Structure & Function of Enzymes in Two Uncharacterized Gene Clusters from Pseudomonas Brassicacearum & Streptomyces Griseofuscus
Date of Award
Doctor of Philosophy
Nicholas NRS Silvaggi
Arsenio AAP Pacheco, Alan AWS Schwabacher, Sonia SLB Bardy, David DNF Frick
L-arginine oxidase, L-end, Mannopeptimycine, MppP, Pseudomonas brassicacaerum, Streptomyces griceofuscus
Pyridoxal 5’-phosphate (PLP)-dependent enzymes harness this versatile cofactor to catalyze a variety of reactions including transamination, decarboxylation, racemization and various elemination/subsitution reactions. Several years ago, a new class of PLP-dependent enzymes was discovered that uses PLP and molecular oxygen to catalyze the 4-electron oxidation of L-arginine to 4-hydroxy-2-ketoarginine. Work with the prototypical enzyme of this class, MppP from Streptomyces wadayamensis (SwMppP), showed that the dioxygen consumed during the reaction is reduced to hydrogen peroxide, and that the hydroxyl group installed in the product derives from water. Thus, SwMppP is an L-arginine oxidase, and not an oxygenase. This was surprising given that the hydroxylation step occurs at an un-activated methylene group of the substrate, L-arginine. SwMppP is part of the mannopeptimycin biosynthetic cluster and works together with MppR and MppQ to synthesize the non-proteinogenic amino acid L-enduracididine (L-End). A BLASTp search of the NR database using the sequence of S. wadayamensis MppP revealed close homologs in a number of species of pseudomonads, including Pseudomonas brassicacearum, P. syringae, P. amygdali, and P. aeruginosa.
The first section of this thesis is focused on the genomic context of the pseudomonad MppP homologs. None of the pseudomonad genomes examined contained other key mannopeptimycin biosynthetic genes like the nonribosomal peptide synthetases mppA and mppB, or the other two L-End biosynthetic genes mppQ and mppR. The lack of MppQ and MppR homologs in these organisms suggests that the arginine oxidase activity of MppP homologs is used in a distinct biochemical context. We cloned, expressed, and purified the MppP homolog from Pseudomonas brassicacearum, a soil-dwelling bacterium associated with the roots of plants in the mustard/cabbage family that protects the plants from several bacterial and fungal pathogens. The genomic context of the gene encoding this MppP homolog (PbrMppP) consists of a regulatory gene, the MppP homolog, and four additional open reading frames annotated as hypothetical protein (PbrHYP), dihydrodipicolinate synthase family protein (PbrDHPS), Mononuclear Fe-dependent oxygenase; (PbrOX), and 2Fe2S-binding protein ferredoxin (PbrFD). All these genes were annotated from sequence analysis. The observations that (1) the genes are overlapping, (2) that the open reading frame includes a luxR-type regulatory gene, and (3) 5’-untranslated region contains a putative promoter sequence suggests that these genes may constitute an operon, which we will refer to as the P. brassicacaerum MppP containing operon (PbrMPCO). Understanding the biochemical context of PbrMppP requires assigning functions for the other 4 non-regulatory gene products. To begin, we confirmed that the structure and activity of PbrMppP are both identical to the prototypical SwMppP. Next, we analyzed the X-ray crystal structures of PbrHYP, PbrDHPS and PbrOx. We also identified condition that activates the promoter of this gene cluster. Since the structures were not sufficient to deduce the functions of these enzymes, we turned to a metabolomic strategy to determine the final product of theiv operon. Knowing the final product would limit the possibilities for substrate of these putative enzymes, we show here a comparative metabolomic analysis by knocking out the entire operon and searching for “missing” metabolite(s) by multiple MS. The second section focuses on a second MppP homolog from Streptomyces griseofuscus. The gene encoding this MppP homolog, SgrMppP, is flanked by 5 other genes whose products are annotated as a putative biotin carboxylase (SgrLIG), flavin-dependent monooxygenase (SgrOX), S-adenosyl methionine (SAM)-dependent methyltransferase (SgrMT), amidinohydrolase (SgrAH), and GNAT-family acetyltransferase (SgrNAT). The goal of these studies is to determine the activities of all the enzymes in this gene cluster and to identify the final product. So far I have shown, in the presence of Mg(II) and SAM, SgrMT catalyzes the transfer of a methyl group from SAM to carbon 4 of the 4-hydroxy-2-ketoarginine produced by SgrMppP. We also determined that SgrMT products are used by SgrAH as substrate and the turned over products by SgrAH are ornithine derivatives and urea. Succeeding research will involve structural and functional determination of rest of the gene products, which will eventually lead to the identification of final product of these gene-contexts.
Badhon, Lamia Tabassum, "Structure & Function of Enzymes in Two Uncharacterized Gene Clusters from Pseudomonas Brassicacearum & Streptomyces Griseofuscus" (2021). Theses and Dissertations. 2643.
Available for download on Thursday, May 25, 2023
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