Cloning and Characterization of Indolepyruvate Decarboxylase from Methylobacterium extorquens AM1

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D. N. Fedorov, N. V. Doronina, and Yu. A. Trotsenko^*

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, pr. Nauki 5, 142290 Pushchino, Moscow Region, Russia; fax: (495) 956-3370; E-mail: trotsenko@ibpm.pushchino.ru

^* To whom correspondence should be addressed.

Received April 8, 2010; Revision received September 8, 2010

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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.

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KEY WORDS: Methylobacterium extorquens, indole-3-pyruvate decarboxylase, heterologous expression, kinetic properties, mutants

DOI: 10.1134/S0006297910120035

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