Use of Nanostructure-Initiator Mass Spectrometry to Deduce Selectivity of Reaction in Glycoside Hydrolases.

Kai Deng,Christopher M Bianchetti,Lai F Bergeman,Paul D Adams,Trent R Northen,Brian G Fox,Taichi E Takasuka

doi:10.3389/fbioe.2015.00165

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.

Highlights

The enzymatic hydrolysis of plant cell wall material is a formidable task because of the complexity of the plant cell wall (Himmel et al, 2007)
Clostridium thermocellum enzymes were chosen for this study based on previous transcriptomic and proteomic results (Gold and Martin, 2007; Raman et al, 2011; Riederer et al, 2011) and other biochemical and structural results (Table 1)
The tetra-saccharide is linked to the nanostructure-initiator mass spectrometry (NIMS) probe by a potentially hydrolyzable anomeric linkage

Summary

INTRODUCTION

The enzymatic hydrolysis of plant cell wall material is a formidable task because of the complexity of the plant cell wall (Himmel et al, 2007). The specific gene regulatory and protein secretory patterns of this model consolidated bioprocessing organism have been well described (Brown et al, 2007; Gold and Martin, 2007; Roberts et al, 2010; Feinberg et al, 2011; Raman et al, 2011; Riederer et al, 2011), and many of the enzymes have been characterized Given this state of knowledge, individual enzymes from C. thermocellum have proven useful for the development and testing of new approaches for assignment of GH function. 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 reactive properties This method, which combines high-sensitivity detection of multiple products with quantitative numerical analysis of their time-dependent formation, provides a new approach to enhance the annotation of GH phylogenetic trees with functional measurements

MATERIALS AND METHODS

RESULTS AND DISCUSSION

CONCLUSION