Mechanistic studies on the flavin-dependent N6-lysine monooxygenase MbsG reveal an unusual control for catalysis

The mechanism of Mycobacterium smegmatis G (MbsG), a flavin-dependent L-lysine monooxygenase, was investigated under steady-state and rapid reaction conditions using primary and solvent kinetic isotope effects, substrate analogs, pH and solvent viscosity effects as mechanistic probes. The results suggest that L-lysine binds before NAD(P)H, which leads to a decrease in the rate constant for flavin reduction. L-lysine binding has no effect on the rate of flavin oxidation, which occurs in a one-step process without the observation of a C4a-hydroperoxyflavin intermediate. Similar effects were determined with several substrate analogs. Flavin oxidation is pH independent while the k(cat)/K-m and k(red)/K-D pH profiles for NAD(P)H exhibit single pK(a) values of similar to 6.0, with increasing activity as the pH decreases. At lower pH, the enzyme becomes more uncoupled, producing more hydrogen peroxide and superoxide. Hydride transfer is partially rate-limiting at neutral pH and becomes more rate-limiting at low pH. An inverse solvent viscosity effect on k(cat)/K-m for NAD(P)H was observed at neutral pH whereas a normal solvent viscosity effect was observed at lower pH. Together, the results indicate a unique mechanism where a rate-limiting and pH-sensitive conformational change occurs in the reductive half-reaction, which affects the efficiency of lysine hydroxylation. (C) 2014 Elsevier Inc. All rights reserved.