Radical aminomutases are pyridoxal 5'-phosphate (PLP, a B6 vitamer)-dependent enzymes that require the generation of a 5'-deoxyadenosyl radical to initiate the catalytic cycle, to perform a 1,2 amino group shift reaction. The role of the nitrogen atom of PLP in radical aminomutases has not been investigated extensively yet. We report an alternative synthetic procedure to provide easy access to 1-deazaPLP (dAPLP), an isosteric analog of PLP which acts as a probe for studying the role of the nitrogen atom. Our results revealed that lysine 5,6-aminomutase (5,6-LAM), a radical aminomutase, reconstituted with dAPLP cannot turn over a substrate, demonstrating that the nitrogen atom is essential for radical aminomutases. In contrast, biochemical and spectroscopic studies on the S238A variant reconstituted with PLP revealed a minuscule loss of activity. This apparent anomaly can be explained by a water-mediated rescue of activity in S238A, as if mimicking the active site of lysine 2,3-aminomutase. This study leads to a better comprehension of how enzymes harness the optimum capability of PLP to realize catalysis.
Keywords: DFT; EPR; PLP; aminomutase; coenzyme B12; dAdoCbl; mutagenesis.