Reversal of the Substrate Specificity of CMP N-Glycosidase to dCMP

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 in both sequence and function to MilB. These two enzymes belong to the nucleoside 2′-deoxyribosyltransferase (NDT) superfamily. NDTs catalyze N-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 sequence comparisons alone. MilB and BcmB that prefer substrates containing 2′-ribosyl groups have a phenylalanine positioned in the active site whereas NDT family members with preference for 2′-deoxyribosyl groups have a tyrosine residue. Further studies show that the phenylalanine is critical for MilB and BcmB specificity towards CMP, and mutation of this phenylalanine residue to tyrosine results in a 1000-fold reversal of substrate specificity from CMP to dCMP.

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

“…As the k cat of MilB for hmCMP in reference (8) is 3.1×10 −3 s −1 , even lower than that for MilB for CMP reported by Megan D. Sikowitz et al. (9), the kinetic data obtained by this study, therefore, look more consistent and convincing (Table 1). These variations might arise by the way for calculating the peak area of the products, type of column and separation conditions used for HPLC as well as protein activity and purity in different preparations.…”

“…Recently, the Steven E. Ealick group reported the crystal structure of MilB in complex with CMP and revealed that the F17Y mutation of MilB completely reversed its substrate specificity from CMP to dCMP (9). However, CMP is not the natural and the most preferred substrate for MilB.…”

The SARM1 Toll/Interleukin-1 Receptor Domain Possesses Intrinsic NAD + Cleavage Activity that Promotes Pathological Axonal Degeneration

“…The SARM1-TIR domain was modeled using the crystal structure of two enzymes identified from our prediction: MilB Cytidine 5’ monophosphate (CMP) Hydrolase (PDB: 4JEM) (Figure 4B) and Nucleoside 2-deoxyribosyltransferase (PDB: 1F8Y) (Figure S4B). Importantly, we found that a glutamic acid E642 in the SARM1-TIR domain aligned with both the key catalytic glutamic acid residue in CMP hydrolase (Sikowitz et al, 2013) and the proposed nucleophilic glutamic acid in the active site of nucleoside 2-deoxyribosyltransferase (Armstrong et al, 1995) (Figure 4A, 4B and S4C). Moreover, glutamic acid residues are also known catalytic residues in other NADases (Ghosh et al, 2010).…”

“…However, in addition to these TIR domains, we also detected a number of nucleotide hydrolase/transferase enzymes (Table S2). For some of these enzymes, residues that contribute to catalytic activity have been established (Sikowitz et al, 2013; Armstrong et al, 1995). We therefore used structural modeling and sequence alignments to identify putative residues in the SARM1-TIR domain that might contribute to enzymatic activity (Figure 4A, 4B, S4A, S4B).…”

Bacterial retrons function in anti-phage defense

“…Point mutations predicted to inactivate the catalytic site of the RT (D216A/D217A in the RT of Ec48, and D189A/D190A in the RT of Ec73) completely abolished defense, indicating that reverse transcription of the retron is essential for defense. In addition, an E117Q point mutation predicted to inactivate the catalytic site of the ribosyltransferase domain of the Ec73 effector 26 , led to a non-functional system, and similarly, deletion of the transmembrane helices of the gene associated with the Ec48 retron also abolished defense (Figure 3, Supplementary Figure 3). Together, these results show that retrons, functioning together with their associated effector genes, form anti-phage defense systems.…”