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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.
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
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.
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
ACP
-- or longer fatty acyl MAT chain
1 3
KS
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)
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
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
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 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
Synthesis of phosphatidylinositol (PI) in ER; phosphatidylglycerol (PG) and cardiolipin (CL) in mitochondria
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
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 Esters Are Formed in Liver For Storage and Transport on the HDL Surface
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
LDL is Released from Receptor by Interactions in Extracellular Receptor Domain Between Receptor Repeats and the !- Propeller Region
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
Receptor recycling