Biosynthesis of Lipids II

Suaib Luqman

Objectives

v To understand the biosynthesis of lipids
v How do fatty acid, triacylglycerol, phospholipid and cholesterol synthesize

Concept Map

Description

Synthesis of Branched Chain FAs

Branched chain FAs are typically saturated and are originated in two divergent families: (i) the iso series and (ii) the ante-iso series. It has been established that Actinomycetales include unique branch chain FA synthesis mechanisms together with tuberculosteric acid.

The branched chain FAs synthesizing system make use of α keto acids (leucine, Isoleucine and Valine) as primers in contrast to the branched chain FAS that exploits short-chain acyl CoA esters as primers. α Keto acid primers are derived from the decarboxylation and transamination of leucine, isoleucine and valine to form 3 methylbutyryl CoA, 2-Methylbutyryl-CoA and 2 methylpropanyl CoA respectively. 2 Methylpropanyl CoA primers resulting from valine are extended to make even-numbered iso-series fatty acids like 14 methyl pentadecanoic (isopalmitic) acid, 3 methylbutyryl CoA primers from leucine is being used to form odd-numbered iso series FAs (e.g. 13-methyl-tetradecanoic acid). 2 Methylbutyryl CoA primers from isoleucine are lengthened to outline ante-iso series FAs enclosing an odd number of carbon atoms such as 12-Methyl tetradecanoic acid. The decarboxylation of the primer precursors occurs via the branched chain α keto acid decarboxylase (BCKA) enzyme. In E. coli, elongation of the fatty acid pursues the identical biosynthetic pathway to produce straight chain FAs where malonyl CoA is used as a chain extender. The key end products are branched chain FAs (12-17 carbon) and their concerto apt to be consistent and trait for several bacterial species.

BCKA decarboxylase and relative activities of α-keto acid substrates

The enzyme BCKA decarboxylase is poised of two sub-units in a tetrameric structure (A2B2) and is indispensable for the synthesis of branchedchain FAs. It is accountable for the decarboxylation of α keto acids produced by the transamination of leucine, isoleucine and valine resulting in the formation of primers used for branched chain FA synthesis. The enzyme activity is exceedingly higher with branched chain α keto acid substrates as compared to straight chain substrates. In Bacillus species its specificity is maximum for the isoleucine derived α keto β methylvaleric acid, trailing by α ketoisocaproate and α ketoisovalerate. The high

affinity of the enzyme for branched-chain α keto acids assign it to function as the primer donating system for

branched chain FAS.

Activity of BCKA	Substrate Used	Product	Vmax	Km (µM)
(%)		(CO2, nM/min/mg)	(nM/min/mg)
25	Pyruvate	04.90	15.20	51.1
38	α-Ketoisocaproate	07.40	05.60	< 1.0
63	α-Ketoisovalerate	12.40	13.30	< 1.0
100	L-α-keto-β-methyl-valerate	19.7	17.80	< 1.0

Source: Adapted from https://en.wikipedia.org/wiki/Fatty_acid_synthesis

Factors affecting Branched FAs chain length and pattern distribution

The α Keto acid primers (12-17 carbon) are utilized to make branched chain FAs and the size tend to be unswerving and uniform among a meticulous bacterial species which may be changed due to malonyl-CoA concentration, heat-stable factors (HSF) present or temperature. Since these factors affect chain length, HSFs modify the specificity of BCKA decarboxylase for a specific α-keto acid substrate, accordingly varying the ratio of branched chain FAs formed. It has been observed that an increase in malonyl CoA concentration result affect a bigger proportion of C17 FAs formed till an optimal concentration of malonyl CoA (~20μM) is accomplished. In Bacillus species, a decrease in temperature also apt to move the FA distribution vaguely toward C17 FAs.

Branch chain FAS

Bacteria that do not have the aptitude to execute the branch chain FA system via α keto primer apply this procedure using short chain carboxylic acids as primers. Distinctive short-chain primers consist of isobutyrate, 2 methyl butyrate and isovalerate. Commonly, the acids required for these primers are taken up from the environment which is frequently seen in ruminal bacteria. The disparity amid straight chain FAS and branch chain FAS is substrate specificity of the enzyme converting acyl CoA to acyl ACP.

