Multifunctional enzyme type

Like all other retroviruses, human immunodeficiency virus type 1 (HIV-1) contains the multifunctional enzyme reverse transcriptase (RT). Retroviral RTs have a DNA polymerase activity that can use either an RNA or a DNA template and an RNase H activity. HIV-1 RT is essential for the conversion of single-stranded viral RNA into a linear double-stranded DNA that is subsequently integrated into the host cell chromosomes [1-4]. In this conversion process HIV-1 RT catalyzes the incorporation of approximately... 

Chen, M.C. Walker, J. Prusoff, W.H. Kinetic studies of herpes simplex virus type 1-encoded thymidine and thymidylate kinase, a multifunctional enzyme. J. Biol. Chem., 254, 10747-10753 (1979)... 

However, this is only one of three types of reactions catalyzed by DNA polymerase I. Its two other activities involve the hydrolysis of phosphodiester bonds. One is a 5 — 3 exonuclease acting on double-stranded DNA, and the other is a 3 - 5 exonuclease acting on a frayed or mismatched terminus of double-stranded DNA. DNA polymerase I is thus a multifunctional enzyme. It consists of a single... 

DNA polymerase III is also a multifunctional enzyme. It resembles DNA polymerase I in catalytic properties however, there are slight differences with respect to the type of template primer preferred for DNA synthesis as well as the preferred substrates for the two exonuclease activities. It contains many polypeptide subunits (a, e, 6, t, y, 8. y, ip and B)- The complex containing all subunits (Mr 900,000) is called the DNA polymerase III holoenzyme, while that comprising just a, e and 8 exhibits the polymerase activity and is referred to as the core enzyme. The holoenzyme carries out most of the DNA synthesis at the replication fork in vivo. 

EPHXs are important multifunctional enzymes from both the deactivation and activation of reactive species. Furthermore, they convert any potentially reactive epoxide formed by the P450s system into a diol metabolite, which is usually less reactive, more water soluble, and more easily cleared by GSTs. There are two major types of EPHX enzymes the microsomal (mEPHX), which uses epoxides of polycyclic aromatics or drugs as substrates (type 1) and which controls hepatic uptake of bile acids (type 2) [67], and the soluble EPHX (sEPHX), which forms diols from many endogenous and exogenous epoxides, including fatty acids and leukotrienes [68],... 

The polyketide synthesis chemically and biochemically resembles that of fatty acids. The reaction of fatty acid synthesis is inhibited by the fungal product ceru-lenin [9]. It inhibits all known types of fatty acid synthases, both multifunctional enzyme complex and unassociated enzyme from different sources like that of some bacteria, yeast, plants, and mammalians [10]. Cerulenin also blocks synthesis of polyketides in a wide variety of organisms, including actinomycetes, fungi, and plants [11, 12]. The inhibition of fatty acid synthesis by cerulenin is based on binding to the cysteine residue in the condensation reaction domain [13]. Synthesis of both polyketide and fatty acids is initiated by a Claisen condensation reaction between a starter carboxylic acid and a dicarboxylic acid such as malonic or methylmalonic acid. An example of this type of synthesis is shown in Fig. 1. An acetate and malonate as enzyme-linked thioesters are used as starter and extender, respectively. The starter unit is linked through a thioester linkage to the cysteine residue in the active site of the enzymatic unit, p-ketoacyl ACP synthase (KS), which catalyzes the condensation reaction. On the other hand, the extender... 

This chapter focuses on the catalytic transformations that result in the cyclic biosynthesis and breakdown of fatty acids. These metabolic pathways will serve as a paradigm for three classes of chemical reactions carbon-carbon bond formation and cleavage, oxidation and reduction, and hydration—dehydration. The most extensively studied reactions are those involved in microbial fatty acid biosynthesis (Type II fatty acid synthase (FAS-II)) and mammalian fatty acid /3-oxidation. In both pathways, the reactions are catalyzed by separate enzymes that have been cloned and overexpressed, thus providing a ready source of material for structural and mechanistic studies. In contrast, mammalian fatty acid biosynthesis and microbial fatty acid breakdown are catalyzed by multifunctional enzymes (MFEs) that have historically been less amenable to analysis. 

