bacillus collagenovorans MO-1, can degrade native type I collagenat 37°C and below, though they have high temperature optima (50to 60°C). Therefore, these S8 collagenolytic proteases can be re-garded as bacterial collagenases.

BACTERIAL SERINE COLLAGENOLYTIC PROTEASES FROMTHE S53 FAMILYDiversity and structure. The proteases of the S53 family, or thesedolisin family, have been identified recently and found in a widevariety of organisms (12, 68). These enzymes usually exhibit max-imum activity at low pH and high temperature. Crystal structuresof some members of this family are available, and these adopt afold that resembles subtilisin but is significantly larger. However,the enzymes of the S53 family have a unique catalytic triad, Ser-Glu-Asp, distinct from that of subtilisin-like proteases (Asp-His-Ser) (68).

Kumamolisin-As, originally named ScpA, is the only knownmember with collagenolytic activity in the sedolisin family (69).Kumamolisin-As was originally identified in the culture filtrate ofa thermoacidophilic soil bacterium, Alicyclobacillus sendaiensisstrain NTAP-1 (70, 71). Mature kumamolisin-As contains 361amino acid residues. Crystal structures of the uninhibited enzymeand its complex with a covalently bound inhibitor, N-acetyl-iso-

FIG 3 Schematic model for stepwise collagen degradation by the S8 protease myroicolsin from Myroides profundi D25. Black arrows indicate the collagenolyticprocess of myroicolsin from collagen fiber to peptides and amino acids. Green arrows indicate the cross-links or bonds destroyed by myroicolsin in each step. Redarrows indicate the cleavage sites of myroicolsin on synthetic peptides. T1, T2, and T3 are the three polypeptide chains in a collagen monomer, and dotted linesindicate the hydrogen bonds between the polypeptide chains in a collagen monomer. (Adapted from reference 66 with permission of the publisher [copyright2014, the American Society for Biochemistry and Molecular Biology.]).

Minireview

6102 aem.asm.org September 2015 Volume 81 Number 18Applied and Environmental Microbiology

on March 16, 2021 by guest

http://aem.asm

.org/D

ownloaded from

leucyl-prolyl-phenylalaninal, have been solved (72). The catalytictriad is composed of Ser278, Glu78, and Asp82 (Fig. 2). In recentyears, the catalytic mechanism of kumamolisin-As has been elu-cidated by quantum mechanical/molecular mechanical moleculardynamics and free energy simulations and site-directed mutations(73–77).

Collagenolytic mechanism. Specificity analyses showed thatkumamolisin-As is highly specific for collagen under thermal andacidic conditions and has maximum collagenolytic activity at pH4.0 and 60°C (69, 71). Collagen is denatured under these condi-tions, and thus, kumamolisin-As actually acts on denatured colla-gen. Because strain NTAP-1 is a thermoacidophilic bacterium(70), collagen should be relaxed under the bacterial growth con-ditions before proteolysis can occur (71). These analyses suggestthat kumamolisin-As may not be a true bacterial collagenase.Structural and docking studies have also shown that a groove thatencompasses the active site provides a sufficient space for bindinga relaxed collagen molecule and that the negative charges clusteredon the surface of this groove facilitate the binding of the positivelycharged collagen. The low-pH activity of kumamolisin-As may bedue to the presence of Asp164 in the oxyanion hole. Kumamoli-sin-As shows a strong preference for arginine at the P1 position oncollagen, due to the presence of a negatively charged residue(Asp179) in the S1 pocket of the enzyme (72).

BACTERIAL COLLAGENOLYTIC PROTEASES FROM THE U32FAMILYDiversity and structure. The U32 family contains several collag-enolytic proteases of unknown catalytic type (12). Most of theseproteases are the virulence factors of human-pathogenic bacteria.Protease PrtC from Porphyromonas gingivalis ATCC 53977 (whichis no longer available from the ATCC) was the first reported and isalso the most studied U32 protease (78). The other members ofthis family are from Helicobacter pylori, Salmonella enterica sero-var Typhimurium, Clostridium beijerinckii, Escherichia coli, andBacillus subtilis (79–82). The prtC gene from Porphyromonas gin-givalis ATCC 53977 contains 1,002 bp and encodes a 333-residuePrtC protein (78). The structure of the 10-kDa C-terminal do-main of a putative U32 protease from Geobacillus thermoleovoranshas been solved. This C-terminal domain shows a compact dis-torted open �-barrel made up of eight �-strands and may func-tion in protein binding based on structure similarity analysis (83).If this is true, it can be concluded that the N-terminal domain ofU32 proteases should function as a catalytic domain (Fig. 1).

Collagenolytic mechanism. PrtC degraded soluble and recon-stituted fibrillar type I collagen, heat-denatured type I collagen,and azocoll at temperatures below 37°C but not gelatin or the Pzpeptide (78). This characteristic indicates that PrtC is likely a truebacterial collagenase. The activity of PrtC was enhanced by Ca2�

and inhibited by EDTA, sulfhydryl-blocking agents, and the sali-vary peptide histatin (78). The substrate specificity of other U32proteases has not been characterized in detail. Due to the lack ofstructural information, the protein fold and active site residues ofthe catalytic domain for any member of this family are unclear.However, because most U32 proteases are the virulence factors ofhuman-pathogenic bacteria, it is important to understand thestructures and collagen degradation mechanisms of these virulentproteases, as such information is helpful for the development oftherapies for the diseases caused by relevant pathogenic bacteria.

