Cloning, purification, and characterization of diaminobutyrate acetyltransferase from the halotolerant methanotroph Methylomicrobium alcaliphilum 20Z.
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 degrees C, and its activity increased threefold in the presence of 0.1-0.2 M NaCl or 0.2 M KCl. The Km values of recombinant DABAcT measured at the optimal pH and temperature in the presence of 0.2 M KCl were 460 and 36.6 microM 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.
Reshetnikov
A S
AS
Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia.
Mustakhimov
I I
II
Khmelenina
V N
VN
Trotsenko
Yu A
YA
D000123:Acetyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000613:Aminobutyrates / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002104:Cadmium / Q000494:pharmacology; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008772:Methylococcaceae / Q000201:enzymology*; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity; D013696:Temperature; D015032:Zinc / Q000494:pharmacology
D000613:Aminobutyrates; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002104:Cadmium; C005959:2,4-diaminobutyric acid; C106232:2,4-diaminobutyric acid acetyltransferase; D000123:Acetyltransferases; D015032:Zinc