In general, the overall reaction is as under:

Isobutyryl CoA + 6 malonyl CoA + 12 NADPH + 12H+

Isopalmitic acid + 6 CO2 + 12 NADP + 5 H2O + 7 CoA

Omega Alicyclic FAs

In numerous species of bacteria, omega alicyclic FAs (structure shown below) are the foremost membrane FAs, classically enclosing an omega terminal propyl or butyryl cyclic group. The FAS employed to build omega-alicyclic FAs is also used to make membrane branched chain FAs. The regale of cyclic carboxylic acid CoA esters is a lot superior to that of branched-chain primers in bacteria with omega alicyclic FAs. The cyclic primers synthesis is not well-known but it has been recommended that procedure engrosses the conversion of sugars to shikimic acid followed by cyclohexylcarboxylic acid CoA esters that dole out as primers for omega alicyclic FA synthesis.

Synthesis of Tuberculostearic acid

Tuberculostearic acid chemically known as D-10 Methylstearic acid is a saturated FA well-known to be produced by Mycobacterium spp. and two species of Streptomyces. Oleic acid (a monosaturated FA) is the precursor that forms the Tuberculostearic acid by esterification to a phospholipid (S-adenosyl-methionine) that donates a methyl group to the double bond of oleic acid. The methylation results in the formation of intermediate 10 methylene octadecanoyal which on successive reduction of the residue with NADPH as a cofactor results in the formation of 10-methylstearic acid.

Fatty Acid Synthase (FAS): The Charismatic Enzyme Complex

In humans, FAS is encoded by the FASN gene. It is a multi-enzyme protein that catalyzes FA synthesis. FAS is not a solitary enzyme though a whole enzymatic system poised of two identical 272 kDa multifunctional polypeptides where substrates are handed from one functional domain to the subsequent domain catalyzing the synthesis of palmitate (C16:0, a long-chain saturated FA) from acetyl-CoA and malonyl-CoA, in the charisma of cofactor NADPH.

Metabolic Function & Classes

FAs are aliphatic acids essential to energy formation and storage, cellular structure and as intermediates in hormone biosynthesis and other biologically significant molecules. They are synthesized from acetyl CoA and malonyl CoA by a series of decarboxylative Claisen condensation reactions. Subsequent to every round of elongation the β-keto group is abridged to the effusive saturated carbon chain by the chronological action of a ketoreductase (KR), dehydratase (DH) and enol reductase (ER). The budding FA chain is carried among these active sites while fasten covalently to the phosphopantetheine prosthetic group of an acyl carrier protein (ACP) followed by its release through the accomplishment of a thioesterase (TE) abreast of reaching a 16C chain length (palmitic acid).

There are two major classes of FAS namely Type I and Type II.

· Type I FAS systems make the most of a single large, multifunctional polypeptide and are widespread to both mammals and fungi (even if the structural arrangement of mammalian and fungus FAS vary). This FAS system is also found in the CMN group of bacteria (corynebacteria, mycobacteria and nocardia) where it produces palmitic acid and collaborates with FAS II system to produce a superior range of lipid products.

· Type II FAS system is present in archaea and bacteria and is exemplified by the use of distinct monofunctional enzymes for FAS. Investigators are searching for the inhibitors of this pathway (FAS II) as probable antibiotics.

The mechanism of elongation and reduction in both FAS I and FAS II is similar because the domains of the FAS

II enzymes are mostly homologous to their domain counterparts in FAS I multienzyme polypeptides. Conversely, the disparity in the organization of the enzymes i.e. coordinated in FAS I and distinct in FAS II gives rise to a lot of essential biochemical differences.

The evolutionary history of FAS is very much disheveled with that of polyketide synthases (PKS). PKS make use of an analogous mechanism and homologous domains to produce lipids (secondary metabolite). Besides, PKS also reveal a Type I and Type II organization. In animals, the FAS system I is thought to have arisen through alteration of PKS I in fungi, whereas in fungi and the CMN group of bacteria, FAS I appear to have arisen discretely through the fusion of FAS II genes.

Structure of FAS

The mammalian FAS embodies a homodimer of two identical protein subunits, in which three catalytic domains in the N-terminal section namely dehydrase (DH), ketoacyl synthase (KS) and malonyl/acetyltransferase (MAT) are alienated by a core region of 600 residues from four C-terminal domains i.e. acyl carrier protein (ACP), enoyl reductase (ER), ketoacyl reductase (KR) and thioesterase (TE). The traditional model for organization of FAS (the head-to-tail model on the right) is basically based on the observations that the bifunctional reagent 1,3- dibromopropanone (DBP) is competent to crosslink the cysteine thiol (active site) of the KS domain in one FAS monomer with the phosphopantetheine (prosthetic group) of the ACP domain in the other monomer. Complementation analysis of FAS dimers haulage diverse mutations on each monomer has established that the KS and MAT domains can work together with the ACP of either monomer and a reinvestigation of the DBP cross linking experiments discovered that the KS Cys161 thiol (active site) could be cross linked to the ACP 4′-phosphopantetheine thiol (prosthetic group) of either monomer. It has also been revealed that a heterodimeric FAS having only one competent monomer is proficient of palmitate synthesis.

