Structural studies of enzymes involved in propylene and acetone metabolism in Xanthobacter autotrophicus
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Date
2007
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Montana State University - Bozeman, College of Letters & Science
Abstract
X-ray crystallography has been an indispensable tool in understanding the mechanism of the enzymes of the epoxide carboxylation pathway in Xanthobacter autotrophicus. The main focus of this dissertation involves providing the structural basis for the stereoslectivity of the two stereospecific dehydrogenases of the pathway namely R- and S-HPCDH. The crystal structure of R- HPCDH cocrystallized with the substrate has been determined. The key elements of interactions between the enzyme and substrate are electrostatic interactions between the sulfonate oxygen atoms and two arginine residues (Arg152 and Arg196) of R- HPCDH. The comparison of the structure of R- HPCDH with a homology model of the S-HPCDH provides a structural basis for a mechanism of substrate specificity in which the binding of the substrate sulfonate moiety at distinct sites on each stereoselective enzyme directs the orientation of the appropriate substrate enantiomer for hydride abstraction. Moreover, crystal structures of the two methionine mutants of R-HPCDH have revealed that they have a role in shielding electrostatic interactions between the enzyme and the substrate from the surroundings. The structure of the presumed product bound state reveals that binding interactions between the substrate and the enzyme have striking similarities to the ones observed in the previously determined structure of 2-KPCC highlighting the utility of coenzyme M as a carrier molecule in the pathway. Extensive comparative structural analyses of the enzymes of the pathway reveal a common structural signature for coenzyme M binding. Coenzyme M, when conjugated with the substrates that lack innate chemical groups, such as short chain alkenes and epoxides, provide these substrates a handle to specifically bind and interact with the enzymes, thereby orienting them in a proper fashion for catalysis. Finally, exhaustive amount of ground work is laid towards the determination of the three dimensional structure of acetone carboxylase, the enzyme which converts acetone to acetoacetate in Xanthobacter. Initial electron density maps calculated by the phase information obtained from a number of moderately good heavy atom derivatives show a clear protein-solvent envelope, providing the first glimpse into the three dimensional structure of acetone carboxylase.