Streptomyces wadayamensis MppP is a PLP-Dependent Oxidase, Not an Oxygenase

Abstract

The PLP-dependent l-arginine hydroxylase/deaminase MppP from Streptomyces wadayamensis (SwMppP) is involved in the biosynthesis of l-enduracididine, a nonproteinogenic amino acid found in several nonribosomally produced peptide antibiotics. SwMppP uses only PLP and molecular oxygen to catalyze a 4-electron oxidation of l-arginine to form a mixture of 2-oxo-4(S)-hydroxy-5-guanidinovaleric acid and 2-oxo-5-guanidinovaleric acid. Steady-state kinetics analysis in the presence and absence of catalase shows that one molecule of peroxide is formed for every molecule of dioxygen consumed in the reaction. Moreover, for each molecule of 2-oxo-4(S)-hydroxy-5-guanidinovaleric acid produced, two molecules of dioxygen are consumed, suggesting that both the 4-hydroxy and 2-keto groups are derived from water. This was confirmed by running the reactions using either O or HO and analyzing the products by ESI-MS. Incorporation of O was only observed when the reaction was performed in HO. Crystal structures of SwMppP with l-arginine, 2-oxo-4(S)-hydroxy-5-guanidinovaleric acid, or 2-oxo-5-guanidinovaleric acid bound were determined at resolutions of 2.2, 1.9. and 1.8 Å, respectively. The structural data show that the N-terminal portion of the protein is disordered unless substrate or product is bound in the active site, in which case it forms a well-ordered helix that covers the catalytic center. This observation suggested that the N-terminal helix may have a role in substrate binding and/or catalysis. Our structural and kinetic characterizations of N-terminal variants show that the N-terminus is critical for catalysis. In light of this new information, we have refined our previously proposed mechanism of the SwMppP-catalyzed oxidation of l-arginine.

Emergence of oxygen‐ and pyridoxal phosphate‐dependent reactions

“…The amino acid l ‐arginine has emerged as a common substrate for sequence‐related oxygen‐dependent PLP enzymes from Streptomyces bacteria. These enzymes include Ind4 from the indolmycin biosynthetic pathway [71,72], MppP from enduracididine biosynthesis [73,74], and RohP from the azomycin biosynthetic pathway [75,76]. Indolmycin, enduracididine, and azomycin each contain arginine‐derived heterocycles, for which Ind4, MppP, and RohP are essential upstream enzymes toward the biosynthesis of each heterocycle.…”

“…The branching point between desaturation and hydroxylation is shown with green and purple reaction arrows, respectively. Adapted from Du et al 2016 [72], Hedges et al 2018 [75], and Han et al 2018 [74].…”

“…Genes for making this noncanonical amino acid were proposed during the discovery of the enduracidin gene cluster [86] through comparison with the mannopeptimycin gene cluster [87]; however, the function of these genes was established by Silvaggi through in vitro and crystallographic characterization of enzymes in the pathway [73‐74,88] (Fig. 11).…”

“…11). First, a PLP‐dependent oxidase, MppP/EndP, converts l ‐arginine into 4‐hydroxy‐2‐ketoarginine [73,74], which is dehydrated and cyclized by MppR/EndR [88], and proposed to be aminated by MppQ/EndQ, another PLP‐dependent enzyme. Enduracididine is sometimes further hydroxylated at the β‐position by MppO, as is seen in mannopeptimycin [87,89], before being incorporated into a natural product by a nonribosomal peptide synthetase.…”

“…Enduracididine is sometimes further hydroxylated at the β‐position by MppO, as is seen in mannopeptimycin [87,89], before being incorporated into a natural product by a nonribosomal peptide synthetase. Further studies of MppP [73,74] found that it was an oxygen‐ and PLP‐dependent arginine oxidase that catalyzes a two‐ and four‐electron oxidation to produce 2‐ketoarginine and ( S )‐4‐hydroxy‐2‐ketoarginine, respectively, with the hydroxyl group on the ( S )‐4‐hydroxy‐2‐ketoarginine product coming from water (Fig. 10).…”