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 kcat/Km and kred/KD pH profiles for NAD(P)H exhibit single pKa values of ∼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 kcat/Km 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.
Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA; Fralin Life Science Institute, Virginia Tech, Blacksburg, VA 24061, USA.
Reeder M
RM
Robinson
Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA; Fralin Life Science Institute, Virginia Tech, Blacksburg, VA 24061, USA.
Pedro J
PJ
Rodriguez
Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA; Fralin Life Science Institute, Virginia Tech, Blacksburg, VA 24061, USA; Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, VA 24061, USA. Electronic address: psobrado@vt.edu.
Pablo
P
Sobrado
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D004138:Dinitrocresols / Q000378:metabolism*; D005415:Flavins; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020102:Mycobacterium smegmatis / Q000737:chemistry* / Q000201:enzymology; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism
D001426:Bacterial Proteins; C531259:C4a-hydroperoxyflavin; D004138:Dinitrocresols; D005415:Flavins; D011994:Recombinant Proteins; D013481:Superoxides; C024132:4,6-dinitro-o-cresol; D009249:NADP; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; C100031:lysine monooxygenase; D008239:Lysine
Conformational changes; Flavin-dependent monooxygenases; Rapid reaction kinetics; Siderophores; Viscosity effects; pH studies