Expression in Escherichia coli and refolding of the malonyl-/acetyltransferase domain of the multifunctional animal fatty acid synthase.
A cDNA encoding residues 429-815 of the multifunctional rat fatty acid synthase has been expressed in Escherichia coli and the recombinant protein refolded in vitro as a catalytically active malonyl-/acetyltransferase. Kinetic properties of the refolded recombinant enzyme were indistinguishable from those of a transferase preparation derived from the natural fatty acid synthase by limited proteolysis, indicating that the transferase domain is capable of folding correctly as an independent protein. Replacement of the active site Ser-581 (full-length fatty acid synthase numbering) with alanine completely eliminated catalytic activity, whereas replacement with cysteine resulted in retention of about 1% activity. The wild type transferase was extremely susceptible to inhibition by diethyl pyrocarbonate, and protection against inhibition was afforded by both malonyl- and acetyl-CoA. Replacement of the highly conserved residue His-683 with Ala reduced activity by 99.95%, and the residual activity was relatively unaffected by diethyl pyrocarbonate. The rate of acylation of the active site serine residue was also reduced by several orders of magnitude in the His-683 --> Ala mutant. These results indicate that His-683 plays an essential role in catalysis, likely by accepting a proton from the active site serine, thus increasing its nucleophilicity.
Rangan
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Children's Hospital Oakland Research Institute, Oakland, California 94609, USA. ssmith@lanminds.com
Smith
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D000105:Acetyl Coenzyme A / Q000494:pharmacology; D000101:Acetyl-CoA C-Acetyltransferase / Q000737:chemistry*; D051080:Acyl-Carrier Protein S-Malonyltransferase; D000217:Acyltransferases / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D054889:Fatty Acid Synthase, Type II; D064429:Fatty Acid Synthases / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D051381:Rats; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D029968:Escherichia coli Proteins; D008316:Malonyl Coenzyme A; D000105:Acetyl Coenzyme A; D000217:Acyltransferases; D051080:Acyl-Carrier Protein S-Malonyltransferase; C494968:fabD protein, E coli; D064429:Fatty Acid Synthases; D000101:Acetyl-CoA C-Acetyltransferase; D054889:Fatty Acid Synthase, Type II; D004047:Diethyl Pyrocarbonate