Cloning, Purification, and Characterization of Diaminobutyrate Acetyltransferase from the Halotolerant Methanotroph Methylomicrobium alcaliphilum 20Z

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A. S. Reshetnikov^1,2, I. I. Mustakhimov^1,2, V. N. Khmelenina¹, and Yu. A. Trotsenko^1*

¹Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia; fax: (7-095) 956-3370; E-mail: trotsenko@ibpm.pushchino.ru

²Pushchino State University, 142290 Pushchino, Moscow Region, Russia

^* To whom correspondence should be addressed.

Received July 27, 2004; Revision received August 17, 2004

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L-2,4-Diaminobutyrate (DAB) acetyltransferase (DABAcT) catalyzes one of the key reactions of biosynthesis of the bacterial osmoprotectant ectoine--acetylation of L-2,4-DAB yielding Ngamma-acetyl-2,4-DAB. Gene ectA encoding DABAcT was cloned from DNA of the halotolerant methanotroph Methylomicrobium alcaliphilum 20Z and expressed in Escherichia coli with an additional six His residues at the C-terminus. Homogeneous enzyme preparation with specific activity 200 U/mg was obtained by affinity metal-chelating chromatography. DABAcT was found to be a homodimer with molecular mass 40 kD. The enzyme is most active at pH 9.5 and 20°C, and its activity increased threefold in the presence of 0.1-0.2 M NaCl or 0.2 M KCl. The K_m values of recombinant DABAcT measured at the optimal pH and temperature in the presence of 0.2 M KCl were 460 and 36.6 µM for L-2,4-DAB and acetyl-CoA, respectively. The enzyme is specific for L-2,4-DAB and acetyl-CoA and is also active against propionyl-CoA (20%). Zn²⁺ and Cd²⁺ at 1 mM concentration completely inhibit the recombinant enzyme; 10 mM ATP inhibits 26% of the enzyme activity, whereas EDTA, o-phenanthroline, ADP, NAD(P), and NAD(P)H do not significantly effect the enzyme activity. The possible participation of DABAcT in regulation of ectoine biosynthesis in M. alcaliphilum 20Z is discussed.

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KEY WORDS: ectoine, osmoprotectants, halotolerant alkaliphilic methanotrophs, Methylomicrobium alcaliphilum, L-2,4-diaminobutyrate acetyltransferase

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