ROLES IN HUMAN DISEASES AND ENVIRONMENTALNITROGEN CYCLING AND BIOTECHNOLOGICAL ANDMEDICAL APPLICATIONSRoles in human diseases. Bacterial collagenolytic proteases frompathogens have been of concern mainly because they are potentialvirulence factors. The collagenases from several pathogenic Clos-tridium species are suggested to be involved in the degradation ofspecific cell membrane or extracellular matrix components, whichhave been documented in a 2009 review by Popoff and Bouvet(84). The collagenase of Vibrio vulnificus, a pathogen which cancause cellulitis or septicemia, was reported to have a potentialcontribution to the invasiveness of the bacteria into human tissuethrough open wounds (85, 86). Many collagenases from Clostrid-ium and Vibrio pathogens are supposed to have similar roles.Some S8 proteases with high collagenolytic activity that are se-creted by the pathogens Stenotrophomonas maltophilia (opportu-nistic agent of hemorrhagic pneumonia) and Acanthamoeba spp.(causative agent of amebic keratitis and granulomatous amebicencephalitis) probably function as important pathogenic factorsand may serve as targets for the development of therapeutic agents(60–62).

The U32 family collagenolytic proteases from several humanpathogens, such as Porphyromonas gingivalis, Aeromonas veronii,and Helicobacter pylori, are also suggested to be virulence factors(78, 81, 87). Although a lot of pathogens are found to secretecollagenolytic proteases, the roles and mechanisms of the pro-teases involved in their pathogenic processes are still largely un-known. The exploration of the mechanisms of human pathogencollagenolytic proteases on collagen degradation is essential forestablishing treatments for related human diseases. More detailedinformation on bacterial collagenases related to human diseasescan be found in a recent review (88).

Roles in environmental nitrogen cycling. Besides pathogens,a lot of collagenolytic-protease-secreting bacteria have been iso-lated from terrestrial soil and marine sediments. Thus, it is rea-sonable to believe that collagen degradation by extracellular col-lagenolytic proteases from various environmental bacteria is animportant biological process for the release of fixed nitrogen intothe global nitrogen cycle. It is well known that Clostridium spp. areinvolved in carcass putrefaction, and their collagenases play obvi-ous roles in this process. Collagenolytic proteases from other en-vironmental bacteria can be supposed to play similar roles in de-grading various collagens from dead or even living animals.Revealing the mechanisms of environmental bacteria on collagendegradation is helpful for the study of the global nitrogen cycle.Moreover, bacterial collagenases have shown much biotechnolog-ical potential, which is documented in detail in the following sec-tion.

Biotechnological and medical applications. Clostridium col-lagenase has been used in laboratories to dissociate tissues andisolate cells (89) and used as a therapeutic drug for the removal ofnecrotic wound tissues for several decades (90, 91). It is success-fully applied in the treatment of third-degree burns (92, 93), softtissue and pressure ulcers (94–97), diabetic ulcers (98), ischemicarterial ulcers (99–101), and Dupuytren’s disease (102). Recently,clostridial collagenases (Xiaflex/Xiapex) were approved by boththe FDA (December 2013) and the European Union (February2015) to be used as nonsurgical treatment for Peyronie’s disease. Ithas been shown that both Clostridium collagenase itself and its

Minireview

September 2015 Volume 81 Number 18 aem.asm.org 6103Applied and Environmental Microbiology

on March 16, 2021 by guest

http://aem.asm

.org/D

ownloaded from

collagen degradation products can promote cell migration (103).In addition, the serine collagenolytic protease from Pseudoaltero-monas sp. SM9913 has shown good potential in meat taste im-provement (104) and meat tenderization (105). These examplesindicate that bacterial collagenolytic proteases may have wide ap-plications in research, medicine, and food processing in the fu-ture.

SUMMARY AND PROSPECTSSummary. Due to their essential role in global nitrogen cyclingand/or their vital virulent role in some diseases, bacterial collag-enolytic proteases have drawn increasing attention. During thelast decade, the intact or partial structures of some bacterial col-lagenolytic proteases, including ColG, ColH, and ColT from M9,deseasin MCP-01 from S8, and kumamolisin-As from S53, havebeen solved, and their mechanisms for collagen degradation havebeen revealed by structural and biochemical studies. Several kindsof noncatalytic domains, including PKD-like domains, PPC do-mains, and CBDs, are found at the C termini of some bacterialcollagenolytic proteases. Unlike PKD-like domains and PPC do-mains, which are identified by sequence homology, CBD is a func-tional name of the domains in bacterial collagenolytic proteasesthat have collagen-binding ability. The CBDs from different bac-terial collagenolytic proteases may have little sequence homology.Except for the M9 Vibrio collagenase and the S1 protease SOT,whose cleavage patterns on collagen are similar to those of MMPs,the other bacterial collagenolytic proteases (the M9 Clostridiumcollagenase, the S8 protease, and the S53 protease) have multiplecleavage sites on collagen and can degrade collagen into smallpeptides and amino acids. Therefore, these bacterial proteases arevery effective at environmental collagen degradation.

