Poly synthetase

A new pathway was recently engineered in vitro employing purified His6-tag-ged PhaCl and PhaC2 from P. aeruginosa plus a commercially available acyl-CoA synthetase allowing in vitro de novo PHA granules and in vitro synthesis of poly(3HD) from 3-hydroxydecanoate [74]. 

Fig. 1. The metabolic cycle for the synthesis and degradation of poly(3HB). (1) 3-ketothiolase (2) NADPH-dependent acetoacetyl-CoA reductase (3) poly(3HB) synthase (4) NADH-dependent acetoacetyl-CoA reductase (5), (6) enolases (7) depolymerase (8) d-(-)-3-hydroxybutyrate dehydrogenase (9) acetoacetyl-CoA synthetase (10) succinyl-CoA transferase (11) citrate synthase (12) see Sect. 3...

Banasik M, Komura H, Shimoyama M, Ueda K (1992) Specific inhibitors of poly(ADP-ribose) synthetase and mono(ADP-ribosyl)transferase. J Biol Chem 267 1569-1575... 

Lin SJ, Defossez PA, Guarente L (2000) Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae. Science 289 2126—2128 Luo J, Nikolaev AY, Imai S, Chen D, Su F, Shiloh A, Guarente L, Gu W (2001) Negative control of p53 by Sir2alpha promotes cell survival under stress. Cell 107 137-148 Mabley JG, Suarez-Pinzon WL, Hasko G, Salzman AL, Rabinovitch A, Kun E, Szabo C (2001) Inhibition of poly (ADP-ribose) synthetase by gene disruption or inhibition with 5-iodo-6-amino-l,2-benzopyrone protects mice from multiple-low-dose-streptozotocin-induced diabetes. Br J Pharmacol 133 909-919... 

Szabo C (1998) Role of poly(ADP-ribose)synthetase in inflammation. Eur J Pharmacol 350 1-19 Tanuma S, Johnson GS (1983) ADP-ribosylation of nonhistone high mobility group proteins in intact cells. J Biol Chem 258 4067-4070... 

Zingarelli B, Salzman AL, Szabo C (1998) Genetic disruption of poly (ADP-ribose) synthetase inhibits the expression of P-selectin and intercellular adhesion molecule-1 in myocardial ischemia/reperfusion injury. Circ Res 83 85-94... 

The enzyme catalyzing the addition of ADP-ribose units onto the histones and itself is poly(ADP-ribose) polymerase or synthetase. Poly(ADP-ribose) polymerase is a nuclear, DNA-dependent enzyme that is stimulated by DNA breaks [302]. This property of the enzyme would target its action to sites that have DNA strand breaks (regions of the genome involved in replication, repair, recombination). The enzyme is associated with chromatin areas and perichromatin regions in interphase Chinese hamster ovary cells [312]. Degradation of the ADP-ribose polymer is catalyzed by the nuclear enzyme poly(ADP-ribose) glycohydrolase and ADP-ribosyl protein lyase. 

This enzyme [EC 6.3.2.17], which is also known as folyl-poly-y-glutamate synthetase, catalyzes the sequential ATP-dependent addition of glutamyl groups onto tetra-hydrofolyl-[Glu] to yield tetrahydrofolyl-[Glu] +i plus ADP and orthophosphate. 

NAD glycohydrolases from rat liver nuclei, 66, 151 poly(ADP-ribose) synthetase from rat liver nuclei, 66, 154 poly(ADP-ribose) synthetase from calf thymus, 66, 159 extraction and quantitative determination of larger than tetrameric endogenous polyadenosine diphosphoribose from animal tissues, 66, 165 covalent modification of proteins by metabolites of NAD, 66, 168 coenzyme activity of NAD bound to polymer supports through the adenine moiety, 66, 176 use of differently immobilized nucleotides for binding NAD -dependent dehydrogenases, 66, 192. 

OCTANOL DEHYDROGENASE OCTOPINE DEHYDROGENASE PHENYLALANINE DEHYDROGENASE POLY(ADP-RIBOSE) SYNTHETASE (or, SYNTHASE)... 

The enzyme catalyzing the transfer of D-apiose from UDP-apiose to 4, 5-dihydroxyflavon-7-yl /3-D-glucopyranoside is commonly called apiin synthetase.7 Activity is measured by the formation of [14C]apiin from UDP-[U-14C]apiose. Apiin synthetase can be measured by the rapid separation and isolation, by poly(ethylenimine)-paper chromatography, of a product of the reaction, namely, [14C] apiin, from UDP-D-[U-14C]xylose and degradation products of UDP-[U-14C]apiose.31 There are reports of the isolation and purification of apiin synthetase from parsley leaves,31 from cell-suspension cultures of parsley,121 and from foxglove (Digitalis purpurea).31 Apiin synthetase isolated from parsley does not require metal ions, NAD+, or other cofactors, and is soluble. It is inhibited by several heavy metals, but not by tetra-N-... 

