Solution structure of the nucleotide hydrolase BlsM: Implication of its substrate specificity

Abstract

Biosynthesis of the peptidyl nucleoside antifungal agent blasticidin S in Streptomyces griseochromogenes requires the hydrolytic function of a nucleotide hydrolase, BlsM, to excise the free cytosine from the 5′‐monophosphate cytosine nucleotide. In addition to its hydrolytic activity, interestingly, BlsM has also been shown to possess a novel cytidine deaminase activity, converting cytidine, and deoxycytidine to uridine and deoxyuridine. To gain insight into the substrate specificity of BlsM and the mechanism by which it performs these dual function, the solution structure of BlsM was determined by multi‐dimensional nuclear magnetic resonance approaches. BlsM displays a nucleoside deoxyribosyltransferase‐like dimeric topology, with each monomer consisting of a five‐stranded β‐sheet that is sandwiched by five α‐helixes. Compared with the purine nucleotide hydrolase RCL, each monomer of BlsM has a smaller active site pocket, enclosed by a group of conserved hydrophobic residues from both monomers. The smaller size of active site is consistent with its substrate specificity for a pyrimidine, whereas a much more open active site, as in RCL might be required to accommodate a larger purine ring. In addition, BlsM confers its substrate specificity for a ribosyl‐nucleotide through a key residue, Phe19. When mutated to a tyrosine, F19Y reverses its substrate preference. While significantly impaired in its hydrolytic capability, F19Y exhibited a pronounced deaminase activity on CMP, presumably due to an altered substrate orientation as a result of a steric clash between the 2′‐hydroxyl of CMP and the ζ‐OH group of F19Y. Finally, Glu105 appears to be critical for the dual function of BlsM.

Structural basis of Qng1-mediated salvage of the micronutrient queuine from queuosine-5′-monophosphate as the biological substrate

“…Qng1 adopts an HhH-glycosylase fold, prefers Q-5′MP as substrate, is metal independent and functions as a monomer; while QueK is a homotrimeric protein with a preference for Q as substrate and belongs to the Ca 2+ -dependent nucleoside hydrolase (NH) superfamily which use a Rossmann-fold catalytic core and a bound Ca 2+ ion to coordinate the substrate ribose and nucleophilic water molecule (single-displacement mechanism) ( 70 ). Further, Qng1 is structurally distinct from all known nucleotide hydrolases, which are all Rossmann fold-containing multimeric proteins, including the ppnN family of pyrimidine/purine-5′-nucleotide nucleosidases ( 71 ), the LOG family of cytokinin riboside-5′-monophosphate phosphoribohydrolases ( 72 ), 2′-deoxyguanosine-5′-monophosphate hydrolase DNPH1 ( 73 ), and cytosine-5′-monophosphate hydrolase Blsm ( 74 ) and hydroxyl-methyl cytosine-5′-monophosphate hydrolase MilB ( 75 ) involved in the biosynthesis of the peptidyl nucleoside antifungal agents blasticidin and mildiomycin, respectively.…”

Identification and Characterization of Enzymes Catalyzing Early Steps in Miharamycin and Amipurimycin Biosynthesis

“…Therefore, the low content of free bases usually cannot meet the precursor requirements for natural product biosynthesis; thus dedicated enzymes have evolved to cleave nucleosides or nucleotides. In the CGA pathway, CMP hydrolases such as BlsM and ArgD are responsible for hydrolyzing CMP to free cytosine, 15,18,19 but their homologues are not encoded in mih or apm. Among the untapped open reading frames (ORFs), BLAST analysis showed that mihD/ apmD encodes the TIGR10730 family Rossmann fold protein with unknown function.…”