11-Acetyltransferase

Chloramphenicol (Cm) is a broad spectrum antibiotic that acts by inhibiting the function of bacterial ribosomes. Cm binds the 50 subunit of bacterial (70S) ribosomes and inhibits peptidyltransferase (Kj 3 ixM), effectively blocking prokaryotic protein synthesis. Because of its broad spectrum activity on ribosomal function, Cm also causes some serious side effects in eukaryotic hosts. (In humans 

FIGURE 8.7 Principal reaction catalyzed by chloramphenicol acetyltransferase. R = -COCHCI2 (Cm), -COCH2OH (2-hydroxyacetamido-Cm), -COCH3 (2-acetamido-Cm). 

The high level resistance of certain bacteria to Cm is due to the enzyme chloramphenicol acetyltransferase (CAT) which modifies the Cm to a biologically inactive derivative. CAT is an intracellular, trimeric enzyme with an average monomer size of 25 kDa. CAT catalyzes the transfer of an acetyl group from donor acetyl-CoA to the primary (C-3) hydroxyl of Cm, generating chloramphenicol 3-acetate and CoA-SH as products (Fig. 8.7). The acetylated Cm is incapable of binding to bacterial ribosome and is devoid of antimicrobial activity. 

Type I CAT (or CAT]), which is typically encoded by transposon Tn9, has been widely used both as a selection marker and as a reporter for studying gene expression in eukaryotic cells. The CAT reporter system is a powerful and sensitive tool for such studies since eukaryotic cells frequently used in such experiments do not have endogenous CAT or CAT-like activities. Furthermore, CAT activity can be detected and quantitated with relative ease and at high sensitivities. 

In this section, type I CAT (and the cat gene) will be described as a typical example of a dual function marker/reporter system, although much of the available biochemical information on CAT comes from the CATm that is specified by plasmid R387, a non-F enteric plasmid. 

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The neurotransmitter must be present in presynaptic nerve terminals and the precursors and enzymes necessary for its synthesis must be present in the neuron. For example, ACh is stored in vesicles specifically in cholinergic nerve terminals. It is synthesized from choline and acetyl-coenzyme A (acetyl-CoA) by the enzyme, choline acetyltransferase. Choline is taken up by a high affinity transporter specific to cholinergic nerve terminals. Choline uptake appears to be the rate-limiting step in ACh synthesis, and is regulated to keep pace with demands for the neurotransmitter. Dopamine [51 -61-6] (2) is synthesized from tyrosine by tyrosine hydroxylase, which converts tyrosine to L-dopa (3,4-dihydroxy-L-phenylalanine) (3), and dopa decarboxylase, which converts L-dopa to dopamine. 

Acetylcholine. Acetylcholiae (ACh) (1) is a crystalliae material that is very soluble ia water and alcohol. ACh, synthesized by the enzyme choline acetyltransferase (3), iateracts with two main classes of receptor ia mammals muscarinic (mAChR), defiaed oa the basis of the agonist activity of the alkaloid muscarine (4), and nicotinic (nAChR), based on the agonist activity of nicotine (5) (Table 1). m AChRs are GPCRs (21) n AChRs are LGICs (22). 

E Wolf, A Vassilev, Y Makmo, A Sail, Y Nakatam, SK Burley. Crystal structure of a GCN5-related N-acetyltransferase Serratia marcescens aminoglycoside 3-N-acetyltransferase. Cell 94 51-61, 1998. 

Ketone body synthesis occurs only in the mitochondrial matrix. The reactions responsible for the formation of ketone bodies are shown in Figure 24.28. The first reaction—the condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA—is catalyzed by thiolase, which is also known as acetoacetyl-CoA thiolase or acetyl-CoA acetyltransferase. This is the same enzyme that carries out the thiolase reaction in /3-oxidation, but here it runs in reverse. The second reaction adds another molecule of acetyl-CoA to give (i-hydroxy-(i-methyl-glutaryl-CoA, commonly abbreviated HMG-CoA. These two mitochondrial matrix reactions are analogous to the first two steps in cholesterol biosynthesis, a cytosolic process, as we shall see in Chapter 25. HMG-CoA is converted to acetoacetate and acetyl-CoA by the action of HMG-CoA lyase in a mixed aldol-Claisen ester cleavage reaction. This reaction is mechanistically similar to the reverse of the citrate synthase reaction in the TCA cycle. A membrane-bound enzyme, /3-hydroxybutyrate dehydrogenase, then can reduce acetoacetate to /3-hydroxybutyrate. 

