Substrate promiscuity of the Escherichia coli xanthine oxidase

PpnN is a cytosolic nucleosidase that cleaves nucleotide monophosphates to nucleobases and ribose-5-phosphate and plays a crucial role in regulating bacterial competitive fitness and persistence. To quantify the PpnN reaction, here we developed an enzyme-coupled assay, wherein PpnN hydrolyzes IMP to hypoxanthine, which is then converted by xanthine oxidase (XO) into xanthine, uric acid and hydrogen peroxide. The detection of hydrogen peroxide via horseradish peroxidase and Amplex Red provides a measure of PpnN enzymatic activity. Surprisingly, we found that in addition to IMP, other nucleotides like GMP significantly increased the signal, suggesting that the corresponding nucleobases might also be substrates for XO. Direct tests by using guanine confirmed XOs capacity to use it as a substrate, albeit less effectively than hypoxanthine. These findings suggest a potential guanine aminohydrolase activity of E. coli XO and broaden our understanding of nucleobase metabolism in bacterial systems.