Functional Analysis of Keto-Acid Reductoisomerase ILVC in the Entomopathogenic Fungus Metarhizium robertsii

Abstract

Ketol-acid reductoisomerase (ILVC) is the second enzyme in the branched-chain amino acid (BCAA) biosynthesis, which regulates many physiological activities in a variety of organisms from bacteria to fungi and plants. In this work, function mechanisms of ILVC in Metarhizium robertsii Metchnikoff (Hypocreales: Clavicipitaceae) were explored with site-directed mutagenesis, reductase activity assays and transcriptomics analysis. The reductase activity assays showed that ILVC from phytopathogenic fungi exhibited significantly higher activities than those from entomopathogenic fungi but lower than those from yeast. Site-directed mutagenesis and enzymatic activities of MrILVC with different active-site mutants (Arg-113, Ser-118, Asp-152, Asp-260, and Glu-264) confirmed that active sites of MrILVC are conserved with plant and bacterial ILVCs. Deleting MrilvC causes the complete failures of vegetative growth and conidial germination, feeding with branched-chain amino acids (BCAAs) recovers the fungal growth but not conidial germination, while both characteristics are restored when supplemented with yeast extract. Compared to ΔMrilvC cultured in czapek agar (CZA), plenty of genes involved in the biosynthesis of antibiotics and amino acids were up- or down-regulated in the wild type or ΔMrilvC feeding with either BCAAs or yeast extract. Further analysis showed some genes, such as catalase A, participate in mycelial growth and conidial germination was down-regulated in ΔMrilvC from CZA, revealing that MrILVC might control the fungal development by gene regulation and BCAAs or yeast extract could play partial roles of MrILVC. This study will advance our understanding of ILVC function mechanisms in fungi.

Regulation of the Leucine Metabolism in Mortierella alpina

“…An inter-kingdom phylogenetic analysis revealed that BatA is part of a cluster among other fungal BATs (Figure S1, Table S8). In contrast, BatC grouped together with plant counterparts, and its gene is most likely plant-derived by HGT, as suggested for the other fungal leucine biosynthetic genes [4,31].…”

Neisseria gonorrhoeae Ketol-Acid Reductoisomerase Is a Potential Therapeutic Target

“…Upon the binding of cofactors, NADP(H) and Mg 2+ , the N-terminal domain of KARI undergoes large local conformational changes, only in the NADP(H) binding site. Four Mg 2+ -binding residues are also identified (D190, E194, E226, and E230) [19]. The side chains of these residues rotate upon metal ion binding.…”

“…Ketol-acid reductor-isomerase (KARI) is the second enzyme in the branched-chain amino acid (BCAA) biosynthesis, which regulates many physiological activities in a variety of organisms from bacteria to fungi and plants. The conservation in fungi but absence in mammals of the BCAA biosynthetic pathway makes it the target for herbicides, fungicides, and antimicrobial compounds [19]. KARI catalyzes the conversion of 2-acetolactate and 2-aceto-2-hydroxybutyrate to 2,3-dihydroxyisoverate and 2,3-dihydroxy-3-methyl valerate, respectively.…”

“…KARI catalyzes the conversion of 2-acetolactate and 2-aceto-2-hydroxybutyrate to 2,3-dihydroxyisoverate and 2,3-dihydroxy-3-methyl valerate, respectively. ILVC is a bifunctional enzyme that catalyzes two quite different reactions, but occurs at a common active site, acting both as an isomerase and as a reductase [19].…”