Step 1 of Figure 27.7 Claisen Condensation The first step in mevalonate biosynthesis is a Claisen condensation (Section 23.7) to yield acetoacetyl CoA, a reaction catalyzed by acetoacetyl-CoA acetyltransferase. An acetyl group is first bound to the enzyme by a nucleophilic acyl substitution reaction with a cysteine —SH group. Formation of an enolate ion from a second molecule of acetyl CoA, followed by Claisen condensation, then yields the product. 

Acetyltransferase is an enzyme that catalyses the transfer of an acetyl group from one substance to another. 

JV-Acetyltransferases (NATs) catalyze the conjugation of an acetyl group from acetyl-CoA on to an amine, hydrazine or hydroxylamine moiety of an aromatic compound. NATs are involved in a variety of phase II-diug metabolizing processes. There are two isozymes NAT I and NAT II, which possess different substrate specificity profiles. The genes encoding NAT I and NAT II are both multi-allelic. Especially for NAT II, genetic polymoiphisms have been shown to result in different phenotypes (e.g., fast and slow acetylators). 

The preferred substrates of acetyltransferases are amino-groups of antibiotics, like chloramphenicol, strepto-gramin derivatives, and the various aminoglycosides. The modification is believed to block a functional group involved in the drug-target-interaction. All acetyltransferases use acetyl-coenzyme A as cofactor. 

The major mechanism of resistance to chloramphenicol is mediated by the chloramphenicol acetyltransferases (CAT enzymes) which transfer one or two acetyl groups to one molecule of chloramphenicol. While the CAT enzymes share a common mechanism, different molecular classes can be discriminated. The corresponding genes are frequently located on integron-like structures and are widely distributed among Gramnegative and - positive bacteria. 

Enzymes transferring an acetyl moiety to one specific of several amino-groups of the aminocyclitol-aminoglycoside antibiotics (e.g. gentamicin, amikacin, kanamycin) are called aminoglycoside acetyltransferases... 

The metabolism of foreign compounds (xenobiotics) often takes place in two consecutive reactions, classically referred to as phases one and two. Phase I is a functionalization of the lipophilic compound that can be used to attach a conjugate in Phase II. The conjugated product is usually sufficiently water-soluble to be excretable into the urine. The most important biotransformations of Phase I are aromatic and aliphatic hydroxylations catalyzed by cytochromes P450. Other Phase I enzymes are for example epoxide hydrolases or carboxylesterases. Typical Phase II enzymes are UDP-glucuronosyltrans-ferases, sulfotransferases, N-acetyltransferases and methyltransferases e.g. thiopurin S-methyltransferase. 

A/-acetyltransferase 2 Low activity in about 60% of Caucasian populations. High incidence of adverse events from the drug isoniazide in slow acetylators. 

Coactivators enhancing the transcriptional activity of steroid hormone receptors activators include SRC-1 (steroid-receptor co-activator 1) or TEF2 (transcriptional intermediary factor 2), which are recruited by the DNA/ steroid hormone receptor complex. Their main role is to attract other transcriptional coactivators with histone acetyltransferase activity in order to decondense chromatin and allow for the binding of components of the general transcription apparatus. 

Figure 3. Mitochondrial fatty acid oxidation. Long-chain fatty acids are converted to their CoA-esters as described in the text, and their fatty-acyl-groups transferred to CoA in the matrix by the concerted action of CPT 1, the acylcarnitine/carnitine exchange carrier and CPT (A) as described in the text. Medium-chain and short-chain fatty acids (Cg or less) diffuse directly into the matrix where they are converted to their acyl-CoA esters by a acyl-CoA synthase. The mechanism of p-oxidation is shown below (B). Each cycle of P-oxidation removes -CH2-CH2- as an acetyl unit until the fatty acids are completely converted to acetyl-CoA. The enzymes catalyzing each stage of P-oxidation have different but overlapping specificities. In muscle mitochondria, most acetyl-CoA is oxidized to CO2 and H2O by the citrate cycle (Figure 4) some is converted to acylcamitine by carnitine acetyltransferase (associated with the inner face of the inner membrane) and exported from the matrix. Some acetyl-CoA (if in excess) is hydrolyzed to acetate and CoASH by acetyl-CoA hydrolase in the matrix. Enzymes ...

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