Abstract
Thermotoga neapolitana 1,4-beta-d-glucan glucohydrolase A preferentially hydrolyzes cello-oligomers, such as cellotetraose, releasing single glucose moieties from the reducing end of the cello-oligosaccharide chain. Using directed evolution techniques of error-prone PCR and mutant library screening, a variant glucan glucohydrolase has been isolated that hydrolyzes the disaccharide, cellobiose, at a 31% greater rate than its wild type (WT) predecessor. The mutant library, expressed in Escherichia coli, was screened at 85 degrees C for increased hydrolysis of cellobiose, a native substrate rather than a chromogenic analog, using a continuous, thermostable coupled enzyme assay. The V(max) for the mutant was 108 +/- 3 units mg(-1), whereas that of the WT was 75 +/- 2 units mg(-1). The K(m) for both proteins was nearly the same. The k(cat) for the new enzyme increased by 31% and its catalytic efficiency (k(cat)/K(m)) for cellobiose also rose by 31% as compared with the parent. The nucleotide sequence of two positive clones and two null clones identified 11 single base shifts. The nucleotide transition in the most active clone caused an isoleucine to threonine amino acid substitution at position 170. Structural models for I170T and WT proteins were derived by sequence homology with Protein Data Bank code 1BGA from Paenibacillus polymyxa. Analysis of the WT and I170T model structures indicated that the substitution in the mutant enzyme repositioned the conserved catalytic residue Asn-163 and reconfigured entry to the active site.
Highlights
The multistep transformation of cellulosic biomass from the biopolymer to its glucose components is mediated in nature by the enzymatic hydrolysis of the polymer and its glucan intermediates
The kcat for the new enzyme increased by 31% and its catalytic efficiency for cellobiose rose by 31% as compared with the parent
To screen for an evolved -glucosidase (GghA) with increased activity toward a native substrate, cellobiose, we developed a thermostable (85 °C) coupled enzyme assay with glucokinase and glucose-6-phosphate dehydrogenase from Thermotoga maritima [20]
Summary
GghA, 1,4--D-glucan glucohydrolase; TEA, triethanolamine; WT, wild type; pNPG, p-nitrophenyl--Dglucoside. In the absence of a published crystal structure for WT GghA, a three-dimensional molecular model, based on sequence homology to Paenibacillus polymyxa [21], was derived for both wild type (WT) and mutant GghA proteins. Our analysis of the parent and mutant apoenzymes was facilitated by the breadth of structural data available for family 1 glycosyl hydrolases [22]: the conservation of the eightstranded TIM barrel-fold [23]; the known residues involved in positioning and substrate recognition (24 –26); the mechanism of hydrolysis by general acid catalysis and the condition of specific residues in the active site microenvironment [27, 28]. Comparing the enhanced GghA apoenzyme to known family 1 structures enabled us to suggest how its single amino acid substitution produces the observed functional improvement
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