Reversal of the Substrate Specificity of CMP <i>N</i>‑Glycosidase to dCMP
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Abstract
MilB is a CMP hydrolase involved
in the early steps of biosynthesis of the antifungal compound mildiomycin.
An enzyme from the bacimethrin biosynthetic pathway, BcmB, is closely
related to MilB in both sequence and function. These two enzymes belong
to the nucleoside 2′-deoxyribosyltransferase (NDT) superfamily.
NDTs catalyze <i>N</i>-glycosidic bond cleavage of 2′-deoxynucleosides
via a covalent 2-deoxyribosyl-enzyme intermediate. Conservation of
key active site residues suggests that members of the NDT superfamily
share a common mechanism; however, the enzymes differ in their substrate
preferences. Substrates vary in the type of nucleobase, the presence
or absence of a 2′-hydroxyl group, and the presence or absence
of a 5′-phosphate group. We have determined the structures
of MilB and BcmB and compared them to previously determined structures
of NDT superfamily members. The comparisons reveal how these enzymes
differentiate between ribosyl and deoxyribosyl nucleotides or nucleosides
and among different nucleobases. The 1.6 Å structure of the MilB–CMP
complex reveals an active site feature that is not obvious from comparisons
of sequence alone. MilB and BcmB that prefer substrates containing
2′-ribosyl groups have a phenylalanine positioned in the active
site, whereas NDT family members with a preference for 2′-deoxyribosyl
groups have a tyrosine residue. Further studies show that the phenylalanine
is critical for the specificity of MilB and BcmB toward CMP, and mutation
of this phenylalanine residue to tyrosine results in a 1000-fold reversal
of substrate specificity from CMP to dCMP