This collaborative research award in the Chemistry of Life Processes (CLP) program supports work by Prof. Richard C. Holz of the Department of Chemistry and Biochemistry at Loyola University Chicago to carry out fundamental studies on the catalytic mechanism of iron- and cobalt-type nitrile hydratases (NHase, EC 4.2.1.84), in collaboration with Prof. Brian Bennett at the Medical College of Wisconsin (CHE-1058396). NHase's catalyze the hydration of nitriles to their corresponding commercially valuable amides in a chemo-, regio-, and/or enantio-selective manner at ambient pressures, temperatures, and physiological pH. For these reasons, NHases have attracted substantial interest as biocatalysts in preparative organic chemistry and NHase containing bacteria have found some industrial applications in the large scale production of acrylamide and nicotinamide.
Since little is understood about how NHase enzymes function, a better understanding of the structure and reaction mechanism of NHase enzymes will enable access to nitrile-hydrolyzing materials with broader substrate ranges, higher activities, and greater stabilities. These biomaterials will also provide a "green" alternative to the harsh industrial conditions required for nitrile hydration.
Nitriles are used extensively in the production of specialty chemicals such as polyacrylamide and nylon-66, the latter of which is one of the most important industrial polyamides. Nitrile hydratases (NHases, EC 4.2.1.84) catalyze the hydration of nitriles to their corresponding amides under ambient conditions and physiological pH. Since the currently employed industrial conditions used to hydrate nitriles to amides (either acid or base hydration), are often incompatible with the sensitive structures of many industrially and synthetically relevant compounds, NHases have attracted substantial interest as biocatalysts in preparative organic chemistry. Despite the industrial importance of NHase enzymes, details of their reaction mechanism remain poorly understood. The successful completion of the proposed project will benefit society by facilitating a more intelligent design and manufacture of nitrile based chiral pharmaceuticals and industrially important specialty chemicals such as acrylamide and nicotinamide. A better understanding of the structure and reaction mechanism of NHases, together with advances in protein screening and genetic engineering, will facilitate the exploitation of natural and synthetic biomimetic nitrile-hydrating catalysts with broader substrate ranges, higher activities and higher biocatalyst stabilities, further enhancing the future biotechnological application of these versatile biocatalysts. The data obtained during the three year period of this project (July 2011 – June 2014) allowed the elimination of three of the four possible catalytic mechanisms for NHase enzymes. Stopped-flow and time-resolved EPR spectroscopy identified that the substrate binds directly to the active site metal ion with catalytic competence, ESEEM indicated that the binding is via the nitrile N-atom, and EPR identified two strongly coupled protons in the product complex, consistent with a metal-bound amide product. Interestingly, 1H-ENDOR did not provide any evidence for a metal-bound nucleophile but this observation was rationalized following the X-ray structural studies of the boronic acid complexes, which indicate that the nucleophile is likely derived from an active site sulfenic acid moiety, a previously unknown role for this ligand (Figure 1). The current funding period has also provided the first evidence for a functional eukaryotic NHase. Thus, the collaborative approach used during the current funding period provided solid evidence on which to propose a novel catalytic mechanism for NHases (Figure 2). The profoundly interdisciplinary nature of this effort served as a platform to understand the catalytic mechanism of NHase enzymes and also provide training opportunities for women and classically underrepresented graduate and undergraduate students in biophysics, chemistry, and biochemistry.
