Physiological, Biochemical and Biotechnological Characterization of Glycoside Hydrolases from the Hyperthermophilic Bacterium

Abstract

The genome sequence of Thermotoga maritima MSB8, encodes for the highest number of glycoside hydrolase genes amongst hyperthermophilic Bacterial and Archaeal genome sequences reported to date. The ability of T. maritima to utilize the polysaccharides galactomannan and CM cellulose as carbon sources can be attributed at least in part due to the presence of the genes cel5A (TM1751), man5 (TM1227) and cel74 (TM0305). The encoded proteins Tm Man5 and Tm Cel74 are extracellular marked by the presence of N-terminal signal peptides whereas Tm Cel5A is intracellular. Biochemical properties of recombinant versions of Tm Man5 and Tm Cel74, expressed in Escherichia coli, correlated well with predictions made by sequence comparisons. Thus, Tm Man5 was found to be a strict -mannanase while Tm Cel74 was found to be a strict endoglucanase. In contrast, although Tm Cel5A shows sequence similarity to an endoglucanase, its biochemical characteristics point to dual substrate specificity such that Tm Cel5A was found to hydrolyze both -mannan and -glucan polysaccharides. Glu-137 (proton donor) and Glu-253 (nucleophile) were found to be the catalytic residues in Tm Cel5A while Glu-329 was the catalytic nucleophile in Tm Man5. A mutation of these residues in each protein resulted in a complete loss of hydrolytic activity. Currently, Tm Cel74 is the only endoglucanase in Family 74 of glycoside hydrolases that lacks the presence of a cellulose-binding module at its C- terminus. Fusion of a binding module to the C-terminus of Tm Cel74 allowed the chimeric protein to bind and hydrolyze ii microcrystalline cellulose. Gene expression profiles of cel5A and man5 using Northern hybridizations and cDNA microarrays suggested co-regulation during growth on mannose and -1,4 mannan polysaccharides. Overall expression levels of cel74 were several fold lower than the other extracellular endoglucanase gene cel12A (TM1524) during growth on -1,4 glucan polysaccharides. Global gene expression analysis using a targeted cDNA microarray indicated the presence of tight regulatory mechanisms for glycoside hydrolase expression in T. maritima during growth on different carbon sources. Mixed model data analysis revealed co-regulation of genes within potential operons as well as sets of spatially distant gene strings with similar expression profiles, suggesting the presence of regulons in the T. maritima genome. This information in conjunction with biochemical characteristics of encoded proteins, was used to predict pathways for polysaccharide uptake and utilization in T. maritima. The research presented in this work provides a framework for future studies using full genome microarrays of T. maritima and other hyperthermophiles for the identification of glycoside hydrolases with novel sequences and substrate specificities.