Clavaminate synthase is an Fe(2+)-, O2-, and alpha-ketoglutarate-dependent oxygenase that catalyzes three transformations in the biosynthesis of the important beta-lactamase inhibitor clavulanic acid. The genes from Streptomyces clavuligerus encoding two isoenzymes of clavaminate synthase have been over-expressed in Escherichia coli to give soluble proteins whose reactions, kinetic properties, and molecular masses are in excellent agreement with the wild-type isozymes. Preliminary investigation of the active site of clavaminate synthase was undertaken using diethyl pyrocarbonate and N-ethylmaleimide. Each was inhibitory to catalytic activity. Protection from inactivation in the presence of these reagents by Fe2+, O2, and alpha-ketoglutaric acid was thwarted by the rapid self-inactivation of the enzyme in the absence of substrate. However, protection was achieved when Co2+, a potent competitive inhibitor of clavaminate synthase 2 with respect to Fe2+, was substituted. This is consistent with the presence of histidine and cysteine, respectively, at or near the active site and possibly involved in iron binding. In the course of constructing the expression vector, a simply applied general error analysis of the polymerase chain reaction was formulated to calculate the proportion of correctly replicated DNA and guide the design of experiments using this method.

The starch-hydrolysing enzyme GA (glucoamylase) from Rhizopus oryzae is a commonly used glycoside hydrolase in industry. It consists of a C-terminal catalytic domain and an N-terminal starch-binding domain, which belong to the CBM21 (carbohydrate-binding module, family 21). In the present study, a molecular model of CBM21 from R. oryzae GA (RoGACBM21) was constructed according to PSSC (progressive secondary structure correlation), modified structure-based sequence alignment, and site-directed mutagenesis was used to identify and characterize potential ligand-binding sites. Our model suggests that RoGACBM21 contains two ligand-binding sites, with Tyr32 and Tyr67 grouped into site I, and Trp47, Tyr83 and Tyr93 grouped into site II. The involvement of these aromatic residues has been validated using chemical modification, UV difference spectroscopy studies, and both qualitative and quantitative binding assays on a series of RoGACBM21 mutants. Our results further reveal that binding sites I and II play distinct roles in ligand binding, the former not only is involved in binding insoluble starch, but also facilitates the binding of RoGACBM21 to long-chain soluble polysaccharides, whereas the latter serves as the major binding site mediating the binding of both soluble polysaccharide and insoluble ligands. In the present study we have for the first time demonstrated that the key ligand-binding residues of RoGACBM21 can be identified and characterized by a combination of novel bioinformatics methodologies in the absence of resolved three-dimensional structural information.

Multiple sequence alignment is often used to locate consensus sequence stretches with evolutionary and functional conservation. However, when sequence similarity among the queries becomes low, sequence alignment tools generate extremely diverse results. The aim of this study is to incorporate relevant biological knowledge and assumptions to improve quality of general alignment on low similarity sequences. Since recognition of key features in carbohydrate binding module (CBM) family does not apply to general models, a more accurate weighted entropy function employing secondary-structure-based and key-residueweighted algorithms for alignment was designed to approach this goal. The results indicate that the proposed method is able to detect the known ligand-binding residues and to predict unknown functional residues in cellulose binding domains (CBDs) and xylooligosaccharides binding domains (XBDs) in terms of three-dimensional structures. Our results contribute molecular basis of CBDs and XBDs and potential application in development of alternative energy for future needs.