Molecular cloning and sequencing of cDNAs encoding the entire rat fatty acid synthase.

Abstract

Overlapping cloned cDNAs representing the entire sequence of the rat fatty acid synthase mRNA have been isolated from a cDNA library and sequenced. Authenticity of the cDNA clones was supported by hybridization to fatty acid synthase mRNA and by amino-terminal sequencing of 39 fatty acid synthase CNBr fragments. The full-length fatty acid synthase mRNA is 9156 nucleotides long and includes an 84-nucleotide 5' noncoding region, a 7515-nucleotide coding sequence, and a 1537-nucleotide 3' noncoding region; a second mRNA species containing a shortened 3' noncoding sequence is also transcribed in the rat. The encoded fatty acid synthase subunit contains 2505 amino acids and has a molecular weight of 272,340. Active sites and substrate binding sites were located within the sequence, thus establishing the order of domains on the multifunctional animal fatty acid synthase as condensing enzyme-transferase-dehydrase-enoyl reductase-ketoreductase-acyl carrier protein-thioesterase.The synthesis of fatty acids from malonyl-CoA de novo requires several enzymatic activities (1). In most bacteria and plants the activities exist as discrete monofunctional polypeptides, whereas in animals they are integrated into a single multifunctional polypeptide (2). Partial sequences for animal fatty acid synthases have been reported (3-6). In this paper we report the complete amino acid sequencer of an animal fatty acid synthase and the ordering of the seven functional domains on the multifunctional subunit. MATERIALS AND METHODSIsolation and Sequencing of Fatty Acid Synthase cDNA Clones. Clones characterized in this study were isolated from Agtll cDNA libraries constructed from poly(A) RNA obtained from the mammary glands of lactating Long-Evans rats (7) or the livers of fasted-refed Long-Evans rats (8). To maximize the probability of including cDNA sequences corresponding to the 5' end of the fatty acid synthase in the liver library, we added a specific primer [nucleotides (nt) 3183-3200 of fatty acid synthase, antisense direction, 50 ng/ml] to the reaction mixture for first-strand synthesis.Asymmetrical adaptors with dephosphorylated EcoRI overhangs (Pharmacia) were used in the ligation reaction, eliminating the need for EcoRI methylase treatment. The liver library yielded, in Escherichia coli Y1090r-, 1.2 X 107 plaque-forming units/,ug of DNA and was amplified 7.6 x 104-fold (68% white plaques) before screening.Probes for library screening were derived from established fatty acid synthase cDNAs and were labeled with [32P]dCTP by random-priming (9). Inserts were subcloned (10) using pUC12 or pUC19 vectors and E. coli DH5a cells (Bethesda Research Laboratories). Nested deletions were constructed using BAL-31 nuclease (11). Double-stranded plasmid DNA was used directly in dideoxynucleotide sequencing reactions with purified synthetic oligonucleotide primers (3, 12).Amino Acid Sequencing. Fatty acid synthase was purified from the livers of Long-Evans rats. The thioesterase domains were removed with trypsin, and the core polypeptides...

6‐Deoxyerythronolide‐B synthase 2 from Saccharopolyspora erythraea

“…Each of the N-terminal and C-terminal halves of the Orf B protein shows striking sequence similarity to their counterparts in Orf A. The alignment of these four 'half-proteins' with each other, with the sequence of rat fatty-acid synthase [27] and with the sequence of 6-methylsalicylate synthase from Penicillium patulum [21], is shown in Fig. 3.…”

Structure and function of eukaryotic fatty acid synthases

“…By that time, the multifunctional enzymes still were thought to be stable assemblies of individual enzymes, and only in the early 1970s it became clear that the type I FASs in fact consist of multifunctional polypeptide chains encoded by large genes, and not of assembled individual enzymes (Schweizer et al 1973 ;Stoops et al 1975). Domain mapping, genetic studies and the publication of the primary structures of animal and yeast FAS in the 1980s showed that although both systems are classified as ' FAS type I ', the animal and yeast enzymes have a radically different domain organization and therefore evolved along two unrelated lines (Amy et al 1989 ;Mohamed et al 1988;Schweizer et al 1986). …”

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

“…Construction of the Transferase Expression Plasmids-The plasmid pFAS54, which consists of nucleotides 642-2544 of the fatty acid synthase cDNA (5) and is believed to encode the entire transferase domain of the fatty acid synthase flanked by regions encoding part of the ␤-ketoacyl synthase and possibly the dehydrase domains (2), was used as template DNA for the PCR-based engineering of two different length constructs both encoding the transferase (Fig. 1).…”