MppP from <i>Streptomyces wadayamensis</i> (SwMppP) is a pyridoxal-5’-phosphate (PLP)- and oxygen-dependent enzyme that oxidizes L-arginine to 4<i>S</i>-hydroxy-2-ketoarginine in the biosynthesis of the non-proteinogenic amino acid L-enduracididine (L-End). L-End is a key building block of several nonribosomally produced peptide antibiotics, such as the cyclic glycopeptide mannopeptimycin. Our previous work established a plausible mechanism for this unusual reaction, and that MppP is an oxidase rather than an oxygenase. We then began to look at other MppP homologs to see in what other biochemical contexts this activity appears. A PHI-BLAST search identified a group of approximately 200 MppP-like proteins, most of which are associated with different genomic contexts from SwMppP (i.e. they are not L-End biosynthetic enzymes). We have examined homologs from several of the major clades in this group and found that they seem to fall into one of two classes. About one third of the sequences are adjacent to an NAD(P)H-dependent reductase gene, while the remaining two thirds comprise clades with widely varying genomic contexts. A relatively small number of these sequences are true MppP homologs, occurring in the context of the other L-eEnd biosynthesis genes. We have characterized multiple MppP-like proteins from both classes and found that those co-occurring with the reductase have a subtly different activity, producing 4,5-dehydro-2-ketoarginine. The Ryan group at the University of British Columbia has made the identical observation. For convenience, we call enzymes in this latter group “desaturases”, and those that hydroxylate L-Arg “hydroxylases”. Structure and sequence comparisons suggest that the hydroxylases and desaturases all share essentially identical tertiary structures and active sites. Surprisingly, we have found that the hydroxylase mechanism does not include a 4,5-unsaturated intermediate. Thus, the dehydrated product of the desaturases does not arise from a simple loss of the ability to hydroxylate a common intermediate, but rather from mechanisms that branch from a common intermediate. Structural and kinetics studies of representatives from both classes have suggested several possible explanations for this mechanistic wrinkle, but the more we learn about the structures and substrate-binding modes in these two groups of enzymes, the more similar the hydroxylases and desaturases appear to be.

Searching for mechanistic differences in the hydroxylating and desaturating L-Arg oxidases

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ACS Fall 2022

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