Expression in Escherichia coli and Refolding of the Malonyl-/Acetyltransferase Domain of the Multifunctional Animal Fatty Acid Synthase

Abstract

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 3 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.The animal fatty acid synthase is a multifunctional protein consisting of two identical head-to-tail oriented subunits, each carrying seven functional domains (1). It is generally believed that multifunctional proteins such as the fatty acid synthase have evolved by the fusion of genes encoding their monofunctional counterparts (2). Thus, each of the catalytic components of the fatty acid synthase is envisioned to be an independently folded domain. However, as yet, only one of the components of the animal fatty acid synthase, the thioesterase, has been expressed as an independent, catalytically active recombinant protein (3). In this study we sought to develop a system for expression of the malonyl-/acetyltransferase domain of the animal fatty acid synthase that would allow us to define the boundaries of this domain and ultimately would facilitate a detailed analysis of the catalytic mechanism of the enzyme through the construction and characterization of specific mutants. EXPERIMENTAL PROCEDURESMaterials-The pET17b and pET23a expression vectors were obtained from Novagen, Madison, WI. Rabbit anti-(rat)-fatty acid synthase antibodies were prepared as described previously (4) and purified by affinity chromatography on Sepharose-antigen columns, essentially as recommended by Pharmacia Biotech Inc. Alkaline phosphatasecoupled goat anti-rabbit IgG antibodies and SDS-PAGE 1 standards were purchased from Bio-Rad. Acetyl-[1-14 C]CoA (54 Ci/mol) and malonyl-[2-14 C]CoA (57 Ci/mol) were obtained from Moravek Biochemicals (Brea, CA). Baker-flex cellulose thin layer chromatography sheets were obtained from J. T. Baker Inc., and DEPC was purchased from SERVA feinbiochemica (New York). Synthe...

Cloning, Expression, Characterization, and Interaction of Two Components of a Human Mitochondrial Fatty Acid Synthase

“…5A). Thus, the mitochondrial MT closely resembles typical type II MTs, as exemplified by that of E. coli (32), that exhibit marked specificity for malonyl moieties and is distinctly different from the transferase domains of cytosolic type I FASs that are equally active in the transfer of malonyl and acetyl moieties (29,33,34).…”

“…5). In contrast, the transferase domain of the cytosolic type I animal FAS has a dual donorsubstrate specificity, transferring both acetyl and malonyl moieties with equal efficiency (29), and in vitro is capable of utilizing either type I or type II ACPs as acceptor substrate (Fig. 5).…”

“…1A). In addition, all of these sequences contained three positionally conserved features that are characteristic of malonyl and malonyl/acetyltransferases: a Gly-Xaa-Ser-XaaGly serine esterase active site motif, a conserved Arg residue that interacts with the 3-carboxylate of the malonyl substrate, and a conserved His residue that is required for activation of the Ser nucleophile (27)(28)(29)(30). On the basis of this analysis, we identified expressed sequence tag cDNA clones encoding the putative human mitochondrial MT protein for further study.…”

Alteration of the Substrate Specificity of the Malonyl-CoA/Acetyl-CoA:Acyl Carrier ProteinS-Acyltransferase Domain of the Multifunctional Fatty Acid Synthase by Mutation of a Single Arginine Residue

“…Our rationale is first to identify from multiple sequence alignments conserved residues that might be candidates for specific roles in catalysis and then to examine the effect of mutating these residues. Using this approach, we have previously identified His-683 2 of the rat FAS as playing an essential role in activation of the catalytic residue Ser-581 (10). Only one other basic residue, Arg-606 in the rat FAS, is universally conserved in the transacylases associated with multifunctional and monofunctional forms of FAS and polyketide synthase (10).…”

“…Using this approach, we have previously identified His-683 2 of the rat FAS as playing an essential role in activation of the catalytic residue Ser-581 (10). Only one other basic residue, Arg-606 in the rat FAS, is universally conserved in the transacylases associated with multifunctional and monofunctional forms of FAS and polyketide synthase (10). Since all of these transacylases can utilize either malonyl-CoA or methylmalonyl-CoA as a substrate, we hypothesized that this residue might facilitate substrate binding by interaction with the free carboxyl group of the malonyl or methylmalonyl moiety and that its replacement might compromise binding of these substrates.…”