Steve Ealick's Research Group
Saccharomyces cerevisiae MilB
4JEL with sulfate
4JEM with cytodine 5´-monophosphate
Saccharomyces rimofaciens MilB (MilB) is a member of the 2´-deoxynucleoside ribosyltransferase (NDT) superfamily, which includes purine deoxyribosyltransferase (PTD) and nucleoside 2-deoxyribosyltransferase (NDRT) that we previously studied. MilB preferentially hydrolizes the N-glycosidic bond of 5-hydroxymethyl cytidine 5´-monophosphate (hmCMP) to release 5-hydroxymethyl cytosine, which is later incorporated into mildiomycin. Other NDT family members prefer 2´-deoxyriboxyl groups. MilB has 47% sequence identity to Clostridium botulinum BcmB, which we concurrently studied in this project.
There is one protomer of MilB per asymmetric unit, which consists of an α/β fold with a five-stranded parallel β-sheet with a flavodoxin-like strand order (21345).
|Chains A and B form a dimer by twofold noncrystallographic symmetry with predominant interactions between helices α4, α5, and α6.|
A comparison of the active sites of MilB (green, with CMP) and BcmB (blue) is shown at the right. Unlike other NDT family members, MilB and BcmB family members that prefer 2´-ribosyl groups have a critical phenylalanine (Phe6 in BcmB and Phe17 in MilB) positioned in the active site. Mutation of this phenylalanine residue to tyrosine results in a 1000-fold reversal of substrate specificity from CMP to deoxy CMP.
Sikowitz MD, Cooper LE, Begley TP, Kaminski PA, and Ealick SE. Reversal of Substrate Specificity of CMP N-glycosidase to dCMP. Biochemistry 52:4037–4047 (2013). PubMed