With more and more serine collagenolytic proteases beingstudied, common characteristics of these proteases from differentfamilies can be observed. The catalytic domains of serine collag-enolytic proteases have activity toward collagen, independently ofwhether they have a collagen-binding domain. Serine collageno-lytic proteases have an enlarged and negatively charged substrate-binding pocket for collagen recognition compared to their ho-mologs that have no collagenolytic activity. Serine collagenolyticproteases usually have a preference for basic residues at the P1position on collagen. The basic residue Arg is abundant in humancollagens. The numbers of Arg residues in the sequences of chains�1 and �2 of human type I collagen are 71/1,464 and 72/1,366,respectively, and the numbers of Arg residues in the sequences ofsubunits �1 to �6 of collagen IV are 45/1,669, 79/1,721, 65/1,672,70/1,690, 37/1,685, and 47/1,691, respectively. The peptide bondswith a C-terminal Arg residue in human collagens are all potentialcleavage sites for serine collagenolytic proteases with a strongspecificity for Arg at P1.

Prospects. It is most likely that there are still many undiscov-ered bacterial collagenolytic proteases hidden in various environ-mental or pathogenic bacteria. Some of these proteases may havenovel structures and collagenolytic mechanisms. The explorationof these proteases could provide further understanding of bacte-rial degradation of collagen globally and could uncover promisingproteases with biotechnological potential. In addition, under-standing the structures and collagen degradation mechanisms ofthe collagenolytic proteases from human pathogens, such asVibrio proteases and the U32 proteases, will be helpful for the

development of therapies for the diseases caused by relevantpathogenic bacteria.

ACKNOWLEDGMENTS

The work was supported by the National Natural Science Foundation ofChina (grants 31290230, 31290231, 91228210, and 41276149), the Hi-Tech Research and Development Program of China (grant2014AA093509), and the China Ocean Mineral Resources R & D Associ-ation (COMRA) Special Foundation (grant DY125-15-T-05).

REFERENCES1. Ricard-Blum S. 2011. The collagen family. Cold Spring Harb Perspect

Biol 3:a004978. http://dx.doi.org/10.1101/cshperspect.a004978.2. Gordon MK, Hahn RA. 2010. Collagens. Cell Tissue Res 339:247–257.3. Khoshnoodi J, Pedchenko V, Hudson BG. 2008. Mammalian colla-

gen IV. Microsc Res Tech 71:357–370. http://dx.doi.org/10.1002/jemt.20564.

4. Ricard-Blum S, Ruggiero F. 2005. The collagen superfamily: from theextracellular matrix to the cell membrane. Pathol Biol (Paris) 53:430 –442. http://dx.doi.org/10.1016/j.patbio.2004.12.024.

5. Khoshnoodi J, Cartailler JP, Alvares K, Veis A, Hudson BG. 2006.Molecular recognition in the assembly of collagens: terminal noncollag-enous domains are key recognition modules in the formation of triplehelical protomers. J Biol Chem 281:38117–38121. http://dx.doi.org/10.1074/jbc.R600025200.

6. Kadler KE, Holmes DF, Trotter JA, Chapman JA. 1996. Collagen fibrilformation. Biochem J 316:1–11.

7. Brodsky B, Persikov AV. 2005. Molecular structure of the collagen triplehelix. Adv Protein Chem 70:301–339. http://dx.doi.org/10.1016/S0065-3233(05)70009-7.

8. Kadler KE, Baldock C, Bella J, Boot-Handford RP. 2007. Collagens ata glance. J Cell Sci 120:1955–1958.

9. Visse R, Nagase H. 2003. Matrix metalloproteinases and tissue inhibitors ofmetalloproteinases: structure, function, and biochemistry. Circ Res 92:827–839. http://dx.doi.org/10.1161/01.RES.0000070112.80711.3D.

10. Lauer-Fields JL, Juska D, Fields GB. 2002. Matrix metalloproteinasesand collagen catabolism. Biopolymers 66:19 –32. http://dx.doi.org/10.1002/bip.10201.

11. Fields GB. 2013. Interstitial collagen catabolism. J Biol Chem 288:8785–8793. http://dx.doi.org/10.1074/jbc.R113.451211.

12. Rawlings ND, Barrett AJ, Bateman A. 2010. MEROPS: the peptidasedatabase. Nucleic Acids Res 38:D227–D233. http://dx.doi.org/10.1093/nar/gkp971.

13. Harrington DJ. 1996. Bacterial collagenases and collagen-degrading en-zymes and their potential role in human disease. Infect Immun 64:1885–1891.

14. MacLennan JD, Mandl I, Howes EL. 1953. Bacterial digestion of colla-gen. J Clin Invest 32:1317–1322. http://dx.doi.org/10.1172/JCI102860.

15. Yoshida E, Noda H. 1965. Isolation and characterization of collagenasesI and II from Clostridium histolyticum. Biochim Biophys Acta 105:562–574. http://dx.doi.org/10.1016/S0926-6593(65)80239-9.

16. Yoshihara K, Matsushita O, Minami J, Okabe A. 1994. Cloning andnucleotide sequence analysis of the colH gene from Clostridium histolyti-cum encoding a collagenase and a gelatinase. J Bacteriol 176:6489 – 6496.

17. Matsushita O, Jung CM, Katayama S, Minami J, Takahashi Y, OkabeA. 1999. Gene duplication and multiplicity of collagenases in Clostridiumhistolyticum. J Bacteriol 181:923–933.

