Studies of the in vitro Nalpha-acetyltransferase activities of E. coli RimL protein.
Although the Escherichia coli N(alpha)-acetyltransferase RimL catalyzing the N-terminal acetylation of L12 have been identified through mutant analysis, little is known about its enzymatic activity and auxiliary subunit requirement. This study was to investigate the enzymatic activities of RimL and its substrate specificity. RimL, its substrate L12, and two mutant substrates L12S1A and L12I2D were overexpressed and purified from E. coli. In vitro experimental results revealed that RimL itself can convert L12 to L7 by acetylation of the N-terminal serine residue. The K(m) value for L12 was 0.55 microM and the V(max) was 25.71 min(-1) as determined by a spectrophotometrical method. We also found that RimL acetylated the L12S1A mutant with an N-terminal alanine residue instead of the native serine residue, suggesting RimL can acetylate other N-terminal residues. Furthermore, when the second N-terminal residue isoleucine was replaced by aspartic acid, the mutant L12I2D was also acetylated by RimL but under a much lower rate.
Miao
Lin
L
Institute of Biotechnology, Academy of Military Medical Sciences, 20 Dong Da Street, Feng Tai District, Beijing 100071, PR China.
Fang
Hongqing
H
Li
Yanying
Y
Chen
Huipeng
H
D000107:Acetylation; D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D000123:Acetyltransferases; C521051:RimL protein, E coli