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694:40 7/115: 5121 - Mo lec ul ar Bi ol o gy a nd Bi oc he mi st ry Lect ure# 3, Part 2 - - Li pid Bi os ynt hes is Nov. 11, 2008 1.

Fatty acid biosynthesis a.) Sources of acetyl-CoA and NADPH for fatty acid biosynthesis b.) Acetyl-CoA activated to malonyl-CoA via biotin-linked carboxylation c.) Multifunctional fatty acid synthase catalyzes: -- Transacylation of malonyl-CoA and acetyl-CoA to ACP derivatives -- Condensation to acetoacetyl-ACP -- NADPH-linked reduction to D-!-hydroxy derivative -- Dehydration to trans-"2-derivative -- NADPH-linked reduction to butyryl-ACP -- Condensation with malonyl-ACP and 6 more cycles -- Palmitoyl-ACP as product d.) Elongation via CoA derivatives e.) Structure of the multifunctional fatty acid synthase complex -intertwined dimer with two lateral semicircular reaction chambers, each with a full set of catalytic domains f.) Desaturation driven by 02 and electron transfer from NADPH which, together with e- derived from desaturation of substrate ! 2H2O g.) How fatty acid levels are regulated Acetly-CoA carboxylase as first committed step in FA biosynthesis subjected to feed-back inhibition by Palmitoyl-CoA product, allosteric activation by citrate and hormone dependent phosphorylation/dephosphorylation 2. Phospholipid biosynthesis a.) Basic phospholipid biosynthetic patterns b.) Important eukaryotic pathways in ER and mitochondria -- Synthesis from CDP-diacylglycerol as activated species: phosphatidylinositol, phosphatidylglycerol and cardiolipin -- Synthesis from CDP-head-group and diacylglycerol: phosphatidylcholine and phosphatidylethanolamine -- Head-group exchange: Interconversion of phosphatidylserine and phosphatidylethanol -- Methylation of attached headgroup: phosphatidylcholine formation from phosphatidylethanolamine in liver

3. Cholesterol biosynthesis a.) Synthesized in four stages: -- Conversion of acetyl-CoA to mevalonate (C6) -- Conversion of mevalonate to activated isoprene unit -- Polymerization of isoprenes to from squalene (C30) -- Squalene cyclization and conversion to cholesterol (C27) b.) Esterified with fatty acid by ACAT or LCAT c.) Cholesterol transport and utilization: -- Transported by plasma lipoproteins (chylomicrons, VLDL, LDL, HDL) that are synthesized mainly in ER -- Cellular uptake of LDL by receptor-mediated endocytosis -- LDL is released from LDL receptors into acidic endosomes by interaction between N-terminal LDL receptor repeats and !-propeller region of extracellular receptor domain -- LDL receptors are recycled -- LDL apoprotein B-100 is degraded, cholesterol esters are stored in cell -- Free cholesterol from LDL monolayer is released and esterified as needed, or acts as inhibitor of HMG-CoA reductase and LDL receptor synthesis Reading: Nelson, D.L. and Cox, M.M. (2008) Lehninger Principles of Biochemistry 5th Edition, Chapter 21 (p. 805-815; 820-844.

Subcellular Localization of Lipid Metabolic Events

Lipid Biosynthesis
Fatty acid biosynthesis Phospholipid biosynthesis Cholesterol biosynthesis Cholesterol transport and utilization

Fatty acid, isoprenoid, and sterol synthesis take place in cytosol where NADPH concentration is high Early steps of phospholipid synthesis using diacylglycerol also occur in the cytoplasm

Mitochondria

Phospholipid synthesis

Phospholipid synthesis (early stages)

Fatty Acid Biosynthesis is Not Merely a Reversal of Fatty Acid Oxidation


Occurs in cytosol rather than in the mitochondrial matrix Reductases are NADPH linked After first step (acetyl-CoA carboxylase, ACC): -- Intermediates are activated by linkage to acyl carrier protein (ACP) rather than to CoA -- Reactions are catalyzed in mammals by a single multifunctional fatty acid synthase protein containing ACP and all requisite catalytic sites (ACP acts as a flexible arm delivering substrates to enzyme active sites during modification reactions)

