Biosynthetic enzymes acyltransferases

A short-term regulation mechanism for cholesterol 7a-hydroxylase activity has been investigated recently in rat liver. The enzyme appears to exist in two forms, which are interconverted by cytosolic fiictors (K12). These foctors may correspond to a protein kinase and a phosphatase, which have been proposed to regulate cholesterol 7a-hydroxylase activity by a phosphorylation (active form)-dephosphorylation (inactive form) mechanism (S9). Another enzyme utilizing cholesterol as substrate, acyl-CoA cholesterol O-acyltransferase (EC 2.3.1.26), may also be regulated in this way, while the biosynthetic enzyme, HMC-CoA reductase, is inhibited in the phosphory-lated form (SIO). Thus, short-term regulation of the concentration of un-esterified cholesterol in the liver may be achieved by coordinate control of these three key enzymes in cholesterol metabolism by reversible phosphorylation (SIO). 

Several catalytic elements are required for the biosynthetic process acyltransferases that load the primer and extender substrates onto the FAS complex a posttranslationally phosphopantetheinylated acyl carrier protein (ACP) that translocates the various thioester intermediates between catalytic sites a P-ketoacyl synthase (KS) that performs the condensation reaction the p-ketoacylreductase, dehydrase, and enoylreductase enzymes that are responsible for the beta-carbon-processing reactions and a chain-terminating enzyme... 

Essential non-steroidal isoprenoids, such as dolichol, prenylated proteins, heme A, and isopentenyl adenosine-containing tRNAs, are also synthesized by this pathway. In extrahepatic tissues, most cellular cholesterol is derived from de novo synthesis [3], whereas hepatocytes obtain most of their cholesterol via the receptor-mediated uptake of plasma lipoproteins, such as low-density lipoprotein (LDL). LDL is bound and internalized by the LDL receptor and delivered to lysosomes via the endocytic pathway, where hydrolysis of the core cholesteryl esters (CE) occurs (Chapter 20). The cholesterol that is released is transported throughout the cell. Normal mammalian cells tightly regulate cholesterol synthesis and LDL uptake to maintain cellular cholesterol levels within narrow limits and supply sufficient isoprenoids to satisfy metabolic requirements of the cell. Regulation of cholesterol biosynthetic enzymes takes place at the level of gene transcription, mRNA stability, translation, enzyme phosphorylation, and enzyme degradation. Cellular cholesterol levels are also modulated by a cycle of cholesterol esterification mediated by acyl-CoA cholesterol acyltransferase (ACAT) and hydrolysis of the CE, by cholesterol metabolism to bile acids and oxysterols, and by cholesterol efflux. 

Since the PKS (polyketide synthase) gene cluster for actinorhodin (act), an antibiotic produced by Streptomyces coelicolor[ 109], was cloned, more than 20 different gene clusters encoding polyketide biosynthetic enzymes have been isolated from various organisms, mostly actinomycetes, and characterized [98, 100]. Bacterial PKSs are classified into two broad types based on gene organization and biosynthetic mechanisms [98, 100, 102]. In modular PKSs (or type I), discrete multifunctional enzymes control the sequential addition of thioester units and their subsequent modification to produce macrocyclic compounds (or complex polyketides). Type I PKSs are exemplified by 6-deoxyerythronolide B synthase (DEBS), which catalyzes the formation of the macrolactone portion of erythromycin A, an antibiotic produced by Saccharopolyspora erythraea. There are 7 different active-site domains in DEBS, but a given module contains only 3 to 6 active sites. Three domains, acyl carrier protein (ACP), acyltransferase (AT), and P-ketoacyl-ACP synthase (KS), constitute a minimum module. Some modules contain additional domains for reduction of p-carbons, e.g., P-ketoacyl-ACP reductase (KR), dehydratase (DH), and enoyl reductase (ER). The thioesterase-cyclase (TE) protein is present only at the end of module 6. 

Lipid biosynthetic enzymes catalyzing the production of TAG building blocks (FA and the glycerol backbone) and acyltransferases catalyzing TAG bioassembly have... 

Kroon, J.T.M., Wei, W.X., Simon, W.J. and Slabas, A.R. 2006. Identification and functional expression of a type 2 acyl-CoA diacylglycerol acyltransferase (DGAT2) in developing castor bean seeds which has high homology to the major triglyceride biosynthetic enzyme of fungi and animals. Phytochemistry 67 2541-2549. 

