Structural insights into thermophilic chaperonin complexes.

Group I chaperonins are dual heptamer protein complexes that play significant roles in protein homeostasis. The structure and function of the Escherichia coli chaperonin are well characterized. However, the dynamic properties of chaperonins, such as large ATPase-dependent conformational changes by binding of lid-like co-chaperonin GroES, have made structural analyses challenging, and our understanding of these changes during the turnover of chaperonin complex formation is limited. In this study, we used single-particle cryogenic electron microscopy to investigate the structures of GroES-bound chaperonin complexes from the thermophilic hydrogen-oxidizing bacteria Hydrogenophilus thermoluteolus and Hydrogenobacter thermophilus in the presence of ATP and AMP-PNP. We captured the structure of an intermediate state chaperonin complex, designated as an asymmetric football-shaped complex, and performed analyses to decipher the dynamic structural variations. Our structural analyses of inter- and intra-subunit communications revealed a unique mechanism of complex formation through the binding of a second GroES to a bullet-shaped complex.

Biochemical characterization of a novel thermostable chitinase from Hydrogenophilus hirschii strain KB-DZ44.

An extracellular acido-thermostable endo-chitinase (called ChiA-Hh59) from thermophilic Hydrogenophilus hirschii strain KB-DZ44, was purified and characterized. The maximum chitinase activity recorded after 36-h of incubation at 60°C was 3000U/ml. Pure enzyme was obtained after heat and acidic treatment, precipitation by ammonium sulphate and acetone, respectively, followed by sequential column chromatographies on Sephacryl S-200 and Mono Q-Sepharose. Based on Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis, the purified enzyme is a monomer with a molecular mass of 59103.12-Da. The 22 residue NH2-terminal sequence of the enzyme showed high homology with family-18 bacterial chitinases. The optimum pH and temperature values for chitinase activity were pH 5.0 and 85°C, respectively. The pure enzyme was completely inhibited by p-chloromercuribenzoic acid (p-CMB) and N-ethylmaleimide (NEM). The obtained results suggest that ChiA-Hh59 might be an endo-chitinase. The studied chitinase exhibited high activity towards colloidal chitin, chitin azure, glycol chitin, while it did not hydrolyse chitibiose and amylose. Its Km and kcat values were 0.298mg colloidal chitin/ml and 14400s-1, respectively. Its catalytic efficiency was higher than those of chitodextrinase and ChiA-65. Additionally, Thin-layer chromatography (TLC) analysis from chitin-oligosaccharides showed that ChiA-Hh59 acted as an endo-splitting enzyme. In conclusion, this chitinase may have great potential for the enzymatic degradation of chitin.

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.