Acetyl-CoA is Delivered to the Cytosol For Fatty Acid Biosynthesis by the Citrate-Malate-Pyruvate Shuttle
1. Acetyl-CoA (arising from oxidation of pyruvate and amino acids) + oxaloacetate are condensed to citrate by citrate synthase in matrix 2. Inner membrane tricarboxylate transporter carries citrate to cytosol 3. Citrate lyase converts cytosolic citrate back to oxaloacetate + acetylCoA for FA biosynthesis 4. Oxaloacetate is unable to return and is converted to malate and finally back to oxaloacetate to complete the shuttle (5.) 6. In addition to malic enzyme, pentose-P pathway is source of NADPH

2 1 3

4 5 6

The mitochondrial inner membrane is impermeable to AcetylCoA and this indirect route is used to shuttle acetyl groups out to the cytoplasm, disguised in citrate.

The Acetyl-CoA Carboxylase Reaction


Three functional regions of mulifunctional mammalian acetyl-CoA carboxylase catalyze malonylCoA formation First committed and rate-limiting step in fatty acid biosynthesis, and its control point
Biotinyl prosthetic group ATP-dependent activation of CO2 by attachment to N in biotin ring

Fatty Acid Synthesis Proceeds by Addition of Two Carbons to a Growing Fatty Acyl Chain
1.In reaction calalyzed by bifuctional Malonyl-Acetyl Transferase (MAT), acetyl group of acetyl-CoA is transferred to a Cys-SH group in the !-Ketoacyl Synthase (KS) catalytic site 2. Malonyl group of malonyl-CoA is transferred to the -SH group of ACP in a reaction also catalyzed by Malonyl-Acetyl Transferase (MAT) 3. KS catalyzes condensation and releases CO2, which pulls the reaction 4. ! - Ketoacyl-ACP Reductase (KR) reduces !-keto group to an alcohol in an NADPH linked reaction

MAT

Two thiol groups on complex are charged with acyl groups:

ACP
-- or longer fatty acyl MAT chain
1 3

KS

Transfer of activated CO2 to Acetyl-CoA

KR

C-terminal domain struct- Structure of E. coli biotin ure of E. coli biotin carboxlase; active site pocket carrier protein is in C-terminal domain (pink)

Fatty Acid Synthesis -- Last Two Steps of First Round


5. ! - Hydroxyacyl-ACP dehydrase (DH) creates double bond through H2 elimination 6. Enoyl-ACP reductase (ER) reduces double bond to form saturated fatty acyl group (butyryl-ACP) in an NADPH linked reaction to complete first cycle (Steps 3-6 are essential a reversal of ! oxidation)
DH
5

The Overall Process of Palmitate Synthesis

ER

The fatty acyl chain grows by two-carbon units donated by activated malonate, with loss of CO2 at each step After first round, butyrl-ACP is translocated to KS Cys-SH Overall reaction:

Butyrl-ACP

8 Acetyl-CoA + 7ATP + 14(NADPH + H+) ! palmitate + 7(ADP + Pi) + 14NADP+ + 6H2O

Structure of E. coli ACP

Architecture of Mammalian Fatty Acid Synthase Determined by X-ray Crystallography at 3.2- Resolution
Pseudo 2-fold dimer axis

Bound NADP+ is shown in blue "KR /" KR and "ME / " ME = noncatalytic pseudoketoreductase and pseudomethyltransferase, probably a remnants of ancestral methyltransferase domains ACP and Thioesterase (TE), that releases final FA product, are missing from the structure because of their apparent inherent mobility LD = linker domains

(Maier et al. Science 321:1315, 2008)

