title: Use of Nanostructure-Initiator Mass Spectrometry to Deduce Selectivity of Reaction in Glycoside Hydrolases.
abstract: Chemically synthesized nanostructure-initiator mass spectrometry (NIMS) probes derivatized with tetrasaccharides were used to study the reactivity of representative Clostridium thermocellum β-glucosidase, endoglucanases, and cellobiohydrolase. Diagnostic patterns for reactions of these different classes of enzymes were observed. Results show sequential removal of glucose by the β-glucosidase and a progressive increase in specificity of reaction from endoglucanases to cellobiohydrolase. Time-dependent reactions of these polysaccharide-selective enzymes were modeled by numerical integration, which provides a quantitative basis to make functional distinctions among a continuum of naturally evolved catalytic properties. Consequently, our method, which combines automated protein translation with high-sensitivity and time-dependent detection of multiple products, provides a new approach to annotate glycoside hydrolase phylogenetic trees with functional measurements.
authors.affiliation: US Department of Energy Joint BioEnergy Institute , Emeryville, CA , USA ; Sandia National Laboratories , Livermore, CA , USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Deng
authors.affiliation: US Department of Energy Great Lakes Bioenergy Research Center , Madison, WI , USA.
authors.forename: Taichi E
authors.initials: TE
authors.lastname: Takasuka
authors.affiliation: US Department of Energy Great Lakes Bioenergy Research Center , Madison, WI , USA ; Department of Chemistry, University of Wisconsin-Oshkosh , Oshkosh, WI , USA.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Bianchetti
authors.affiliation: US Department of Energy Great Lakes Bioenergy Research Center , Madison, WI , USA.
authors.forename: Lai F
authors.initials: LF
authors.lastname: Bergeman
authors.affiliation: US Department of Energy Joint BioEnergy Institute , Emeryville, CA , USA ; Lawrence Berkeley National Laboratory , Berkeley, CA , USA ; Department of Bioengineering, University of California Berkeley , Berkeley, CA , USA.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Adams
authors.affiliation: US Department of Energy Joint BioEnergy Institute , Emeryville, CA , USA ; Lawrence Berkeley National Laboratory , Berkeley, CA , USA.
authors.forename: Trent R
authors.initials: TR
authors.lastname: Northen
authors.affiliation: US Department of Energy Great Lakes Bioenergy Research Center , Madison, WI , USA ; Department of Biochemistry, University of Wisconsin-Madison , Madison, WI , USA.
authors.forename: Brian G
authors.initials: BG
authors.lastname: Fox
keywords: Nimzyme; assay; biofuels; cellulase; kinetics; mass spectrometry; protein engineering