Mammalian Fatty Acid Synthase Research Articles

A super-family of medium-chain dehydrogenases/reductases (MDR). Sub-lines including zeta-crystallin, alcohol and polyol dehydrogenases, quinone oxidoreductase enoyl reductases, VAT-1 and other proteins.

The protein super-family of medium-chain alcohol dehydrogenases (and glutathione-dependent formaldehyde dehydrogenase), polyol dehydrogenases, threonine dehydrogenase, archaeon glucose dehydrogenase, and eye lens reductase-active zeta-crystallins also includes Escherichia coli quinone oxidoreductase, Torpedo VAT-1 protein, and enoyl reductases of mammalian fatty acid and yeast erythronolide synthases. In addition, two proteins with hitherto unknown function are shown to belong to this super-family of medium-chain dehydrogenases and reductases (MDR). Alignment of zeta-crystallins/quinone oxidoreductases/VAT-1 reveals 38 strictly conserved residues, of which approximately half are glycine residues, including those at several space-restricted turn positions and critical coenzyme-binding positions in the alcohol dehydrogenases. This indicates a conserved three-dimensional structure at the corresponding parts of these distantly related proteins and a conserved binding of a coenzyme in the two proteins with hitherto unknown function, thus ascribing a likely oxidoreductase function to these proteins. When all forms are aligned, including enoyl reductases, a zeta-crystallin homologue from Leishmania and the two proteins with hitherto unknown function, only three residues are strictly conserved among the 106 proteins characterised within the superfamily, and significantly these residues are all glycines, corresponding to Gly66, Gly86 and Gly201 of mammalian class I alcohol dehydrogenase. Notably, these residues are located in different domains. Hence, a distant origin and divergent functions, but related forms and interactions, appear to apply to the entire chains of the many prokaryotic and eukaryotic members. Additionally, in the zeta-crystallins/quinone oxidoreductases, a highly conserved tyrosine residue is found. This residue, in the three-dimensional structure of the homologous alcohol dehydrogenase, is positioned at the subunit cleft that contains the active site and could therefore be involved in catalysis. If so, this residue and its role may resemble the pattern of a conserved tyrosine residue in the different family of short-chain dehydrogenases/reductases (SDR).

A Super‐Family of Medium‐Chain Dehydrogenases/Reductases (MDR)

The protein super‐family of medium‐chain alcohol dehydrogenases (and glutathione‐dependent formaldehyde dehydrogenase), polyol dehydrogenases, threonine dehydrogenase, archaeon glucose dehydrogenase, and eye lens reductase‐active ζ‐crystallins also includes Escherichia coli quinone oxidoreductase, Torpedo VAT‐1 protein, and enoyl reductases of mammalian fatty acid and yeast erythronolide synthases. In addition, two proteins with hitherto unknown function are shown to belong to this super‐family of medium‐chain dehydrogenases and reductases (MDR). Alignment of ζ‐crystallins/quinone oxidoreductases/VAT‐1 reveals 38 strictly conserved residues, of which approximately half are glycine residues, including those at several space‐restricted turn positions and critical coenzyme‐binding positions in the alcohol dehydrogenases. This indicates a conserved three‐dimensional structure at the corresponding parts of these distantly related proteins and a conserved binding of a coenzyme in the two proteins with hitherto unknown function, thus ascribing a likely oxidoreductase function to these proteins. When all forms are aligned, including enoyl reductases, a ζ‐crystallin homologue from Leishmania and the two proteins with hitherto unknown function, only three residues are strictly conserved among the 106 proteins characterised within the super‐family, and significantly these residues are all glycines, corresponding to Gly66, Gly86 and Gly201 of mammalian class I alcohol dehydrogenase. Notably, these residues are located in different domains. Hence, a distant origin and divergent functions, but related forms and interactions, appear to apply to the entire chains of the many prokarotic and eukaryotic members. Additionally, in the ζ‐crystallins/quinone oxidoreductases, a highly conserved tyrosine residue is found. This residue, in the three‐dimensional structure of the homologous alcohol dehydrogenase, is positioned at the sub‐unit cleft that contains the active site and could therefore be involved in catalysis. If so, this residue and its role may resemble the pattern of a conserved tyrosine residue in the different family of short‐chain dehydrogenases/reductases (SDR).

Intron-exon organization of the gene for the multifunctional animal fatty acid synthase.

The complete intron-exon organization of the gene encoding a multifunctional mammalian fatty acid synthase has been elucidated, and specific exons have been assigned to coding sequences for the component domains of the protein. The rat gene is interrupted by 42 introns and the sequences bordering the splice-site junctions universally follow the GT/AG rule. However, of the 41 introns that interrupt the coding region of the gene, 23 split the reading frame in phase I, 14 split the reading frame in phase 0, and only 4 split the reading frame in phase II. Remarkably, 46% of the introns interrupt codons for glycine. With only one exception, boundaries between the constituent enzymes of the multifunctional polypeptide coincide with the location of introns in the gene. The significance of the predominance of phase I introns, the almost uniformly short length of the 42 introns and the overall small size of the gene, is discussed in relation to the evolution of multifunctional proteins.

Regulation of expression of the fatty acid synthase gene in 3T3-L1 cells by differentiation and triiodothyronine.

