title: Cloning and characterization of indolepyruvate decarboxylase from Methylobacterium extorquens AM1.
abstract: For the first time for methylotrophic bacteria an enzyme of phytohormone indole-3-acetic acid (IAA) biosynthesis, indole-3-pyruvate decarboxylase (EC 4.1.1.74), has been found. An open reading frame (ORF) was identified in the genome of facultative methylotroph Methylobacterium extorquens AM1 using BLAST. This ORF encodes thiamine diphosphate-dependent 2-keto acid decarboxylase and has similarity with indole-3-pyruvate decarboxylases, which are key enzymes of IAA biosynthesis. The ORF of the gene, named ipdC, was cloned into overexpression vector pET-22b(+). Recombinant enzyme IpdC was purified from Escherichia coli BL21(DE3) and characterized. The enzyme showed the highest k(cat) value for benzoylformate, albeit the indolepyruvate was decarboxylated with the highest catalytic efficiency (k(cat)/K(m)). The molecular mass of the holoenzyme determined using gel-permeation chromatography corresponds to a 245-kDa homotetramer. An ipdC-knockout mutant of M. extorquens grown in the presence of tryptophan had decreased IAA level (46% of wild type strain). Complementation of the mutation resulted in 6.3-fold increase of IAA concentration in the culture medium compared to that of the mutant strain. Thus involvement of IpdC in IAA biosynthesis in M. extorquens was shown.
authors.affiliation: Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia.
authors.forename: D N
authors.initials: DN
authors.lastname: Fedorov
authors.forename: N V
authors.initials: NV
authors.lastname: Doronina
authors.forename: Yu A
authors.initials: YA
authors.lastname: Trotsenko
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002262:Carboxy-Lyases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D017077:Culture Media, Conditioned; D057075:Enzyme Assays; D017353:Gene Deletion; D016680:Genome, Bacterial; D020601:Methylobacterium extorquens / Q000201:enzymology* / Q000235:genetics; D016366:Open Reading Frames; D010802:Phylogeny; D055550:Protein Stability; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D017077:Culture Media, Conditioned; D011994:Recombinant Proteins; C068832:indolepyruvate decarboxylase; D002262:Carboxy-Lyases