EPR-kinetic isotope effect study of the mechanism of radical-mediated dehydrogenation of an alcohol by the radical SAM enzyme DesII

Abstract

The radical S-adenosyl-L-methionine enzyme DesII from Streptomyces venezuelae is able to oxidize the C3 hydroxyl group of TDP-D-quinovose to the corresponding ketone via an α-hydroxyalkyl radical intermediate. It is unknown whether electron transfer from the radical intermediate precedes or follows its deprotonation, and answering this question would offer considerable insight into the mechanism by which the small but important class of radical-mediated alcohol dehydrogenases operate. This question can be addressed by measuring steady-state kinetic isotope effects (KIEs); however, their interpretation is obfuscated by the degree to which the steps of interest limit catalysis. To circumvent this problem, we measured the solvent deuterium KIE on the saturating steady-state concentration of the radical intermediate using electron paramagnetic resonance spectroscopy. The resulting value, 0:22 ± 0:03, when combined with the solvent deuterium KIE on the maximum rate of turnover (V) of 1:8 ± 0:2, yielded a KIE of 8 ± 2 on the net rate constant specifically associated with the α-hydroxyalkyl radical intermediate. This result implies that electron transfer from the radical intermediate does not precede deprotonation. Further analysis of these isotope effects, along with the pH dependence of the steady-state kinetic parameters, likewise suggests that DesII must be in the correct protonation state for initial generation of the α-hydroxyalkyl radical. In addition to providing unique mechanistic insights, this work introduces a unique approach to investigating enzymatic reactions using KIEs.T he enzyme-catalyzed dehydrogenation of an alcohol to a carbonyl is one of the most common and fundamental reactions of both primary metabolism and secondary metabolism. In the majority of cases studied, these reactions proceed via hydride transfer to an organic cofactor, such as the nicotinamide moiety of NAD(P) + or the isoalloxazine group of FAD among others (1, 2). In contrast, much less is understood regarding radical-mediated dehydrogenation of an alcohol, because only a handful of enzyme examples have been described. Of the radical alcohol dehydrogenases, galactose oxidase is the most extensively studied, and it is known to use a Cu II ion coordinated to a proteinderived 3′-(S-cysteinyl)tyrosyl radical to catalyze dehydrogenation of the C6 hydroxyl group of galactose (3, 4). In contrast, BtrN and the anaerobic sulfatase maturating enzymes (anSMEs) are radical S-adenosyl-L-methionine (SAM) enzymes (5-7) responsible for the dehydrogenation of the C3 hydroxyl group of 2-deoxy-scylloinosamine (8, 9) and the hydroxyl/sulfhydryl moieties of serine/ cysteine residues (10-12), respectively. In addition to the primary [4Fe-4S] 1+ cluster, which reduces SAM (1) to methionine (2) and the 5′-deoxyadenosyl radical (17; see Fig. 4) to initiate radical catalysis, BtrN (13,14) and anSMEs (15-18) possess one or more auxiliary [4Fe-4S] aux clusters, which have been proposed to coordinate the hydroxyl group to be oxidized in the substrate.The enzyme...

Theory and Application of the Relationship Between Steady-State Isotope Effects on Enzyme Intermediate Concentrations and Net Rate Constants

“…These three models, which are shown in Fig. 4, also take into account the observed pH-dependence of the DesII catalyzed dehydrogenation reaction that implicated titratable forms of the enzyme with decreasing activity as the pH is lowered (Ruszczycky et al, 2013). …”

“…The solvent deuterium isotope effect D 2 O V on the DesII-catalyed dehydrogenation of 3 was measured by direct comparison and found to be 2.7 ± 0.4 at the suboptimal pH 8.0 Hepes buffer ratio, 9 where the α -hydroxylalkyl substrate radical could be observed by EPR (Ruszczycky et al, 2013). This isotope effect seemed to suggest that there was indeed a hydron transfer reaction taking place during the catalytic cycle.…”

“…In the case of DesII, it was possible to reduce the value of f ES • H by running the reaction at the more optimal pH of 10 where the substrate radical could no longer be observed by EPR (Ruszczycky et al, 2013). Consequently, the value of D 2 O V decreased to 1.8±0.2 at this pH, thereby further supporting models A and C. This approach is identical to that first proposed by Cook and Cleland (1981a,b), whereby the degree to which an elementary reaction is rate-limiting can be modulated by examining the isotope effect at suboptimal pH.…”

“…This was controlled for by both measuring the ratio at different freeze-quench times and also examining control quenches by HPLC to determine the extent of reaction in each system. The DesII enzyme also had a number of additional peculiarities requiring attention such as substrate inhibition and ensuring proper integration of the EPR signals, and these aspects are carefully addressed in the original work (Ruszczycky et al, 2013). …”

X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry

“…In BtrN, E189 forms a hydrogen bond/salt bridge with R152 ( Fig. 2A) After or concerted with deprotonation, collapse of the •C3-Oradical to the C3=O product necessitates loss of an electron, most likely to one of BtrN's [4Fe-4S] clusters (5,17,18,34). The AdoMet and Aux clusters are 8.6 and 9.6 Å away, respectively, from the C3 position of DOIA, making them both within range for a suitable electron transfer partner (35) (Fig.…”