To investigate the function of the gene penF in the pentostatin and vidarabine (Ara-A) biosynthetic gene cluster in Streptomyces antibioticus 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 Mg2+ and Mn2+ significantly enhanced enzyme activity, whereas Zn2+, Cu2+, and Ca2+ inhibited it. For vidarabine 5'-monophosphate, the Km and kcat values were determined to be 71.5 μM and 33.9 min-1, respectively. The kcat/Km value was 474.1 mM-1·min-1 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.
Keywords: 5′-nucleotidase; Streptomyces; arabinoside; phosphohydrolase.