Discovery, Functional Genomics and Biochemical Characterization of Alpha-specific Glycosyl Hydrolases from Hyperthermophilic Microorganisms

Abstract

Glycosyl hydrolases — biocatalysts that are capable of hydrolyzing linkages between sugars — comprise a significant fraction of the current industrial enzyme market and are used in applications ranging from baking and brewing, to bioenergy, to oil⁄gas recovery. Biocatalysts for these applications often require stability and functional activity at elevated temperatures. As such, heterotrophic hyperthermophilic microorganisms (growth Topt > 80°C), capable of using a variety of carbohydrates as carbon and energy sources, have been examined as sources of thermally stable enzymes. The biochemical and functional attributes of the glycosyl hydrolases from these and other organisms can often be inferred from primary amino acid sequence information. The current challenges are to fully understand the known glycosyl hydrolases, through biochemical and biophysical investigations, and to identify and characterize glycosyl hydrolases not yet annotated within genome sequences.

Conserved domains allow for classification of glycosyl hydrolases into families, which have conserved structural features and catalytic residues. Clan-D glycoside hydrolases (GH27 and GH36) were compared structurally and shown to share a conserved retaining reaction mechanism, catalytic residues, and structural features. Structural alignment and biochemical analysis of site-directed mutants of the family 36 α-galactosidase from Thermotoga maritima identified the catalytic residues as D327 and D387 for the nucleophile and acid-base residues, respectively. Azide rescue of D327G showed a concentration dependent increase in activity and Brønsted analysis showed a change in the rate-limiting step at pH 8-9. When the acid-base mutant enzyme, D387G, was rescued with external anions azide and formate, a strong increase of activity was shown for galactoside substrates with a good leaving group (2,4-dinitrophenyl, DNP-Gal) over a poor leaving group (4-nitrophenyl, PNP-Gal). Similar increases in activity were not noted for the wild-type enzyme. pH curves of catalytic rate and catalytic efficiency for D387G shows no decrease in activity at higher pH, indicative of an acid-base mutant. Thermal denaturation of the mutant enzymes showed no change in the melting temperature for D327G and a 5.5°C decrease in melting temperature for D387G. These structural, biochemical, and biophysical analyses confirm the catalytic residues and the relationship between the clan-D glycoside hydrolases.

In the post-genomics era, functional annotation of biocatalysts encoded in genome sequences represents an important step in biotechnological applications. Despite the large number of sequenced genomes available and extensive characterization of the enzymes encoded therein, roughly 40% of genes are still annotated as "hypothetical proteins". Using a functional genomics approach, combined with bioinformatic techniques and biochemical characterization, the genome of the hyperthermophilic archaeon Pyrococcus furiosus was probed to identify new glycosyl hydrolases. The open reading frame (ORF) PF0870 was identified by bioinformatic analysis to encode a glycosyl hydrolase that was found to hydrolyze pNP-alpha-maltopyranoside and maltotriose, thereby defining it as a novel b-amylase. Transcriptional response of P. furiosus grown on pullulan, starch, glycogen, maltose, cellobiose, trehalose, and maltose/cellobiose suggested an alternative biochemical role for a previously characterized exo-alpha-glucanase (Costantino et al., J. Bacteriol., 172:3654-3660, 1990), the native form of which had been previously purified from P. furiosus cell extracts. Purification and mass spectroscopy indicated that the native alpha-glucosidase was encoded in PF0132, an ORF annotated as a hypothetical protein, but now known to represent a new family of glycosyl hydrolases. Strong transcriptional response to growth on pullulan suggested activity on alpha-1,6-glucosidic linkages, and hydrolysis of the compounds maltose, isomaltose, panose, turanose, and maltotriose confirmed activity against this broad range of glucosidic linkages. At 90°C, hydrolytic efficiency on panose (Glc-alpha-1,6-Glc-alpha-1,4-Glc; kcat 278 s-1, KM 3.2 mM, and kcat/KM 85.6 s-1*mM-1) was similar to that on maltose (kcat 1,600 s-1, KM 7.1 mM, and kcat/KM 225 s-1*mM-1). This broad substrate specificity and high temperature optimum makes this an especially appealing enzyme for the final hydrolysis step in bioethanol production, which converts remaining maltooligosaccharides into glucose for fermentation. This result demonstrates how functional genomics approaches, when combined with bioinformatics analysis and biochemical characterization, can be utilized to determine the role of proteins encoded in unannotated ORFs of genome sequences.