An essential histidine residue required for fatty acylation and acyl transfer by myristoyltransferase from luminescent bacteria.
The lux-specific acyltransferases are serine esterases responsible for preferential diversion of myristic acid from fatty acid biosynthesis to the luminescent system. In contrast to other acyltransferases, an acylated enzyme intermediate can readily be detected making it ideal for the study of the mechanism of acyl transfer. Although the transferase readily cleaves acyl carrier protein and acyl-CoA, an alternate more rapid and convenient assay involving the cleavage of p-nitrophenyl acyl esters was developed and applied in these studies. The cleavage of the oxyesters by the transferase was shown to have a similar dependence on fatty acid chain length and organic solvents as the cleavage of thioesters. Using this assay, it could be demonstrated that the Photobacterium phosphoreum transferase was inactivated at pH 6 with diethyl pyrocarbonate at a rate (73 M-1 s-1, 10 degrees C) even faster than that reported for other enzymes with reactive histidyl residues at their active site. Spectral changes during chemical modification as well as restoration of activity by neutral hydroxylamine showed that the loss of activity was associated with modification of a single histidine residue. Replacement of the four histidine residues, conserved in all lux-specific acyltransferases, by asparagine demonstrated that cleavage of both thioesters and oxyesters by the P. phosphoreum acyltransferase as well as acylation of the enzyme was blocked on mutation of His-244 but not the other three conserved histidines (His-12, -52, and -75). These results suggest that the histidine residue near the carboxyl terminus (His-244) may be part of a catalytic triad essential for cleavage of acyl esters and transfer of the acyl group to the enzyme.
Ferri
S R
SR
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Meighen
E A
EA
D000215:Acylation; D000217:Acyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D017124:Conserved Sequence; D015870:Gene Expression; D005798:Genes, Bacterial; D006639:Histidine; D007700:Kinetics; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010776:Photobacterium / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012997:Solvents; D013379:Substrate Specificity
D011994:Recombinant Proteins; D012997:Solvents; D006639:Histidine; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase