Kinetic and mechanistic analysis of the Escherichia coli ribD-encoded bifunctional deaminase-reductase involved in riboflavin biosynthesis.
Riboflavin is biosynthesized by most microorganisms and plants, while mammals depend entirely on the absorption of this vitamin from the diet to meet their metabolic needs. Therefore, riboflavin biosynthesis appears to be an attractive target for drug design, since appropriate inhibitors of the pathway would selectively target the microorganism. We have cloned and solubly expressed the bifunctional ribD gene from Escherichia coli, whose three-dimensional structure was recently determined. We have demonstrated that the rate of deamination (370 min (-1)) exceeds the rate of reduction (19 min (-1)), suggesting no channeling between the two active sites. The reductive ring opening reaction occurs via a hydride transfer from the C 4- pro-R hydrogen of NADPH to C'-1 of ribose and is the rate-limiting step in the overall reaction, exhibiting a primary kinetic isotope effect ( (D) V) of 2.2. We also show that the INH-NADP adduct, one of the active forms of the anti-TB drug isoniazid, inhibits the E. coli RibD. On the basis of the observed patterns of inhibition versus the two substrates, we propose that the RibD-catalyzed reduction step follows a kinetic scheme similar to that of its structural homologue, DHFR.
Magalhães
Maria L B
ML
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Argyrou
Argyrides
A
Cahill
Sean M
SM
Blanchard
John S
JS
D002384:Catalysis; D003641:Deamination; D041961:Deuterium Exchange Measurement; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D009709:Nucleotide Deaminases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D010428:Pentosephosphates / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D012256:Riboflavin / Q000096:biosynthesis* / Q000737:chemistry*; D012545:Schiff Bases; D012997:Solvents; D013379:Substrate Specificity / Q000235:genetics; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; D010428:Pentosephosphates; D012545:Schiff Bases; D012997:Solvents; C050562:ribose-1,5-bisphosphate; D009249:NADP; D013401:Sugar Alcohol Dehydrogenases; C104887:RibD protein, E coli; D009709:Nucleotide Deaminases; D012256:Riboflavin