Abstract
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 Mg(2+) and Mn(2+) significantly enhanced enzyme activity, whereas Zn(2+), Cu(2+), and Ca(2+) inhibited it. For vidarabine 5'-monophosphate, the K(m) and k(cat) values were determined to be 71.5 μM and 33.9 min(-1), respectively. The k(cat)/K(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.