Poly(ribitol phosphate) synthetase has been found in particulate fractions from Staphylococcus aureus H, and Lactobacillus plantatrum.lt ll-m The bulk of the activity in Lactobacillus plantarum was in crude, cell-wall preparations, and the enzyme is apparently located in the membrane, although intimate association with the wall itself has been suggested. Unlike the natural teichoic acid, the enzymically synthesized ribitol phosphate polymer was readily extracted with phenol hydrolysis by acid and by alkali gave the expected products, and oxidation with periodate indicated a chain length of 5-9 units, a value which compares well with that of 8 units for the natural polymer in the walls of this organism. 

The poly (ribitol phosphate) synthetase and poly (glycerol phosphate) synthetase are inhibited by vancomycin, novobiocin, and Crystal Violet. Other antibiotic substances which interfere with cell-wall synthesis (such as bacitracin, ristocetin, and streptomycin) are almost without effect on the isolated synthetases, and penicillin is inhibitory at high concentrations only. Moreover, penicillin, vancomycin, and bacitracin do not markedly inhibit synthesis of cell-wall glycosaminopeptide in vitro, although the synthetical activity of extracts of cells which have been pretreated with these antibiotics is lowered.Convincing evidence that the primary site of inhibition by antibiotics is the biosynthesis of cell-wall material has been obtained only for the penicillins and cycloserine, and it appears that the action of even those antibiotics may be more complex than was originally supposed. 

Biosynthetic processes Elongation of RNA or DNA chains Other synthetic routes RNA polymerase I DNA polymerase Poly(ADP-ribose)polymerase Carbamoyl phosphate synthetase Anthranilate synthase-phosphoribosyl transferase Glycogen synthetase Methionyl-tRNA synthetase ATP phosphoribosyl transferase Zn2+... 

Zhang J., Dawson V. L., Dawson T. M., and Snyder S. H. (1994). Nitric oxide activation of poly(ADP-ribose) synthetase in neurotoxicity. Science 263 687-689. 

As beta-lactam antibiotics continue to be a major contributor to human health preservation, research on the biosynthesis of penicillin, an almost ancient drug, continues to open up roads to new technologies and perspectives. The provision of precursor peptides to be transformed enzymatically with chemically unachieved efficiency into mono- or bicyclic antibiotics has been termed by Jack Baldwin and colleagues the irreversible commitment of metabolic carbon to the secondary metabolism [1]. The synthesis of such peptides is indeed performed by a remarkable class of synthetases which, in contrast to the protein-synthesizing machinery, have been termed a nonribosomal system or nonribosomal peptide synthetases (NRPS) [2]. These peptide synthetases have been shown to catalyze the irreversible synthesis of peptides differing both in sequence and stmctural variability, thus extending the scope of directly gene-encoded poly-... 

The copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-co-PHV) produced by A eutrophus has generated more interest than poly-(R)-3-hydroxybutyrate (PHB) homopolymer. Since these bacterial polyesters are biodegradable thermoplastics, their mechanical and physical properties have received much attention. PHB is a relatively stiff and brittle material because of its high crystallinity. However, the physiochemi-cal and mechanical properties of [P(HB-HV)] vary widely and depend on the molar percentage of 3-hydroxyvalerate (HV) in the copolymer (4,5) as shown inTable 1. Propionic acid is converted by a synthetase to propionyl-CoA, and the biosynthetic P-ketothiolase catalyzes the condensation of propionyl-CoA with acetyl-CoA to 3-ketovaleryl-CoA by the acetoacetyl-CoA reductase. The hydroxyvaleryl moiety is finally covalently linked to the polyester by the PHA synthase (6). 

The biological function of ADP-ribosylated nuclear proteins is not clear. Poly-(ADP-ribose) synthetase has been reported to be stimulated by histones, but histones themselves do not serve as acceptors in vitro (42). The endogenous acceptor iji vivo might involve proteins other than histones. Recently an enzyme that cleaves the ADP-ribosyl histone linkage has been purified from rat liver (43). 

Fig. 9.11. Organizational overview and summary of protein engineering efforts on the poly-ketide synthetase (PKS) and nonribosomal peptide synthetase (NRPS) framework, a) In both systems, the complete synthetases consist of multiple subunits encoded on individual genes. The subunits themselves are divided in modules which each catalyze the addition of one acylbuilding block (PKS) or amino acid (NRPS). The minimal module for PKSs is made up of three domains - a keto synthase (KS), an acyl transferase (AT), and an acyl carrier protein (ACP). In addition, modules can contain up to three modifying domains to derivatize (S-carbons on the...

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