Biochemical Characterization of an Arabinoside Monophosphate Specific 5'-Nucleotidase-like Enzyme from .

To investigate the function of the gene in the pentostatin and vidarabine (Ara-A) biosynthetic gene cluster in NRRL 3238, PenF was recombinantly expressed and characterized. Enzymatic characterization of the enzyme demonstrated that PenF exhibited metal-dependent nucleoside 5'-monophosphatase activity, showing a substrate preference for arabinose nucleoside 5'-monophosphate over 2'-deoxyribonucleoside 5'-monophosphate and ribonucleoside 5'-monophosphate. Metal ions such as Mg and Mn significantly enhanced enzyme activity, whereas Zn, Cu, and Ca inhibited it. For vidarabine 5'-monophosphate, the and values were determined to be 71.5 μM and 33.9 min, respectively. The value was 474.1 mM·min for vidarabine 5-monophosphate and was 68-fold higher than that for 2'-deoxyadenosine 5'-monophosphate. Comparative sequence alignment and structural studies suggested that residues outside the primary substrate-binding site are responsible for this substrate specificity. In conclusion, PenF's activity toward vidarabine 5'-monophosphate likely plays a role in the dephosphorylation of precursors during Ara-A biosynthesis.