Figure 6 HMGS containing biosynthetic pathways. Portions of the PKS and PKS/NRPS pathways where the HMGS and related enzymes are located. Abbreviations A - Adenylation, AGP - acyl carrier protein, AT - acyltransferase, Cy - cyciization, DH - dehydratase, ER - enoyl reductase, GNAT -CCN5-related N-acetyltransferase, KS - ketosynthase, KR - ketoreductase, MT - methyltransferase. Ox - Oxidase, Oxy - Oxygenase, PGP - peptide carrier protein, PhyH - phytanoyl-CoA dioxygenase, PS - pyrone synthase, TE - thioesterase, - unknown function, - inactive domain.

Figure 10.6. The biosynthesis of endocannabinoids. Biosynthetic pathways for the biosynthesis of the major endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG). Note that the enzymes N-acyltransferase in anandamide biosynthesis. The biosynthesis of 2-AG can proceed via two different routes and is also dependent on an increase in Ca. Figures reprinted from Piomelli (2003) with permission from Nature Publishing Group.

Synthesis of caffeic and ferulic acids also needs hydroxylase and methyltransferase enzymes transformation into hydroxycinnamic tartaric acid esters (HCTA) is operated by an acyltransferase enzyme. The scheme shown in Figure 2.9 summarizes the biosynthetic pathways described. 

Finally, the enzymatic nature of CPIA-cholesterol ester formation will be briefly mentioned. None of the enzyme preparations of three known biosynthetic pathways for cholesterol esters, namely, acyl-CoA cholesterol Q-acyltransferase (ACAT), lecithin cholesterol 0-acyltransferase (LCAT), nor cholesterol esterase, was effective in producing CPIA-cholesterol ester from the Ba isomer or CPIA. In contrast, the 9,000 g supernatant or microsomal fractions from liver or kidney homogenate were found to be capable of producing CPIA-cholesterol ester without the addition of any cofactors. As substrate, only the Ba isomer was effective, and none of the 3 other fenvalerate isomers nor free CPIA was effective. The hepatic enzyme preparation also catalyzed hydrolysis of fenvalerate, and in this case all the 4 isomers were utilized as substrates. These facts imply that CPIA-cholesterol ester is formed from the Ba isomer through a transesterification reaction via intermediary acyl-enzyme complex. 

Enzymes involved in most protein lipidation pathways have been identified, with the exception of members of the acyltransferase and thioesterase family involved in S-acylation and its turnover. Although many PATs and a single cytoplasmic APT have been identified, many more remain to be discovered. For example, new APTs will have to be identified in order to understand the acylation cycle-dependent trafficking of 5-acylated proteins like Ras. Also, proteins/enzymes have been assigned to all steps of the GPI biosynthetic pathway... 

Fig. 5. Biosynthesis of membrane phospholipids from alkyldihydroxyacetone-P (alkyl-DHAP), the first detectable intermediate formed in the biosynthetic pathway for ether-linked glycerolipids. Enzymes catalyzing the reactions are (1) NADPH alkyl-DHAP oxidoreductase, (II) acyl-CoA 1 -alkyl-2-lyso-sn-glycero-3-phosphate acyltransferase, (III) l-alkyl-2-acyl-in-glycero-3-phosphate phosphohydrolase, (IV) ATP 1-alkyl- /i-glycerol phosphotransferase, (V) CDP-choline l-alkyl-2-acyl-sn-glycerol cholinephosphotransferase (dithiothreitol-sensitive), (VI) CDP-ethanolamine l-alkyl-2-acyI-sn-glycerol ethanolaminephosphotransferase, and (VII) acyl-CoA 1 -alkyl-2-acy 1-OT-glycerol acyltransferase.

Penicillin Formation by Penicillium Chrysogenum. The first reactions of the penicillin biosynthetic pathway are identical to the ones in A. chrysogenum (Figure 1.1-1). IPN, however, is not epimerized to penicillin N instead it is converted to 6-aminopenicillanic acid (6-APA) by removal of the L-a-aminoadipic acid side chain, which is substituted by a hydrophobic acyl group. Both steps are catalyzed by the same enzyme, the acyl coenzyme A IPN acyltransferase (IAT). The enzymatic activity of lAT is believed to be the result of the processing of a 40-kD monomeric precursor into a dimeric form consisting of two subunits with MWs of 11 and 29 kD. Due to the broad substrate specifity of lAT, various penicillin derivatives are synthesized naturally by attachment of different acyl-CoA derivatives to the 6-APA-core. For industrial purposes, to facilitate extraction by organic solvents, synthesis usually is directed to the less hydrophilic penicillin V or penicillin G. This is by addition of phenoxyacetic acid or phenylacetic acid, respectively, as precursors to the culture broth. 

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