Substrate Shuttling Mechanism

The solved structures of FAS (both yeast and mammalian) illustrate two distinct organizations of extremely conserved catalytic domains/enzymes in the multienzyme cellular mechanism. Mammalian FAS has an open lithe structure with only two reaction chambers while yeast FAS has an exceedingly well-organized rigid barrel-like structure with 6 reaction chambers that synthesize FA independently. Nevertheless, the conserved ACP in both the cases acts as the mobile domain liable for shuttling the intermediate FA substrates to different catalytic sites. Using cryo EM analysis, a first direct structural insight into the substrate shuttling mechanism was acquired where ACP is experiential bound to the different catalytic domains in the barrel-shaped yeast FAS. The cryo EM results imply that the binding of ACP to diverse sites is asymmetric and stochastic, as also signified by computer-simulation studies.

Regulation of FAS

The homeostasis and metabolism of FAS is transcriptionally regulated by Upstream Stimulatory Factors (USF1 and USF2) and sterol regulatory element binding protein-1c (SREBP-1c) in retort to feeding insulin in living animals. Even though, liver X receptor (LXRs) alter the expression of SREBP-1c in feeding, regulation of FAS by SREBP-1c is USF-dependent. Acylphloroglucinols isolated from the fern (Dryopteris crassirhizoma) demonstrated a FAS inhibitory activity.

Clinical Significance of FAS

The gene that codes for FAS has been examined as a possible oncogene, up regulated in breast cancers as well as being criteria of poor prognosis might also be valuable as a chemotherapeutic target. It could also be implicated in the assembly of an endogenous ligand for the nuclear receptor PPARα, the target of the fibrate drugs for hyperlipidemia. It is also being investigated as a possible drug target for treating the metabolic syndrome. In several cancer cell lines, this protein has been found to be complexed with ERα (estrogen receptor alpha) where the N-terminus of FAS is fused in frame with the C-terminus of ERα. The association of FAS with uterine leiomyomata has alos been reported.

Summary

In this lecture we learnt about:

The Biosynthesis of Lipids II

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Weblinks

Books

1. Lehninger Principles of Biochemistry by David L. Nelson, ‎Albert L. Lehninger, ‎Michael M. Cox. 2008. https://books.google.co.in/books?isbn=071677108X

2. Lipid Synthesis and Manufacture by F. D. Gunstone. 1999. https://books.google.co.in/books?isbn=0849397375

3. Lipid Biochemistry: An Introduction by Michael I. Gurr, Professor John L. Harwood, ‎Professor Keith N. Frayn. 2008 https://books.google.co.in/books?isbn=1405172703

4. Factors Affecting the Biosynthesis of Branched-chain Fatty …by Luiz Ronaldo de Abreu. 1993. https://books.google.co.in/books?id=obpnAAAAMAAJ

5. Plant Lipid Biochemistry: The Biochemistry of Fatty Acids … by Christopher Hitchcock, ‎Brian William Nichols. 1971. https://books.google.co.in/books?id=xQETETaVTaQC

Journals

T Kaneda. Iso- and anteiso-fatty acids in bacteria: biosynthesis, function, and taxonomic significance. Microbiol Rev. 1991 Jun; 55 (2): 288-302.
Smirnova N, Reynolds KA. Branched-chain fatty acid biosynthesis in Escherichia coli. J Ind Microbiol Biotechnol. 2001 Oct; 27 (4): 246-51.
Hirosuke OkuS, Toshi Kaneda. Biosynthesis of Branched-chain Fatty Acids in Bacillis subtilis. The Journal of Biological Chemistry 236 (34): 18386-18396.
A B Kroumova, Z Xie, G J Wagner. A pathway for the biosynthesis of straight and branched, odd- and even-length, medium-chain fatty acids in plants. Proceedings of the National Academy of Sciences, USA 91 (24): 11437-11441.
Dreher R, Poralla K, König WA. Synthesis of omega-alicyclic fatty acids from cyclic precursors in Bacillus subtilis. J Bacteriol. 1976 Sep; 127 (3): 1136-40.