Catalytic Mechanisms for Cofactor-Free Oxidase-Catalyzed Reactions: Reaction Pathways of Uricase-Catalyzed Oxidation and Hydration of Uric Acid

Abstract

First-principles quantum mechanical/molecular mechanical (QM/MM)-free energy calculations have been performed to uncover how uricase catalyzes metabolic reactions of uric acid (UA), demonstrating that the entire reaction process of UA in uricase consists of two stages–oxidation followed by hydration. The oxidation consists of four steps: (1) chemical transformation from 8-hydroxyxythine to an anionic radical via a proton transfer along with an electron transfer, which is different from the previously proposed electron-transfer mechanism that involves a dianion intermediate (UA2−) during the catalytic reaction process; (2) proton transfer to the O2− anion (radical); (3) diradical recombination to form a peroxo intermediate; (4) dissociation of H2O2 to generate the dehydrourate. Hydration, for the most favorable pathway, is initiated by the nucleophilic attack of a water molecule on dehydrourate, along with a concerted proton transfer through residue Thr69 in the catalytic site. According to the calculated free energy profile, the hydration is the rate-determining step, and the corresponding free energy barrier of 16.2 kcal/mol is consistent with that derived from experimental kinetic data, suggesting that the computational insights into the catalytic mechanisms are reasonable. The mechanistic insights not only provide a mechanistic base for future rational design of uricase mutants with improved catalytic activity against uric acid as an improved enzyme therapy, but also are valuable for understanding a variety of other cofactor-free oxidase-catalyzed reactions involving an oxygen molecule.

Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis

“…The protonation state observed in our UOX-8AZA-W1 complex is directly relevant for the hydration stage of the reaction mechanism. A recent theoretical study (Wei et al, 2017) proposed the existence of a strong hydrogen-bonding network between charged Lys10*, Thr57*, W1 and DHU N7 (note that in their paper the authors use protein numbering for B. subtilis UOX in which Lys9* and Thr69* correspond to A. flavus Lys10* and Thr57*, respectively). As W1 O approaches DHU C5 in the nucleophilic attack, W1 H1 comes close to Thr57* OG1 while HG1 gradually forms a bond with DHU N7 changing the C5 N7 double bond to a single bond (Fig.…”

“…Mechanistically, UOX-mediated degradation of UA follows two sequential steps: (1) an initial oxidation step whereby UA reacts with O 2 to yield dehydroisourate (DHU) via a 5peroxoisourate (5PIU) intermediate and (2) a hydration step in which DHU is hydroxylated to 5HIU [ Fig. 1(a)] (Kahn, 1999;Wei et al, 2017). The latter is ultimately transformed to the more soluble allantoin either enzymatically or nonenzymatically (Ramazzina et al, 2006).…”

Mechanisms and regioselectivities of DABCO/DMAP‐catalyzed [2+4] annulation reactions of allenoate with α,β‐unsaturated cyclic ketimine: A DFT study

“…In the past decades, density functional theory (DFT) has been extensively used as a powerful method to explore the detailed reaction mechanisms, and predict the regioselectivity as well as chemoselectivities in organocatalytic and biological reactions . It is worth mentioning that the DABCO‐catalyzed [2+4] cycloaddition of ethyl allenoate with arylidenoxindole has been theoretically investigated, but the observed regioselectivity is remarkably different from the reaction in this paper.…”

Different Catabolism Pathways Triggered by Various Methylxanthines in Caffeine-Tolerant Bacterium Pseudomonas putida CT25 Isolated from Tea Garden Soil

“…Uricase catalyzes the first reaction in the three-reaction catabolic pathway from uric acid to allantoin. Only one type of uricase usually exists as a cofactor-free enzyme in the peroxisome of eukaryotes [25,26], whereas three distinctive types of uricase are present in organisms without peroxisomes [27]: the first type is a soluble cofactor-free enzyme similar to that in eukaryotes and encoded by the uox gene in most monoderm prokaryotes [28]; the second type is a soluble flavindependent enzyme and encoded by the hpxO [29] or hpyO gene [30] in some diderm prokaryotes; and the third type is a cytochrome c-coupled membrane integral enzyme and encoded by gene puuD only in diderm prokaryotes [27]. According to our genome sequencing data, uricase in CT25 belongs to the third type (data not shown).…”