18. Matsushita O, Yoshihara K, Katayama S, Minami J, Okabe A. 1994.Purification and characterization of a Clostridium perfringens 120-kilodalton collagenase and nucleotide sequence of the correspondinggene. J Bacteriol 176:149 –156.

19. Brüggemann H, Baumer S, Fricke WF, Wiezer A, Liesegang H, DeckerI, Herzberg C, Martinez-Arias R, Merkl R, Henne A, Gottschalk G.2003. The genome sequence of Clostridium tetani, the causative agent oftetanus disease. Proc Natl Acad Sci U S A 100:1316 –1321. http://dx.doi.org/10.1073/pnas.0335853100.

20. Eckhard U, Schönauer E, Nüss D, Brandstetter H. 2011. Structure ofcollagenase G reveals a chew-and-digest mechanism of bacterial collag-enolysis. Nat Struct Mol Biol 18:1109 –1114. http://dx.doi.org/10.1038/nsmb.2127.

21. Eckhard U, Schönauer E, Brandstetter H. 2013. Structural basis for

Minireview

6104 aem.asm.org September 2015 Volume 81 Number 18Applied and Environmental Microbiology

on March 16, 2021 by guest

http://aem.asm

.org/D

ownloaded from

activity regulation and substrate preference of clostridial collagenases G,H, and T. J Biol Chem 288:20184 –20194.

22. Eckhard U, Brandstetter H. 2011. Polycystic kidney disease-like do-mains of clostridial collagenases and their role in collagen recruitment.Biol Chem 392:1039 –1045.

23. Bauer R, Janowska K, Taylor K, Jordan B, Gann S, Janowski T,Latimer EC, Matsushita O, Sakon J. 2015. Structures of three polycystickidney disease-like domains from Clostridium histolyticum collagenasesColG and ColH. Acta Cryst D 71:565–577. http://dx.doi.org/10.1107/S1399004714027722.

24. Wilson JJ, Matsushita O, Okabe A, Sakon J. 2003. A bacterialcollagen-binding domain with novel calcium-binding motif controlsdomain orientation. EMBO J 22:1743–1752. http://dx.doi.org/10.1093/emboj/cdg172.

25. Bauer R, Wilson JJ, Philominathan STL, Davis D, Matsushita O,Sakon J. 2013. Structural comparison of ColH and ColG collagen-binding domains from Clostridium histolyticum. J Bacteriol 195:318 –327. http://dx.doi.org/10.1128/JB.00010-12.

26. Mallya SK, Mookhtiar KA, Van Wart HE. 1986. Accurate quantitativeassays for the hydrolysis of soluble type I, II and III 3H-acetylated colla-gens by bacterial and tissue collagenases. Anal Biochem 158:334 –345.http://dx.doi.org/10.1016/0003-2697(86)90558-0.

27. French MF, Bhown A, Van Wart HE. 1992. Identification of Clostrid-ium histolyticum collagenase hyperreactive sites in type I, II, and III col-lagens: lack of correlation with local triple helical stability. J ProteinChem 11:83–97. http://dx.doi.org/10.1007/BF01025095.

28. Mookhtiar KA, Steinbrink DR, Van Wart HE. 1985. Mode of hydro-lysis of collagen-like peptides by class I and class II Clostridium histolyti-cum collagenases: evidence for both endopeptidase and tripeptidylcar-boxypeptidase activities. Biochemistry 24:6527– 6533. http://dx.doi.org/10.1021/bi00344a033.

29. Van Wart HE, Steinbrink DR. 1985. Complementary substrate speci-ficities of class I and class II collagenases from Clostridium histolyticum.Biochemistry 24:6520 – 6526. http://dx.doi.org/10.1021/bi00344a032.

30. Van Wart HE. 2004. Clostridium collagenase, p 416 – 419. In Barrett AJ,Rawlings ND, Woessner JF (ed), Handbook of proteolytic enzymes, 2nded. Elsevier, London, United Kingdom.

31. Hu Y, Webb E, Singh J, Morgan BA, Gainor JA, Gordon TD, SiahaanTJ. 2002. Rapid determination of substrate specificity of Clostridiumhistolyticum �-collagenase using an immobilized peptide library. J BiolChem 277:8366 – 8371. http://dx.doi.org/10.1074/jbc.M111042200.

32. Eckhard U, Huesgen PF, Brandstetter H, Overall CM. 2014. Proteomicprotease specificity profiling of clostridial collagenases reveals their in-trinsic nature as dedicated degraders of collagen. J Proteomics 100:102–114. http://dx.doi.org/10.1016/j.jprot.2013.10.004.

33. French MF, Mookhtiar KA, Van Wart HE. 1987. Limited proteolysis oftype I collagen at hyperreactive sites by class I and II Clostridium histo-lyticum collagenases: complementary digestion patterns. Biochemistry26:681– 687. http://dx.doi.org/10.1021/bi00377a004.

34. Breite AG, McCarthy RC, Dwulet FE. 2011. Characterization and func-tional assessment of Clostridium histolyticum class I (C1) collagenasesand the synergistic degradation of native collagen in enzyme mixturescontaining class II (C2) collagenase. Transplant Proc 43:3171–3175.http://dx.doi.org/10.1016/j.transproceed.2011.09.059.

