Kinetic characterization of the N-terminal domain of Malonyl-CoA reductase.

Climate change is driving a search for environmentally safe methods to produce chemicals used in ordinary life. One such molecule is 3-hydroxypropionic acid, which is a platform industrial chemical used as a precursor for a variety of other chemical end products. The biosynthesis of 3-hydroxypropionic acid can be achieved in recombinant microorganisms via malonyl-CoA reductase in two separate reactions. The reduction of malonyl-CoA by NADPH to form malonic semialdehyde is catalyzed in the C-terminal domain of malonyl-CoA reductase, while the subsequent reduction of malonic semialdehyde to 3-hydroxypropionic acid is accomplished in the N-terminal domain of the enzyme. A new assay for the reverse reaction of the N-terminal domain of malonyl-CoA reductase from Chloroflexus aurantiacus activity has been developed. This assay was used to determine the kinetic mechanism and for isotope effect studies. Kinetic characterization using initial velocity patterns revealed random binding of the substrates NADP+ and 3-hydroxypropionic acid. Isotope effects showed substrates react to give products faster than they dissociate and that the products of the reverse reaction, NADPH and malonic semialdehyde, have a low affinity for the enzyme. Multiple isotope effects suggest proton and hydride transfer occur in a concerted fashion. This detailed kinetic characterization of the reaction catalyzed by the N-terminal domain of malonyl-CoA reductase could aid in engineering of the enzyme to make the biosynthesis of 3-hydroxypropionic acid commercially competitive with its production from fossil fuels.

Kinetic characterization of the C-terminal domain of Malonyl-CoA reductase.

Malonyl-CoA reductase utilizes two equivalents of NADPH to catalyze the reduction of malonyl-CoA to 3-hydroxypropionic acid (3HP). This reaction is part of the carbon fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. The enzyme is composed of two domains. The C-terminal domain catalyzes the reduction of malonyl-CoA to malonic semialdehyde, while the N-terminal domain catalyzes the reduction of the aldehyde to 3HP. The two domains can be produced independently and retain their enzymatic activity. This report focuses on the kinetic characterization of the C-terminal domain. Initial velocity patterns and inhibition studies showed the kinetic mechanism is ordered with NADPH binding first followed by malonyl-CoA. Malonic semialdehyde is released first, while CoA and NADP+ are released randomly. Analogs of malonyl-CoA showed that the thioester carbon is reduced, while the carboxyl group is needed for proper positioning. The enzyme transfers the pro-S hydrogen of NADPH to malonyl-CoA and pH rate profiles revealed that a residue with a pKa value of about 8.8 must be protonated for activity. Kinetic isotope effects indicated that NADPH is not sticky (that is, NADPH dissociates from the enzyme faster than the rate of product formation) and product release is partially rate-limiting. Moreover, the mechanism is stepwise with the pH dependent step occurring before or after hydride transfer. The findings from this study will aid in the development of an eco-friendly biosynthesis of 3HP which is an industrial chemical used in the production of plastics and adhesives.