Biochemical and molecular studies of transketolase from rhodobacter sphaeroides and its inactivation by oxygen

by Bobst, Cedric Elmer

Abstract (Summary)
Transketolase is a ubiquitous thiamin diphosphate (ThDP)-dependent enzyme that plays an important role in the oxidative pentose phosphate pathway (OPPP) of virtually all organisms; this enzyme also functions as a key catalyst in the Calvin-Benson-Bassham (CBB) reductive pentose phosphate cycle of autotrophic organisms. Most organisms maintain multiple isoforms of transketolase within their genome; however, all reported research comparing different forms of this enzyme within the same organism originated from genetic and cellular biological studies. In addition, most biochemical studies on transketolase have been focused on eukaryotic or OPPP forms of the enzyme. We therefore set out to biochemically characterize both isoforms of transketolase from the non-sulfur purple bacterium Rhodobacter sphaeroides. The OPPP (tkt) and CBB (cbbT) transketolase genes were over-expressed in E. coli and recombinant proteins were purified to homogeneity. Techniques applied to compare these enzymes included substrate kinetics, molecular modeling, and stability studies. Biochemical differences suggest that each enzyme has a preferred environment for catalysis. Similar to the E. coli enzyme, both enzymes displayed inactivation in the presence of oxygen. Characterization of the inactivated enzyme was performed in order to better understand this type of inactivation. In addition to kinetic studies, spectroscopic methods such as circular dichroism and fluorometry, suggested that inactivation occurred in an irreversible manner and was accompanied by a small conformational change. To investigate the role of a loosely conserved cysteine near the active site of CbbT, this residue was targeted for site-directed mutagenesis. Various methods of quantifying ThDP binding were applied, establishing an important function for this cysteine in the binding and orientation of this cofactor. However, no link in oxygen inactivation could be established for this residue. To further probe the conformational changes that occur upon oxygen inactivation, the kinetics of hydrogen/deuterium exchange was studied utilizing Fourier transform ion cyclotron resonance mass spectrometry. Though confident identification of the CbbT peptides produced by pepsin digestion was not possible based on m/z values alone, differences in exchange rates were observed for several peptides from oxygen inactivated CbbT. The magnitudes of the changes were small, indicating a change in protein dynamics rather than a global conformational change.
Bibliographical Information:


School:The Ohio State University

School Location:USA - Ohio

Source Type:Master's Thesis



Date of Publication:01/01/2004

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