35. Shinoda S, Miyoshi S. 2011. Proteases produced by vibrios. BiocontrolSci 16:1–11. http://dx.doi.org/10.4265/bio.16.1.

36. Kim SK, Yang JY, Cha J. 2002. Cloning and sequence analysis of a novelmetalloprotease gene from Vibrio parahaemolyticus 04. Gene 283:277–286. http://dx.doi.org/10.1016/S0378-1119(01)00882-4.

37. Lee JH, Kim GT, Lee JY, Jun HK, Yu JH, Kong IS. 1998. Isolation andsequence analysis of metalloprotease gene from Vibrio mimicus. BiochimBiophys Acta 1384:1–6. http://dx.doi.org/10.1016/S0167-4838(97)00205-7.

38. Lee CY, Su SC, Liaw RB. 1995. Molecular analysis of an extracellularprotease gene from Vibrio parahaemolyticus. Microbiology 141:2569 –2576. http://dx.doi.org/10.1099/13500872-141-10-2569.

39. Takeuchi H, Shibano Y, Morihara K, Fukushima J, Inami S, Keil B,Gilles AM, Kawamoto S, Okuda K. 1992. Structural gene and completeamino acid sequence of Vibrio alginolyticus collagenase. Biochem J 281:703–708.

40. Luan X, Chen J, Zhang XH, Li Y, Hu G. 2007. Expression and char-acterization of a metalloprotease from a Vibrio parahaemolyticus isolate.Can J Microbiol 53:1168 –1173. http://dx.doi.org/10.1139/W07-085.

41. Hickman FW, Farmer JJ, Hollis DG, Fanning GR, Steigerwalt AG,

Weaver RE, Brenner DJ. 1982. Identification of Vibrio hollisae sp. nov.from patients with diarrhea. J Clin Microbiol 15:395– 401.

42. Thompson FL, Hoste B, Vandemeulebroecke K, Swings J. 2003. Re-classification of Vibrio hollisae as Grimontia hollisae gen. nov., comb. nov.Int J Syst Evol Microbiol 53:1615–1617. http://dx.doi.org/10.1099/ijs.0.02660-0.

43. Teramura N, Tanaka K, Iijima K, Hayashida O, Suzuki K, Hattori S,Irie S. 2011. Cloning of a novel collagenase gene from the Gram-negativebacterium Grimontia (Vibrio) hollisae 1706B and its efficient expressionin Brevibacillus choshinensis. J Bacteriol 193:3049 –3056. http://dx.doi.org/10.1128/JB.01528-10.

44. Lecroisey A, Keil-Dlouha V, Woods DR, Perrin D, Keil B. 1975.Purification, stability and inhibition of the collagenase from Achromo-bacter iophagus. FEBS Lett 59:167–172. http://dx.doi.org/10.1016/0014-5793(75)80367-X.

45. Lecroisey A, Keil B. 1979. Differences in the degradation of nativecollagen by two microbial collagenases. Biochem J 179:53–58.

46. Lee JH, Ahn SH, Lee EM, Jeong SH, Kim YO, Lee SJ, Kong IS. 2005.The FAXWXXT motif in the carboxyl terminus of Vibrio mimicus met-alloprotease is involved in binding to collagen. FEBS Lett 579:2507–2513.http://dx.doi.org/10.1016/j.febslet.2005.03.062.

47. Perona JJ, Tsu CA, Craik CS, Fletterick RJ. 1997. Crystal structure of anecotin-collagenase complex suggests a model for recognition and cleav-age of the collagen triple helix. Biochemistry 36:5381–5392. http://dx.doi.org/10.1021/bi9617522.

48. Tsu CA, Perona JJ, Fletterick RJ, Craik CS. 1997. Structural basis for thebroad substrate specificity of fiddler crab collagenolytic serine protease 1.Biochemistry 36:5393–5401. http://dx.doi.org/10.1021/bi961753u.

49. Kim JC, Cha SH, Jeong ST, Oh SK, Byun SM. 1991. Molecular cloningand nucleotide sequence of Streptomyces griseus trypsin gene. Biochem JBiophys Res Commun 181:707–713. http://dx.doi.org/10.1016/0006-291X(91)91248-B.

50. Uesugi Y, Arima J, Usuki H, Iwabuchi M, Hatanaka T. 2008. Twobacterial collagenolytic serine proteases have different topological spec-ificities. Biochim Biophys Acta 1784:716 –726. http://dx.doi.org/10.1016/j.bbapap.2008.01.017.

51. Rawlings ND, Barrett AJ. 2004. Introduction: serine peptides and theirclans, p 1425–1427. In Barrett AJ, Rawlings ND, Woessner JF (ed), Hand-book of proteolytic enzymes, 2nd ed. Elsevier, London, United Kingdom.

52. Okamoto M, Yonejima Y, Tsujimoto Y, Suzuki Y, Watanabe K. 2001.A thermostable collagenolytic protease with a very large molecular massproduced by thermophilic Bacillus sp. strain MO-1. Appl Microbiol Bio-technol 57:103–108. http://dx.doi.org/10.1007/s002530100731.

