The pentafunctional FAS1 gene of yeast: its nucleotide sequence and order of the catalytic domains

Abstract

FAS1, the structural gene of the pentafunctional fatty acid synthetase subunit beta in Saccharomyces cerevisiae has been sequenced. Its reading frame represents an intron-free nucleotide sequence of 5,535 base pairs, corresponding to a protein of 1,845 amino acids with a molecular weight of 205,130 daltons. In addition to the coding sequence, 1,468 base pairs of its 5'-flanking region were determined. S1 nuclease mapping revealed two transcriptional initiation sites; 5 and 36 base pairs upstream of the translational start codon. Within the flanking sequences two TATATAAA boxes, several A-rich and T-rich blocks and a TAG...TATGTT...TATGTT...TTT sequence were found and are discussed as transcriptional initiation and termination signals, respectively. The order of catalytic domains in the cluster gene was established by complementation of defined fas1 mutants with overlapping FAS1 subclones. Acetyl transferase (amino acids 1-468) is located proximal to the N-terminus of subunit beta, followed by the enoyl reductase (amino acids 480-858), the dehydratase (amino acids 1,134-1,615) and the malonyl/palmityl transferase (amino acids 1,616-1,845) domains. One major inter-domain region of about 276 amino acids with so far unknown function was found between the enoyl reductase and dehydratase domains. The substrate-binding serine residues of acetyl, malonyl and palmityl transferases were identified within the corresponding domains. Significant sequence homologies exist between the acyl transferase active sites of yeast and animal fatty acid synthetases. Similarly, a putative sequence of the enoyl reductase active site was identified.

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). …”

“…It became clear that the numerous individual proteins previously detected had been the result of unspecific proteolysis during the preparation. At the beginning of the 1980s, the active site peptides of several catalytic domains had been identified and the catalytic activities had been further characterized biochemically (Lynen, 1980), but the location of the catalytic residues within both multifunctional polypeptides and the approximate domain borders could only be reliably established when the primary structures of both yeast FAS subunits were published in the middle of the 1980s (Mohamed et al 1988 ;Schweizer et al 1986), which enabled further mutagenesis experiments (Fichtlscherer et al 2000;Schuster et al 1995). For the understanding of such a complex multienzyme, the knowledge of its threedimensional (3D) structure is of critical importance, and consequently structural studies on fungal FAS were pursued in parallel with its biochemical and genetic characterization.…”

Plastid‐localised seed acyl‐carrier protein of Brassica napus is encoded by a distinct, nuclear multigene family

“…In animals [l] and yeast [2] the domains are present on one or two multifunctional polypeptide chains (type I fatty acid synthetase), which are localised within the cytoplasm. In contrast, in plants [3] the fatty acid synthetase domains exist as discreet, monofunctional activities (type 11) which are organellar in location.Some insight into the genetic regulation of type I1 fatty acid synthetase systems has recently been obtained through cloning of genes from both yeast [4] and mammals [5]. Our interest lies in understanding the genetic control of fatty acid biosynthesis in plants, in particular within developing oil seeds.…”

“…Some insight into the genetic regulation of type I1 fatty acid synthetase systems has recently been obtained through cloning of genes from both yeast [4] and mammals [5]. Our interest lies in understanding the genetic control of fatty acid biosynthesis in plants, in particular within developing oil seeds.…”

Identification and sequencing of cDNA clones for the rodent negative acute‐phase protein α1‐inhibitor 3

“…For Southern hybridization cDNA inserts of al-inhibitor 3 and c/2-macroglobulin were 32P-labelled either by nick translation [27] or via a random primer DNA sequencing was performed by the dideoxy-chaintermination method of Sanger and Coulson [29] by subcloning restriction fragments of the cDNA clones into the appropriate polylinker restriction sites of M13 vectors, so-called forced cloning [22,30 -321. Experimental details for sequencing have been given by Schweizer et al [33].…”