Biochemical Characterization of an Arabinoside Monophosphate Specific 5′-Nucleotidase-like Enzyme from Streptomyces antibioticus

To investigate the function of the gene <i>penF</i> in the pentostatin and vidarabine (Ara-A) biosynthetic gene cluster in <i>Streptomyces antibioticus</i> 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 <i>K</i>_m and <i>k</i>_cat values were determined to be 71.5 μM and 33.9 min^-1, respectively. The <i>k</i>_cat<i>/K</i>_m 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.