53. Itoi Y, Horinaka M, Tsujimoto Y, Matsui H, Watanabe K. 2006.Characteristic features in the structure and collagen-binding ability of athermophilic collagenolytic protease from the thermophile Geobacilluscollagenovorans MO-1. J Bacteriol 188:6572– 6579. http://dx.doi.org/10.1128/JB.00767-06.

54. Petrova DH, Shishkov SA, Vlahov SS. 2006. Novel thermostable serinecollagenase from Thermoactinomyces sp. 21E: purification and someproperties. J Basic Microbiol 46:275–285. http://dx.doi.org/10.1002/jobm.200510063.

55. Catara G, Ruggiero G, La Cara F, Digilio FA, Capasso A, Rossi M.2003. A novel extracellular subtilisin-like protease from the hyperther-mophile Aeropyrum pernix K1: biochemical properties, cloning, and ex-pression. Extremophiles 7:391–399. http://dx.doi.org/10.1007/s00792-003-0337-4.

56. Ohara T, Makino K, Shinagawa H, Nakata A, Norioka S, Sakiyama F.1989. Cloning, nucleotide sequence, and expression of Achromobacterprotease I gene. J Biol Chem 264:20625–20631.

57. Jang H, Kim B, Pyun Y, Kim Y. 2002. A novel subtilisin-like serineprotease from Thermoanaerobacter yonseiensis KB-1: its cloning, expres-sion, and biochemical properties. Extremophiles 6:233–243. http://dx.doi.org/10.1007/s00792-001-0248-1.

58. Wan MY, Wang HY, Zhang YZ, Feng H. 2009. Substrate specificity andthermostability of the dehairing alkaline protease from Bacillus pumilus.Appl Biochem Biotechnol 159:394 – 403. http://dx.doi.org/10.1007/s12010-008-8497-4.

59. Kurata A, Uchimura K, Shimamura S, Kobayashi T, Horikoshi K.2007. Nucleotide and deduced amino acid sequences of a subtilisin-likeserine protease from a deep-sea bacterium, Alkalimonas collagenimarinaAC40T. Appl Microbiol Biotechnol 77:311–319. http://dx.doi.org/10.1007/s00253-007-1164-9.

Minireview

September 2015 Volume 81 Number 18 aem.asm.org 6105Applied and Environmental Microbiology

on March 16, 2021 by guest

http://aem.asm

.org/D

ownloaded from

60. Windhorst S, Frank E, Georgieva DN, Genov N, Buck F, Borowski P,Weber W. 2002. The major extracellular protease of the nosocomialpathogen Stenotrophomonas maltophilia: characterization of the proteinand molecular cloning of the gene. J Biol Chem 277:11042–11049. http://dx.doi.org/10.1074/jbc.M109525200.

61. Kim HK, Ha YR, Yu HS, Kong HH, Chung DI. 2003. Purification andcharacterization of a 33 kDa serine protease from Acanthamoeba lug-dunensis KA/E2 isolated from a Korean keratitis patient. Korean J Para-sitol 41:189 –196. http://dx.doi.org/10.3347/kjp.2003.41.4.189.

62. Kim WT, Kong HH, Ha YR, Hong YC, Jeong HJ, Yu HS, Chung DI.2006. Comparison of specific activity and cytopathic effects of purified 33kDa serine proteinase from Acanthamoeba strains with different degreeof virulence. Korean J Parasitol 44:321–330. http://dx.doi.org/10.3347/kjp.2006.44.4.321.

63. Zhao GY, Chen XL, Zhao HL, Xie BB, Zhou BC, Zhang YZ. 2008.Hydrolysis of insoluble collagen by deseasin MCP-01 from deep-seaPseudoalteromonas sp. SM9913: collagenolytic characters, collagen-binding ability of C-terminal polycystic kidney disease domain, and im-plication for its novel role in deep-sea sedimentary particulate organicnitrogen degradation. J Biol Chem 283:36100 –36107.

64. Wang YK, Zhao GY, Li Y, Chen XL, Xie BB, Su HN, Zhang YZ. 2010.Mechanistic insight into the function of the C-terminal PKD domain ofthe collagenolytic serine protease deseasin MCP-01 from deep sea Pseu-doalteromonas sp. SM9913: binding of the PKD domain to collagen re-sults in collagen swelling but does not unwind the collagen triple helix. JBiol Chem 285:14285–14291.

65. Ran LY, Su HN, Zhao GY, Gao X, Zhou MY, Wang P, Zhao HL, XieBB, Zhang XY, Chen XL, Zhang YZ. 2013. Structural and mechanisticinsights into collagen degradation by a bacterial collagenolytic serineprotease in the subtilisin family. Mol Microbiol 90:997–1010. http://dx.doi.org/10.1111/mmi.12412.

66. Ran LY, Su HN, Zhou MY, Wang L, Chen XL, Zhang XY, Xie B-B,Song XY, Shi M, Qin QL, Zhou BC, Zhang YZ. 2014. Characterizationof a novel subtilisin-like protease myroicolsin from deep-sea bacteriumMyroides profundi D25 and molecular insight into its collagenolyticmechanism. J Biol Chem 289:6041– 6053. http://dx.doi.org/10.1074/jbc.M113.513861.

67. Chen XL, Xie BB, Lu JT, He HL, Zhang YZ. 2007. A novel type ofsubtilase from the psychrotolerant bacterium Pseudoalteromonas sp.SM9913: catalytic and structural properties of deseasin MCP-01. Mi-crobiology 153:2116 –2125. http://dx.doi.org/10.1099/mic.0.2007/006056-0.

