Structure of the N-glycosidase MilB in complex with hydroxymethyl CMP reveals its Arg23 specifically recognizes the substrate and controls its entry

Hydroxymethylcytosine (5hmC) is present in T- even phage and mammalian DNA as well as some nucleoside antibiotics, including mildiomycin and bacimethrin, during whose synthesis 5hmC is pro- duced by the hydrolysis of 5-hydroxymethyl cytidine 5 � -monophosphate (hmCMP) by an N-glycosidase MilB. Recently, the MilB-CMP complex structure re- vealed its substrate specificity for CMP over dCMP. However, hmCMP instead of CMP is the preferred substrateforMilBassupportedbythatitsKM for CMP is ∼27-fold higher than that for hmCMP. Here, we de- terminedthecrystalstructuresofMilBanditscatalyt- ically inactive E103A mutant in complex with hmCMP. In the structure of the complex, Phe22 and Arg23 are positioned in a cage-like active site resembling the binding pocket for the flipped 5-methylcytosine (5mC) in eukaryotic 5mC-binding proteins. Van der Waals interaction between the benzene ring of Phe22 and the pyrimidine ring of hmCMP stabilizes its bind- ing. Remarkably, upon hmCMP binding, the guani- dinium group of Arg23 was bent ∼65 ◦ toward hm- CMP to recognize its 5-hydroxymethyl group, induc- ing semi-closure of the cage-like pocket. Mutagen- esis studies of Arg23 and bioinformatics analysis demonstrate that the positively charged Arg/Lys at this site is critical for the specific recognition of the 5-hydroxymethyl group of hmCMP.