Steve Ealick's Research Group

Saccharomyces cerevisiae MilB

PDB files:

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). 

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Chains A and B form a dimer by twofold noncrystallographic symmetry with predominant interactions between helices α4, α5, and α6.

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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.


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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

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