Insights into the catalytic mechanism of Grimontia hollisae collagenase through structural and mutational analyses.

Grimontia hollisae collagenase (Ghcol) exhibits high collagen-degrading activity. To explore its catalytic mechanism, its substrate (Gly-Pro-Hyp-Gly-Pro-Hyp, GPOGPO)-complexed crystal structure was determined at 2.0 Å resolution. A water molecule was observed near the active-site zinc ion. Since this water was not observed in the product (GPO)-complexed Ghcol, it was hypothesized that the GPOGPO-complexed Ghcol structure reflects a Michaelis complex, providing a structural basis for understanding the catalytic mechanism. Analyses of the active-site geometry and site-directed mutagenesis of the active-site tyrosine residues revealed that Glu493 and Tyr564 were essential for catalysis, suggesting that Glu493 functions as an acid and base catalyst while Tyr564 stabilizes the tetrahedral complex in the transition state. These results shed light on the catalytic mechanism of bacterial collagenase.

Oxidative phosphorylation in a thermophilic, facultative chemoautotroph, Hydrogenophilus thermoluteolus, living prevalently in geothermal niches.

Hydrogenophilus is a thermophilic, facultative chemoautotroph, which lives prevalently in high temperature geothermal niches. Despite the environmental distribution, little is known about its oxidative phosphorylation. Here, we show that inverted membrane vesicles derived from Hydrogenophilus thermoluteolus cells autotrophically cultivated with H2 formed a proton gradient on the addition of succinate, dl-lactate, and NADH, and exhibited oxidation activity toward these three organic compounds. These indicate the capability of mixotrophic growth of this bacterium. Biochemical analysis demonstrated that the same vesicles contained an F-type ATP synthase. The F1 sector of the ATP synthase purified from H. thermoluteolus membranes exhibited optimal ATPase activity at 65°C. Transformed Escherichia coli membranes expressing H. thermoluteolus F-type ATP synthase exhibited the same temperature optimum for the ATPase. These findings shed light on H. thermoluteolus oxidative phosphorylation from the aspects of membrane bioenergetics and ATPase biochemistry, which must be fundamental and advantageous in the biogeochemical cycles occurred in the high temperature geothermal niches.