Advantages of Multifunctional FAS Complex


Coordinates sequence of synthetic activities Enhanced protein stability when compared to a complex of different enzyme proteins Intermediates can traverse from one active site to another without leaving the complex The multifunctional FAS complex is an evolutionary product of exon shuffling, since each component enzyme is recognizably homologous to a separate bacterial enzyme protein counterpart

Substrate shuttling is facilitated by flexible tethering of acyl carrier protein domain and by limited contact between condensing and modifying portions of FAS multienzyme Active sites are mainly connected by linkers rather than direct interaction

Fatty Acid Biosynthesis is Tightly Regulated

Vertebrate Fatty Acyl-CoA Desaturase Requires Electron Transfer From NADPH


Electron transfer transfer pathway Electron pathway

Allosteric activation Feedback inhibition

Fatty acid levels in vertebrates are controlled by allosteric regulation of acetyl-CoA carboxylase and by hormone-dependent phosphorylation/ dephosphorylation EM showing filaments of acetyl-CoA carboxylase in active, dephosphorylated form -- it is converted to inactive monomers in phosphorylated form Inhibition of carnitine acyltransferase I by malonyl-CoA prevents fatty acid !-oxidation

The two desaturase substrates, NADPH and fatty acyl-CoA, undergo oxidation by molecular oxygen Power of O2 drives cis-double bond formation -- when desaturase is in reduced (Fe2+) state, interacts with O2 and the saturated fatty acyl-CoA substrate Two e- are passed from NADPH through chain involving cytochrome b5 and its reductase Two e- are then derived from fatty acyl substrate to give rise to the 2H2O This mechanism is used in synthesis of palmitoleate (16:1-#9) and oleate (18:1-#9), the 2 most common monounsaturated FAs, from palmitate and stearate

Phospholipid Biosynthesis
Phospholipids functions: -- Structural elements of membranes and plasma lipoproteins -- Second messengers in signal transduction pathways Basic pattern of phospholipid biosynthetic pathways: -- Synthesis of the glycerol backbone molecule -- Fatty acid attachment to glycerol backbone thru ester linkages -- Addition of hydrophilic head-group through phosphodiester linkages -- Alterations and exchange of some head-groups Important eukaryotic pathways -- Synthesis of phosphatidylinositol, phosphatidylglycerol and cardiolipin from CDP-diacylglycerol -- Synthesis of phosphatidylcholine and phosphatidylethanolamine from diacylglycerol and salvaged head-groups -- Interconversion of phosphatidylserine and phosphatidylethanolamine by head group exchange -- Methylation of phosphatidylethanolamine to phosphatidylcholine in liver

Strategies For Attachment of Head-Groups by Phosphodiester Bond Formation

Synthesis of phosphatidylinositol (PI) in ER; phosphatidylglycerol (PG) and cardiolipin (CL) in mitochondria

Synthesis of phosphatidylcholine (PC) and phosphatidylehtanolamine (PE) in ER

Synthesis of Cardiolipin and Phosphatidylinositol in Eukaryotes

Phosphatidlycholine is Synthesized in the Mammalian ER by Salvaging Choline PE is synthesized by a parallel pathway from salvaged ethanolamine

Specific PI kinases convert PI to phosphorylated derivatives that activate signal transduction pathways

Summary of the Phosphatidlycholine and Phosphatidylethanolamine Pathways

Cholesterol Metabolism
Salvage pathways

In liver, PC is synthesized by 3 methylations of PE using S -adenosylmethionine (adoMet) as the methyl donor which gives rise to S-adenoshomocysteine (adoHcy) -- final steps of major path from PS to PE (decarboxylation) to PC in eukaryotes

Component of cell membrane bilayers (fluidity buffer) Component of plasma lipoproteins (outer layer) Precursor of bile acids, steroid hormones, vitamin D3 (7dehydrocholesterol) All the Cs of cholesterol arise from acetate Isoprene units are the essential intermediates in the pathway from acetate to cholesterol