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. 

Zocher R, Keller U, Kleinkauf H. Enniatin synthetase, a novel type of multifunctional enzyme catalyzing depsipeptide synthesis in FusoWum oxysporum. Biochemistry 1982 21 43-48. 

Zocher R, Keller U, Kleinkauf H. Enniatin synthetase, a novel type of multifunctional enzyme catalyzing depsipeptide synthesis in Fusarium oxysporum. Biochem 1982 21 43-48. Zocher R, Nihira T, Paul E, Madry N, Pecters H, Kleinkauf H. Keller U. Biosynthesis of cyclosporin A Partial purification and properties of a mulcifunctional enzyme from Tolypocla-diutn mjkitum. Biochem 1986 25 550-553. 

Acetyl-CoA carboxylase (ACCase) catalyses the ATP-dependant carboxylation of acetyl-CoA to form malonyl-CoA, thus providing the essential substrate for fatty acid biosynthesis. Dicotyledonous plants contain two forms of ACCase a multifunctional enzyme (typel) wich is presumed to be cytosolic, and a multi-subunit complex (typell) located in the plastid wich is responsible for de novo fatty acid synthesis. In prokaryotes, the ACCase is a type II enzyme comprising biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP) and a carboxyl transferase with two subunits (CTa and CTp). The cDNA encoding the B.napus CXp and BCCP have already been cloned (Elborough et a/.,1995) as have the cDNAs encoding the BC and BCCP from tobacco and Arabidopsis respectively (Shorrosh et al, 1995 Choi et al.y 1995). 

Wilson B.J., Ramstad, E., Jansson, I. and Orrenius, S. (1971) Conversion of agroclavine by mammalian cytochrome P450. Biochim. Biophys. Acta, 252, 348-356. Zocher, R., Keller, U. and Kleinkauf, H. (1982) Enniatin synthetase, a novel type of multifunctional enzyme catalyzing depsipeptide synthesis in Fusarium oxysporum. Biochemistry, 21,43—48. 

There are some known inhibitors of fatty acid synthesis. Cerulenin inhibits KAS I completely and irreversibly at 20 j.M [5] and inhibits KAS II but at higher concentrations. Cerulenin also inhibits the condensing enzyme function of multifunctional protein type I fatty acid synthases [6]. For all these enzymes the mechanism of action Is similar, involving the covalent binding of the inhibitor to the cysteine of the active site of the protein [7]. Arsenite, an inhibitor of enzymes containing vicinal thiol groups, inhibits KAS II but no other condensing enzyme. KAS III is insensitive to inhibition by either cerulenin or arsenite but its activity is inhibited by thiolactomycin (TLM). This inhibition has been seen in bacteria [8] and plants [9] but is rather variable as reported in the literature [10-14]. 

Exonuclease III (Exo III) of E. coli is a monomeric multifunctional enzyme (31 kDa) that catalyzes the hydrolysis of at least four different types of phosphoester bonds in dsDNA (Fig. 3.4). The main enzymatic activity of Exo III is the 3 — 5 -exonuclease activity that carries out the successive release of 5 -P-mononucleotides from the 3 ends of dsDNA. The second activity is the DNA 3 -phosphatase activity that hydrolyzes 3 -terminal phosphomonoesters. In fact, Exo III was initially discovered as a DNA 3 -phosphatase in E. coli (1,2). Exo III has a third activity which degrades the RNA strand in a DNA RNA heteroduplex, thus the RNase H activity. The fourth activity of Exo III is an AP endonuclease which cleaves phosphodiester bonds at apurinic or apyrimidinic sites. 

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