Investigation of NADPH and acyl-binding sites on avian fatty acid synthase

Biochemistry ◽  
1982 ◽  
Vol 21 (12) ◽  
pp. 2863-2870 ◽  
Author(s):  
Jeffrey W. Cardon ◽  
Gordon G. Hammes
Keyword(s):  
Fatty Acid ◽  
Binding Sites ◽  
Fatty Acid Synthase

scholarly journals Occupancy and Function of the −150 Sterol Regulatory Element and −65 E-Box in Nutritional Regulation of the Fatty Acid Synthase Gene in Living Animals

2003 ◽  
Vol 23 (16) ◽  
pp. 5896-5907 ◽  
Author(s):  
Maria-Jesus Latasa ◽  
Michael J. Griffin ◽  
Yang Soo Moon ◽  
Chulho Kang ◽  
Hei Sook Sul
Keyword(s):  
Fatty Acid ◽  
Binding Sites ◽  
Fatty Acid Synthase ◽  
Regulatory Element ◽  
Nutritional Regulation ◽  
Fed State ◽  
E Box ◽  
Sterol Regulatory Element ◽  
E Boxes

ABSTRACT Upstream regulatory factor (USF) and sterol regulatory element binding protein (SREBP) play key roles in the transcriptional regulation of the fatty acid synthase (FAS) gene by feeding and insulin. Due to the dual binding specificity of SREBP, as well as the presence of multiple consensus sites for these transcription factors in the FAS promoter, their physiologically relevant functional binding sites have been controversial. Here, in order to determine the occupancy of the putative USF and SREBP binding sites, we examined their protein-DNA interactions in living animals by using formaldehyde cross-linking and immunoprecipitation of chromatin and tested the function of these elements by employing mice transgenic for a reporter gene driven by various 5′ deletions as well as site-specific mutations of the FAS promoter. We show that the −332 and −65 E-boxes are bound by USF in both fasted and refed mice, while the −150 SRE is bound by SREBP-1 only in refed mice. We also found that mutation of either the −150 SRE or the −65 E-box abolishes the feeding-induced activation of the FAS promoter in transgenic mice. Furthermore, in vivo occupancy of the FAS promoter by SREBP in the fed state can be prevented by mutation not only of the −150 SRE but, unexpectedly, of the −65 E-box as well. We conclude that the FAS promoter is activated during refeeding via the induced binding of SREBP to the −150 SRE and that USF binding to the −65 E-box is also required for SREBP binding and activation of the FAS promoter.

scholarly journals Reaction mechanism of recombinant 3-oxoacyl-(acyl-carrier-protein) synthase III from Cuphea wrightii embryo, a fatty acid synthase type II condensing enzyme

1999 ◽  
Vol 345 (1) ◽  
pp. 153-160 ◽  
Author(s):  
Amine ABBADI ◽  
Monika BRUMMEL ◽  
Burkhardt S. SCHüTT ◽  
Mary B. SLABAUGH ◽  
Ricardo SCHUCH ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Binding Sites ◽  
Fatty Acid Synthase ◽  
Catalytic Mechanism ◽  
Higher Plants ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Type Ii ◽  
Acetyl Coa ◽  
Condensing Enzyme

A unique feature of fatty acid synthase (FAS) type II of higher plants and bacteria is 3-oxoacyl-[acyl-carrier-protein (ACP)] synthase III (KAS III), which catalyses the committing condensing reaction. Working with KAS IIIs from Cuphea seeds we obtained kinetic evidence that KAS III catalysis follows a Ping-Pong mechanism and that these enzymes have substrate-binding sites for acetyl-CoA and malonyl-ACP. It was the aim of the present study to identify these binding sites and to elucidate the catalytic mechanism of recombinant Cuphea wrightii KAS III, which we expressed in Escherichia coli. We engineered mutants, which allowed us to dissect the condensing reaction into three stages, i.e. formation of acyl-enzyme, decarboxylation of malonyl-ACP, and final Claisen condensation. Incubation of recombinant enzyme with [1-14C]acetyl-CoA-labelled Cys111, and the replacement of this residue by Ala and Ser resulted in loss of overall condensing activity. The Cys111Ser mutant, however, still was able to bind acetyl-CoA and to catalyse subsequent binding and decarboxylation of malonyl-ACP to acetyl-ACP. We replaced His261 with Ala and Arg and found that the former lost activity, whereas the latter retained overall condensing activity, which indicated a general-base action of His261. Double mutants Cys111Ser/His261Ala and Cys111Ser/His261Arg were not able to catalyse overall condensation, but the double mutant containing Arg induced decarboxylation of [2-14C]malonyl-ACP, a reaction indicating the role of His261 in general-acid catalysis. Finally, alanine scanning revealed the involvement of Arg150 and Arg306 in KAS III catalysis. The results offer for the first time a detailed mechanism for a condensing reaction catalysed by a FAS type II condensing enzyme.

scholarly journals Promoter selective transcriptional synergy mediated by sterol regulatory element binding protein and Sp1: a critical role for the Btd domain of Sp1.

1997 ◽  
Vol 17 (9) ◽  
pp. 5193-5200 ◽  
Author(s):  
J N Athanikar ◽  
H B Sanchez ◽  
T F Osborne
Keyword(s):  
Fatty Acid ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Fatty Acid Synthase ◽  
Present Report ◽  
Regulatory Element ◽  
Critical Role ◽  
Ldl Receptor ◽  
Simian Virus 40 ◽  
Sterol Regulatory Element

Cellular cholesterol and fatty acid levels are coordinately regulated by a family of transcriptional regulatory proteins designated sterol regulatory element binding proteins (SREBPs). SREBP-dependent transcriptional activation from all promoters examined thus far is dependent on the presence of an additional binding site for a ubiquitous coactivator. In the low-density lipoprotein (LDL) receptor, acetyl coenzyme A carboxylase (ACC), and fatty acid synthase (FAS) promoters, which are all regulated by SREBP, the coactivator is the transcription factor Sp1. In this report, we demonstrate that Sp3, another member of the Sp1 family, is capable of substituting for Sp1 in coactivating transcription from all three of these promoters. Results of an earlier study showed that efficient activation of transcription from the LDL receptor promoter required domain C of Sp1; however, this domain is not crucial for activation of the simian virus 40 promoter, where synergistic activation occurs through multiple Sp1 binding sites and does not require SREBP. Also in the present report, we further localize the critical determinant of the C domain required for activation of the LDL receptor to a small region that is highly conserved between Sp1 and Sp3. This crucial domain encompasses the buttonhead box, which is a 10-amino-acid stretch that is present in several Sp1 family members, including the Drosophila buttonhead gene product. Interestingly, neither the buttonhead box nor the entire C domain is required for the activation of the FAS and ACC promoters even though both SREBP and Sp1 are critical players. ACC and FAS each contain two critical SREBP sites, whereas there is only one in the LDL receptor promoter. This finding suggested that buttonhead-dependent activation by SREBP and Sp1 may be limited to promoters that naturally contain a single SREBP recognition site. Consistent with this model, a synthetic construct containing three tandem copies of the native LDL receptor SREBP site linked to a single Sp1 site was also significantly activated in a buttonhead-independent fashion. Taken together, these studies indicate that transcriptional activation through the concerted action of SREBP and Sp1 can occur by at least two different mechanisms, and promoters that are activated by each one can potentially be identified by the number of critical SREBP binding sites that they contain.

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