Mechanistic studies on trans-2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase (Ent A) in the biosynthesis of the iron chelator enterobactin.
The enzyme 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase (2,3-diDHB dehydrogenase, hereafter Ent A), the product of the enterobactin biosynthetic gene entA, catalyzes the NAD(+)-dependent oxidation of the dihydroaromatic substrate 2,3-dihydro-2,3-dihydroxybenzoate (2,3-diDHB) to the aromatic catecholic product 2,3-dihydroxybenzoate (2,3-DHB). The catechol 2,3-DHB is one of the key siderophore units of enterobactin, a potent iron chelator secreted by Escherichia coli. To probe the reaction mechanism of this oxidation, a variety of 2,3-diDHB analogues were synthesized and tested as substrates. Specifically, we set out to elucidate both the regio- and stereospecificity of alcohol oxidation as well as the stereochemistry of NAD+ reduction. Of those analogues tested, only those with a C3-hydroxyl group (but not a C2-hydroxyl group) were oxidized to the corresponding ketone products. Reversibility of the Ent A catalyzed reaction was demonstrated with the corresponding NADH-dependent reduction of 3-ketocyclohexane- and cyclohexene-1-carboxylates but not the 2-keto compounds. These results establish that Ent A functions as an alcohol dehydrogenase to specifically oxidize the C3-hydroxyl group of 2,3-diDHB to produce the corresponding 2-hydroxy-3-oxo-4,6-cyclohexadiene-1-carboxylate (Scheme II) as a transient species that undergoes rapid aromatization to give 2,3-DHB. Stereospecificity of the C3 allylic alcohol group oxidation was confirmed to be 3R in a 1R,3R dihydro substrate, 3, and hydride transfer occurs to the si face of enzyme-bound NAD+.
Sakaitani
M
M
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Rusnak
F
F
Quinn
N R
NR
Tu
C
C
Frigo
T B
TB
Berchtold
G A
GA
Walsh
C T
CT
D001665:Binding Sites; D004755:Enterobacteriaceae / Q000378:metabolism; D004758:Enterobactin / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008956:Models, Chemical; D015394:Molecular Structure; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D012694:Serine / Q000031:analogs & derivatives*; D013237:Stereoisomerism
D029968:Escherichia coli Proteins; D009243:NAD; D004758:Enterobactin; D012694:Serine; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C061899:entA protein, E coli