Greg Prussia
Research Associate
Washington State University
Institute of Biological Chemistry
299 Clark Hall
Pullman, WA 99164
E-mail: gprussia1@gmail.com
Research Interests
I am interested in delineating the determinants of carboxylation in novel microbial-based carboxylase enzymes. The metabolically versatile α-proteobacterium Xanthobacter autotrophicus Py2 utilizes two novel carboxylases, 2-ketopropyl coenzyme M oxidoreductase/carboxylase (2-KPCC) and acetone carboxylase (AC), in propylene and alcohol metabolism, respectively.
2-KPCC is a member of the NAD(P)H-dependent disulfide oxidroreductase (DSOR) family of enzymes. The members of this family catalyze redox reactions and several well-characterized members catalyze the reductive cleavage of disulfide substrate. 2-KPCC performs the reductive cleavage of a thioether bond and subsequently carboxylates it’s intermediate. How 2-KPCC has integrated the paradigms of carboxylation using a scaffold purposed for reductive cleavage is unknown.
Essential to the redox chemistry catalyzed by many DSOR members is a conserved His-Glu catalytic dyad, which also serves to stabilize the electronic interaction between the FAD cofactor and the redox-active cysteine pair in the enzymes reactive state. 2-KPCC has substituted the catalytic His and Glu with Phe and His, respectively. We have shown that the phenylalanine substitution is critical for excluding protons (as competing electrophiles) from the active site and the downstream histidine substitution acts to stabilize the negative charge on the carboxylated product, acetoacetate. Substitution of both catalytic dyad residues affects the protonated and electronic state of the redox-active cysteine pair and FAD cofactor, altering the DSOR active site to accommodate the unique cleavage and CO2-fixation reaction catalyzed by 2-KPCC (Fig 1).
Recently, our lab has published the first crystal structure of acetone carboxylase, allowing insight into the unique mechanism catalyzed by this enzyme. Apo- and AMP-bound structures have led to the proposal of a catalytic mechanism analogous to other carboxylase enzymes. Testing the tenets of this mechanism provides an opportunity to further characterize acetone carboxylases. This is an exciting new front in understanding the determinants of carboxylation in a unique family of enzymes.
