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1.  Cloning, Purification and Characterization of the Collagenase ColA Expressed by Bacillus cereus ATCC 14579 
PLoS ONE  2016;11(9):e0162433.
Bacterial collagenases differ considerably in their structure and functions. The collagenases ColH and ColG from Clostridium histolyticum and ColA expressed by Clostridium perfringens are well-characterized collagenases that cleave triple-helical collagen, which were therefore termed as ´true´ collagenases. ColA from Bacillus cereus (B. cereus) has been added to the collection of true collagenases. However, the molecular characteristics of B. cereus ColA are less understood. In this study, we identified ColA as a secreted true collagenase from B. cereus ATCC 14579, which is transcriptionally controlled by the regulon phospholipase C regulator (PlcR). B. cereus ATCC 14579 ColA was cloned to express recombinant wildtype ColA (ColAwt) and mutated to a proteolytically inactive (ColAE501A) version. Recombinant ColAwt was tested for gelatinolytic and collagenolytic activities and ColAE501A was used for the production of a polyclonal anti-ColA antibody. Comparison of ColAwt activity with homologous proteases in additional strains of B. cereus sensu lato (B. cereus s.l.) and related clostridial collagenases revealed that B. cereus ATCC 14579 ColA is a highly active peptidolytic and collagenolytic protease. These findings could lead to a deeper insight into the function and mechanism of bacterial collagenases which are used in medical and biotechnological applications.
doi:10.1371/journal.pone.0162433
PMCID: PMC5010206  PMID: 27588686
2.  Structural Basis for Activity Regulation and Substrate Preference of Clostridial Collagenases G, H, and T* 
The Journal of Biological Chemistry  2013;288(28):20184-20194.
Background: Bacterial collagenases degrade collagen substrates with high efficiency yet varying specificity.
Results: The newly identified calcium site, aspartate switch, and conformational selectivity filter regulate substrate access to the active sites of these collagenases.
Conclusion: The unanticipated dynamics of the substrate recognition sites plus zinc occupancy combine to tune the enzymatic activity.
Significance: The crystal structures provide a rational framework to understand and optimize the isoform-dependent collagenase activities.
Clostridial collagenases are among the most efficient enzymes to degrade by far the most predominant protein in the biosphere. Here we present crystal structures of the peptidases of three clostridial collagenase isoforms (ColG, ColH, and ColT). The comparison of unliganded and liganded structures reveals a quaternary subdomain dynamics. In the unliganded ColH structure, this globular dynamics is modulated by an aspartate switch motion that binds to the catalytic zinc. We further identified a calcium binding site in proximity to the catalytic zinc. Both ions are required for full activity, explaining why calcium critically affects the enzymatic activity of clostridial collagenases. Our studies further reveal that loops close to the active site thus serve as characteristic substrate selectivity filter. These elements explain the distinct peptidolytic and collagenolytic activities of these enzymes and provide a rational framework to engineer collagenases with customized substrate specificity as well as for inhibitor design.
doi:10.1074/jbc.M112.448548
PMCID: PMC3711286  PMID: 23703618
Protease; Protein Degradation; Protein Structure; Proteolytic Enzymes; X-ray Crystallography; Collagenase; Metal Regulation
3.  Structure of collagenase G reveals a chew and digest mechanism of bacterial collagenolysis 
Nature structural & molecular biology  2011;18(10):1109-1114.
Collagen constitutes one third of the body protein in humans, reflecting its extraordinary role in health and disease. Of similar importance, therefore, are the idiosyncratic proteases that nature evolved for collagen remodeling. Intriguingly, the most efficient collagenases are those that enable clostridial bacteria to colonize their host tissues, but despite intense studies, the structural and mechanistic basis of these enzymes has remained elusive. Here we present the crystal structure of collagenase G from Clostridium histolyticum at 2.55 Å resolution. By combining the structural data with enzymatic and mutagenesis studies, we derive a conformational two-state model of bacterial collagenolysis, in which the recognition and unraveling of collagen microfibrils into triple helices as well as the unwinding of the latter go hand in hand with collagenase opening and closing.
doi:10.1038/nsmb.2127
PMCID: PMC3191118  PMID: 21947205
4.  Crystallization and preliminary X-ray characterization of the catalytic domain of collagenase G from Clostridium histolyticum  
The catalytic domain of collagenase G from C. histolyticum was expressed in E. coli BL21 (DE3) and purified using affinity and size-exclusion column-chromatographic methods. Crystals were obtained at 290 K by the sitting-drop vapour-diffusion method and diffraction data have been collected to 2.75 Å resolution.
The catalytic domain of collagenase G from Clostridium histolyticum has been cloned, recombinantly expressed in Escherichia coli and purified using affinity and size-exclusion column-chromatographic methods. Crystals of the catalytic domain were obtained from 0.12 M sodium citrate and 23%(v/v) PEG 3350 at 293 K. The crystals diffracted to 2.75 Å resolution using synchrotron radiation. The crystals belong to an orthorhombic space group, with unit-cell parameters a = 57, b = 109, c = 181 Å. This unit cell is consistent with the presence of one molecule per asymmetric unit and a solvent content of approximately 53%.
doi:10.1107/S1744309108010476
PMCID: PMC2376405  PMID: 18453715
collagenase G; Clostridium histolyticum
5.  A universal strategy for high-yield production of soluble and functional clostridial collagenases in E. coli 
Clostridial collagenases are foe and friend: on the one hand, these enzymes enable host infiltration and colonization by pathogenic clostridia, and on the other hand, they are valuable biotechnological tools due to their capacity to degrade various types of collagen and gelatine. However, the demand for high-grade preparations exceeds supply due to their pathogenic origin and the intricate purification of homogeneous isoforms. We present the establishment of an Escherichia coli expression system for a variety of constructs of collagenase G (ColG) and H (ColH) from Clostridium histolyticum and collagenase T (ColT) from Clostridium tetani, mimicking the isoforms in vivo. Based on a setup of five different expression strains and two expression vectors, 12 different constructs were expressed, and a flexible purification platform was established, consisting of various orthogonal chromatography steps adaptable to the individual needs of the respective variant. This fast, cost-effective, and easy-to-establish platform enabled us to obtain at least 10 mg of highly pure mono-isoformic protein per liter of culture, ideally suited for numerous sophisticated downstream applications. This production and purification platform paves the way for systematic screenings of recombinant collagenases to enlighten the biochemical function and to identify key residues and motifs in collagenolysis.
doi:10.1007/s00253-009-1953-4
PMCID: PMC3085789  PMID: 19333597
Clostridial collagenases; Expression; Purification; Platform

Results 1-5 (5)