4OHB

Crystal structure of MilB E103A in complex with 5-hydroxymethylcytidine 5'-monophosphate (hmCMP) from Streptomyces rimofaciens

Summary for 4OHB

• Entry DOI: 10.2210/pdb4ohb/pdb
• Related: 4OHR
• Descriptor: CMP/hydroxymethyl CMP hydrolase, 5-(hydroxymethyl)cytidine 5'-(dihydrogen phosphate) (3 entities in total)
• Functional Keywords: hydrolase, protein-hmcmp complex
• Biological source: Streptomyces rimofaciens
• Total number of polymer chains: 1
• Total formula weight: 20823.21

Authors

Zhao, G.,Zhang, Y.,Liu, G.,Wu, G.,He, X. (deposition date: 2014-01-17, release date: 2014-06-25, Last modification date: 2024-03-20)

Primary citation

Zhao, G.,Wu, G.,Zhang, Y.,Liu, G.,Han, T.,Deng, Z.,He, X.
Structure of the N-glycosidase MilB in complex with hydroxymethyl CMP reveals its Arg23 specifically recognizes the substrate and controls its entry
Nucleic Acids Res., 42:8115-8124, 2014

PubMed Abstract: 5-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 produced by the hydrolysis of 5-hydroxymethyl cytidine 5'-monophosphate (hmCMP) by an N-glycosidase MilB. Recently, the MilB-CMP complex structure revealed its substrate specificity for CMP over dCMP. However, hmCMP instead of CMP is the preferred substrate for MilB as supported by that its KM for CMP is ∼27-fold higher than that for hmCMP. Here, we determined the crystal structures of MilB and its catalytically 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 binding. Remarkably, upon hmCMP binding, the guanidinium group of Arg23 was bent ∼65° toward hmCMP to recognize its 5-hydroxymethyl group, inducing semi-closure of the cage-like pocket. Mutagenesis 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.

PubMed: 24920828
DOI: 10.1093/nar/gku486

Experimental method

X-RAY DIFFRACTION (2.4 Å)