Head-group exchange

Cholesterol is Synthesized in Four Stages


(Stages 1 to 3 in cytosol, stage 4 on ER) 1. Condensation of three acetate units to form mevalonate 2. Conversion of mevalonate to activated isoprene units 3. Polymerization of 6 five C isoprenes to form 30 C linear squalene 4. Cyclization of C30 squalene to form the steroid ring nucleus with further oxidations, methyl group removals and migrations to produce the C27 cholesterol product

Formation of Mevalonate From Acetyl-CoA


The first 2 enzymes are cytosolic isozymes of the mitochondrial enzymes that initiate ketone body formation HMG-CoA reductase (97-kDa integral membrane glycoprotein) is 1st committed, ratelimiting step in cholesterol biosynthesis -- Regulatory site (feedback inhibition of activity by cholesterol) -- Inactivated in signal transduction pathway by cAMP-dependent protein kinase phosphorlation -- Undergoes cholesterol-stimulated protein degradation, and cholesterol-mediated trancriptional control of mRNA levels -- Site of inhibition of cholesterol biosynthesis by statins Structure of extramembrane fragment of tetrameric HMG-CoA reductase

Conversion of Mevalonate to Activated Isoprene Units

Cholesterol Esters Are Formed in Liver For Storage and Transport on the HDL Surface

Note pink highlighted leaving groups from hypothetical intermediate

Six of these activated units combine to form squalene Subsequent pathway to squalene involves condensation of 3 dimethylallylPP to yield C15 farnesyl-PP Two farnesyl-PP are condensed with PP release to drive C30 squalene formation Squalene undergoes oxygenase reaction to epoxide, complex cyclization and conversion to C27 cholesterol product in many steps

Reaction in liver is catalyzed by ACAT, where the cholesterol esters are stored, or transported in secreted lipoprotein particles to other tissues LCAT -- part of high density lipoprotein (HDL) apolipoprotein, located on surface of nascent HDL particles, stimulated by Apo-1 of HDL Cholesterol esters synthesized by LCAT form an HDL core which transforms the nascent particles into mature spherical HDL The cholesterol-rich particles return to liver from extrahepatic tissues where the cholesterol is unloaded and is largely excreted in bile salts

Cholesterol and other Lipids are Carried in Plasma Lipoproteins


Low-density lipoprotein (LDL) is synthesized in liver and is major transporter of cholesterol and cholesterol esters from liver to extrahepatic tissues Apolipoprotein B-100 is a 513-kDa polypeptide recognized by LDL receptors on plasma membrane of extrahepatic tissues Beside chylomicrons, very lowdensity lipoprotein (VLDL) is an additional major lipoprotein VLDL delivers endogenously synthesized triacylglycerol from liver to adipose and other tissues -- Breakdown of VLDL remnants by lipoprotein lipase ! intermediateStructural cartoon of low density density lipoproteins (IDL) ! LDL lipoprotein (LDL)

LDL is Released from Receptor by Interactions in Extracellular Receptor Domain Between Receptor Repeats and the !- Propeller Region

Structure of 6-bladed propeller domain and adjacent EGFlike domain


(Rudenko et al., Nature 298:2353, 2002)

Second extracellular domain (red) is homologous to epidermal growth factor (EGF) precursor Note that LDL interacts with receptor repeats R4 and R5 The !-propeller regions displaces bound LDL by acting as alternative substrate for ligand-binding domain in a Ca2+-dependent LDL release

Cholesterol Uptake by Receptor-Mediated Endocytosis

Regulation of Cholesterol Formation Balances Synthesis with Dietary Uptake


Glucagon promotes phosphorylative inactivation of HMG-CoA reductase Insulin promotes dephosphorylative activation High concentrations of cholesterol activate ACAT, increasing cholesterol esterification for storage High cellular cholesterol levels also diminish transcription of the gene encoding LDL receptors, resulting in less cholesterol uptake

Receptor recycling

Incorporation into ER membrane Re-esterification by ACAT

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