Streptomyces wadayamensis MppP: A Novel PLP‐Dependent L‐Arginine Hydroxylase in L‐Enduracididine Biosynthesis

The nonproteinogenic amino acid L‐enduracididine (L‐End) is a critical component of the mannopeptimycins, a family of cyclic glycopeptide antibiotics with potent activities against drug‐resistant pathogens like MRSA. Creating derivatives of mannopeptimycin and other L‐End‐containing natural products is hampered by the limited availability of L‐End. We are investigating the L‐End biosynthetic pathway in order to develop an efficient enzymatic or chemo‐enzymatic route to produce this unusual amino acid. Recently, we found that MppP is a PLP‐dependent L‐Arg hydroxylase in L‐End biosynthesis in Streptomyces wadayamensis. It reacts with L‐Arg and dioxygen to yield two products, 2‐ketoarginine and 4‐hydroxy‐2‐ketoarginine in a ratio of ~1.7:1. Surprisingly, 1 equivalent of H 2 O 2 is produced for each equivalent of O 2 consumed. In addition, the ratio of O 2 consumption to L‐Arg consumption was ~1.4:1, suggesting that one product requires 2 equivalents of O 2 , and the other only 1 equivalent. We have determined the structures of MppP in four states: the internal aldimine, the external aldimine with the substrate, L‐Arg, the product complex with 4‐hydroxy‐2‐ketoarginine, and the product complex with 2‐ketoarginine. According to the structures, the N‐terminal helix is disordered in the internal aldimine and covers the active site only when the substrate is bound. A glutamate residue in the N‐terminal helix, E15, makes a hydrogen bonding interaction with the carboxylate of the substrate, L‐Arg. This observation prompted us to make three N‐terminal variants of SwMppP: E15A, E15Q, and the truncation mutant, SwMppP 23‐276 . Steady state kinetics showed that the two point mutants had no effect on k cat or K M,L‐Arg . The truncation mutant, however, showed an approximately 10‐fold increase in K M and a 3‐fold decrease in k cat , which reduced the pseudo‐second order rate constant by almost 25‐fold. Interestingly, although the steady state kinetics (as measured by dioxygen consumption) are indistinguishable for the E15A and E15Q variants, the E15A variant does not produce 4‐hydroxy‐2‐ketoarginine, only the abortive product 2‐ketoarginine. Likewise, the truncation mutant also produced only 2‐ketoarginine. Our structural and kinetic characterization of the wild‐type and variant forms of SwMppP have allowed us to propose a revised model where the oxygen incorporated in the hydroxy‐arginine product is derived from water rather than from dioxygen. Support or Funding Information National Science Foundation CHE‐1606842