68. Wlodawer A, Li M, Gustchin A, Oyama H, Dunn BM, Oda K. 2003.Structural and enzymatic properties of the sedolisin family of serine-carboxyl peptidases. Acta Biochim Pol 50:81–102.

69. Tsuruoka N, Nakayama T, Ashida M, Hemmi H, Nakao M, MinakataH, Oyama H, Oda K, Nishino T. 2003. Collagenolytic serine-carboxylproteinase from Alicyclobacillus sendaiensis strain NTAP-1: purification,characterization, gene cloning, and heterologous expression. Appl Envi-ron Microbiol 69:162–169. http://dx.doi.org/10.1128/AEM.69.1.162-169.2003.

70. Tsuruoka N, Isono Y, Shida O, Hemmi H, Nakayama T, Nishino T.2003. Alicyclobacillus sendaiensis sp. nov., a novel acidophilic, slightlythermophilic species isolated from soil in Sendai, Japan. Int J Syst EvolMicrobiol 53:1081–1084. http://dx.doi.org/10.1099/ijs.0.02409-0.

71. Nakayama T, Tsuruoka N, Akai M, Nishino T. 2000. Thermostablecollagenolytic activity of a novel thermophilic isolate, Bacillus sp. strainNTAP-1. J Biosci Bioeng 89:612– 614.

72. Wlodawer A, Li M, Gustchina A, Tsuruoka N, Ashida M, Minakata H,Oyama H, Oda K, Nishino T, Nakayama T. 2004. Crystallographic andbiochemical investigations of kumamolisin-As, a serine-carboxyl pepti-dase with collagenase activity. J Biol Chem 279:21500 –21510. http://dx.doi.org/10.1074/jbc.M401141200.

73. Guo H, Wlodawer A, Guo H. 2005. A general acid-base mechanism forthe stabilization of a tetrahedral adduct in a serine-carboxyl peptidase: acomputational study. J Am Chem Soc 127:15662–15663. http://dx.doi.org/10.1021/ja0520565.

74. Okubo A, Li M, Ashida M, Oyama H, Gustchina A, Oda K, Dunn BM,Wlodawer A, Nakayama T. 2006. Processing, catalytic activity and crys-tal structures of kumamolisin-As with an engineered active site. FEBS J273:2563–2576. http://dx.doi.org/10.1111/j.1742-4658.2006.05266.x.

75. Guo H, Wlodawer A, Nakayama T, Xu Q, Guo H. 2006. Catalytic roleof proton transfers in the formation of a tetrahedral adduct in a serine

carboxyl peptidase. Biochemistry 45:9129 –9137. http://dx.doi.org/10.1021/bi060461i.

76. Xu Q, Guo HB, Wlodawer A, Nakayama T, Guo H. 2007. TheQM/MM molecular dynamics and free energy simulations of the acyla-tion reaction catalyzed by the serine-carboxyl peptidase kumamolisin-As. Biochemistry 46:3784 –3792. http://dx.doi.org/10.1021/bi061737p.

77. Xu Q, Li L, Guo H. 2010. Understanding the mechanism of deacylationreaction catalyzed by the serine carboxyl peptidase kumamolisin-As: in-sights from QM/MM free energy simulations. J Phys Chem B 114:10594 –10600. http://dx.doi.org/10.1021/jp102785s.

78. Kato T, Takahashi N, Kuramitsu HK. 1992. Sequence analysis andcharacterization of the Porphyromonas gingivalis prtC gene, which ex-presses a novel collagenase activity. J Bacteriol 174:3889 –3895.

79. Carlson SA, McCuddin ZP, Wu MT. 2005. SlyA regulates the collagenase-mediated cytopathic phenotype in multiresistant Salmonella. Microb Pat-hog 38:181–187. http://dx.doi.org/10.1016/j.micpath.2005.01.004.

80. Wu Q, Li C, Li C, Chen H, Shuliang L. 2010. Purification and charac-terization of a novel collagenase from Bacillus pumilus Col-J. ApplBiochem Biotechnol 160:129 –139. http://dx.doi.org/10.1007/s12010-009-8673-1.

81. Han HJ, Taki T, Kondo H, Hirono I, Aoki T. 2008. Pathogenicpotential of a collagenase gene from Aeromonas veronii. Can J Microbiol54:1–10. http://dx.doi.org/10.1139/W07-109.

82. Xiong N, Brewer MT, Anderson KL, Weeks KE, Carlson SA. 2011.Expression of a collagenase that enables blood-brain barrier penetrationfor Salmonella implicated in bovine encephalopathies. Microb Pathog51:230 –232. http://dx.doi.org/10.1016/j.micpath.2011.04.008.

83. Trillo-Muyo S, Jasilionis A, Domagalski MJ, Chruszcz M, Minor W,Kuisiene N, Arolas JL, Solà M, Gomis-Rüth FX. 2013. Ultratight crystalpacking of a 10 kDa protein. Biol Crystal 69:464 – 470. http://dx.doi.org/10.1107/S0907444912050135.