We have previously reported that fatty acid synthase mRNA levels increase 10-15-fold during the differentiation of 3T3-L1 cells to adipocytes, correlating well with the increase in the relative rate of synthesis for this enzyme. Here we show by transcription run-on assays that fatty acid synthase is highly transcribed in both preadipocytes and adipocytes. Furthermore, the transcription rate of the fatty acid synthase gene increased only 1.5-fold during the adipose conversion, whereas the apparent mRNA half-life increased from 2.5 h in preadipocytes to approximately 20 h in adipocytes. These results indicate that the increase in mRNA level during adipose conversion is not only due to the transcriptional activation of this gene but reflects the post-transcriptional stabilization of the message in adipocytes compared to preadipocytes. As thyroid hormone has been reported to increase lipogenic enzyme activities including fatty acid synthase in adipose tissue and in differentiating adipocytes in vitro, we used fully differentiated 3T3-L1 adipocytes to study T3 regulation of mammalian fatty acid synthase expression. We measured the effect of T3 on the relative rate of protein synthesis, mRNA content, and transcription rate of this gene. When mature adipocytes were treated with 10 nM T3, the relative rate of synthesis of fatty acid synthase increased 1.9-fold at 6 h, and it reached a maximum of 2.9-fold at 12 h. In addition, Northern blot analysis showed that T3 increased the steady-state mRNA level for fatty acid synthase by 2.4-fold at 12 h and 4.5-fold at 24 h. Furthermore, run-on transcription analysis with isolated nuclei from cells treated with T3 showed that the transcription rate of the fatty acid synthase gene increased 4.1-fold after 6 h of T3 treatment and remained at the stimulated level for 24 h. These results demonstrate that the increase in transcription of the fatty acid synthase gene preceded that of the steady-state mRNA level, indicating that T3 regulates expression of fatty acid synthase primarily by modulating the transcription rate of the gene. In conclusion, while the differentiation-dependent increase in fatty acid synthase is mediated by both transcriptional and posttranscriptional processes, T3 regulation is primarily at the transcriptional level.

Molecular cloning of the mammalian fatty acid synthase gene and identification of the promoter region

Rat genomic clones encompassing the entire fatty acid synthase gene have been isolated and characterized. The gene is present in a single copy of approx. 20 kb. Genomic DNA sequencing, direct RNA sequencing and S1 nuclease analysis showed that transcription is initiated primarily 1274 nucleotides upstream from the translation start site and that the 87-nucleotide-long 5'-untranslated mRNA sequence is the same in liver, lung and mammary gland. The 5'-flanking region and first intron contain several sequence elements which may be involved in the transcriptional regulation of this gene.

Mammalian fatty acid synthase: a chimeric protein which has evolved by gene fusion

Mammalian fatty acid synthase: a chimeric protein which has evolved by gene fusion

The multifunctional polypeptide chains of rabbit-mammary fatty-acid synthase. Stoichiometry of active sites and active-site mapping using limited proteolysis.

Several methods have been used to label active centres on the multifunctional polypeptide chains of rabbit mammary fatty acid synthase. Experiments using [14C]acetyl-CoA or [14C]malonyl-CoA have shown that there is a single non-thiol site which binds either acetyl or malonyl groups, present at a stoichiometry of two per enzyme dimer, and representing an intermediate in the acyl transferase reaction. This adds further support to the view that the two subunits are identical and that each polypeptide chain contains up to seven active centres. However, two novel and independent methods for the quantification of the pantetheine thiol demonstrate that this prosthetic group can be present in sub-stoichiometric amounts. By studying intermediates during limited elastase digestion of fatty acid synthase labelled in different active centres, we have been able to map the positions of four active centres within the polypeptide chain. The thioesterase domain is present in a terminal location on both polypeptide chains as previously reported. The acyl carrier domain (pantetheine thiol) is located in a region of molecular weight 9000 immediately adjacent to the thioesterase domain. The acyl transferase (acyl-O-ester site) and the 3-oxoacylsynthase thiol are located in a region of molecular weight 110000 at the opposite end of the polypeptide chain to the thioesterase domain. The relationship between the disposition of the activities on the multifunctional polypeptide chains of yeast and mammalian fatty acid synthase is discussed.

7] Fatty acid synthase from pigeon liver

The fatty acid synthase enzyme systems of pigeon, chicken, and rat liver, and of rat lactating mammary gland have been purified to homogeneity. These systems exist in the soluble portion of the cell as multienzyme complexes with molecular weights of approximately 5 × 10 5 g/mole. The constituent enzymes catalyzing the component reactions of fatty acid synthesis are tightly bound in these complexes, whereas the fatty acid synthesizing enzymes of Escherichia coli are isolated in the dissociated state. Avian and mammalian fatty acid synthase complexes catalyze the conversion of acetyl- and malonyl-CoA to long-chain saturated fatty acids in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH). The products and the stoichiometry for the overall reaction are presented. The overall activity for fatty acid synthesis may be measured either by determining the incorporation of the [1- 14 C]acetyl moiety of labeled acetyl-CoA into long-chain fatty acids or by following the rate of NADPH reduction spectrophotometrically at 340 nm. The former method is reliable for the assay of crude and purified preparations, while the spectrophotometric method is most useful with purified enzyme.