84. Popoff MR, Bouvet P. 2009. Clostridial toxins. Future Microbiol4:1021–1064. http://dx.doi.org/10.2217/fmb.09.72.

85. Smith GC, Merkel JR. 1982. Collagenolytic activity of Vibrio vulnificus:potential contribution to its invasiveness. Infect Immun 35:1155–1156.

86. Miyoshi N, Shimizu C, Miyoshi S-I, Shinoda S. 1987. Purification andcharacterization of Vibrio vulnificus protease. Microbiol Immunol 31:13–25. http://dx.doi.org/10.1111/j.1348-0421.1987.tb03064.x.

87. Kavermann H, Burns BP, Angermüller K, Odenbreit S, Fischer W,Melchers K, Haas R. 2003. Identification and characterization of Heli-cobacter pylori genes essential for gastric colonization. J Exp Med 197:813– 822. http://dx.doi.org/10.1084/jem.20021531.

88. Duarte AS, Correia A, Esteves AC. 2014. Bacterial collagenases—a review.Crit Rev Microbiol http://dx.doi.org/10.3109/1040841X.2014.904270.

89. Suggs W, Van WH, Sharefkin JB. 1992. Enzymatic harvesting of adulthuman saphenous vein endothelial cells: use of a chemically definedcombination of two purified enzymes to attain viable cell yields equal tothose attained by crude bacterial collagenase preparations. J Vasc Surg15:205–213.

90. Mandl I. 1972. Collagenase comes of age, p 1–16. In Mandl I (ed),Collagenase. Gordon & Breach Science Publishers, New York, NY.

91. Mandl I. 1982. Bacterial collagenases and their clinical applications.Arzneimittelforschung 32:1381–1384.

92. Vrabec R, Moserova J, Konickova Z, Behounkova E, Blaha J. 1974.Clinical experience with enzymatic debridement of burned skin with theuse of collagenase. J Hyg Epidemiol Microbiol Immunol 18:496 – 498.

93. Hansbrough JF, Achauer B, Dawson J, Himel H, Luterman A, SlaterH, Levenson S, Salzberg CA, Hansbrough WB, Dore C. 1995. Woundhealing in partial-thickness burn wounds treated with collagenase oint-ment versus silver sulfadiazine cream. J Burn Care Rehabil 16:241–247.http://dx.doi.org/10.1097/00004630-199505000-00004.

94. Varma AO, Bugatch E, German FM. 1973. Debridement of dermalulcers with collagenase. Surg Gynecol Obstet 136:281–282.

95. Lee LK, Ambrus JL. 1975. Collagenase therapy for decubitus ulcers.Geriatrics 30:91–98.

96. Vetra H, Whittaker D. 1975. Hydrotherapy and topical collagenase fordecubitus ulcers. Geriatrics 30:53–58.

97. Rao DB, Sane PG, Georgiev EL. 1975. Collagenase in the treatment ofdermal and decubitus ulcers. J Am Geriatr Soc 23:22–30. http://dx.doi.org/10.1111/j.1532-5415.1975.tb00376.x.

98. Altman MI, Goldstein L, Horowitz S. 1978. Collagenase: an adjunct tohealing trophic ulcerations in the diabetic patient. J Am Podiatry Assoc68:11–15. http://dx.doi.org/10.7547/87507315-68-1-11.

Minireview

6106 aem.asm.org September 2015 Volume 81 Number 18Applied and Environmental Microbiology

on March 16, 2021 by guest

http://aem.asm

.org/D

ownloaded from

99. Boxer AM, Gottesman N, Bernstein H, Mandl I. 1969. Debridement ofdermal ulcers and decubiti with collagenase. Geriatrics 24:75– 86.

100. Záruba F, Lettl A, Brozkova L, Skrdlantova H, Krs V. 1974. Collage-nase in the treatment of ulcers in dermatology. J Hyg Epidemiol Micro-biol Immunol 18:499 –500.

101. Haimovici H, Strauch B. 1972. Use of collagenase in the managementof stasis and ischemic ulcers of the lower extremities, p 177–183. InMandl I (ed), Collagenase. Gordon & Breach Science Publishers, NewYork, NY.

102. Badalamente MA, Hurst LC. 2000. Enzyme injection as nonsurgicaltreatment of Dupuytren’s disease. J Hand Surg Am 25:629 – 636. http://dx.doi.org/10.1053/jhsu.2000.6918.

103. Shi L, Ermis R, Garcia A, Telgenhoff D, Aust D. 2010. Degradation ofhuman collagen isoforms by Clostridium collagenase and the effects ofdegradation products on cell migration. Int Wound J 7:87–95. http://dx.doi.org/10.1111/j.1742-481X.2010.00659.x.

104. He HL, Chen XL, Li JW, Zhang YZ, Gao PJ. 2004. Taste improvementof refrigerated meat treated with cold-adapted protease. Food Chem 84:307–311.

105. Zhao GY, Zhou MY, Zhao HL, Chen XL, Xie BB, Zhang XY, He HL,Zhou BC, Zhang YZ. 2012. Tenderization effect of cold-adapted collageno-lytic protease MCP-01 on beef meat at low temperature and its mechanism.Food Chem 134:1738–1744. http://dx.doi.org/10.1016/j.foodchem.2012.03.118.

Minireview

September 2015 Volume 81 Number 18 aem.asm.org 6107Applied and Environmental Microbiology

on March 16, 2021 by guest

http://aem.asm

.org/D

ownloaded from