Poly substrate specificity

The 3-ketothiolase has been purified and investigated from several poly(3HB)-synthesizing bacteria including Azotobacter beijerinckii [10], Ral-stonia eutropha [11], Zoogloea ramigera [12], Rhodococcus ruber [13], and Methylobacterium rhodesianum [14]. In R. eutropha the 3-ketothiolase occurs in two different forms, called A and B, which have different substrate specificities [11,15]. In the thiolytic reaction, enzyme A is only active with C4 and C5 3-ketoacyl-CoA whereas the substrate spectrum of enzyme B is much broader, since it is active with C4 to C10 substrates [11]. Enzyme A seems to be the main biosynthetic enzyme acting in the poly(3HB) synthesis pathway, while enzyme B should rather have a catabolic function in fatty-acid metabolism. However, in vitro studies with reconstituted purified enzyme systems have demonstrated that enzyme B can also contribute to poly(3HB) synthesis [15]. 

As it has been pointed out above, poly(HA)-degrading microorganisms, in particular the poly(HA)-hydrolyzing enzymes, differ highly in their substrate specificities for various poly(HA). Furthermore the physico-chemistry of the polymer itself also has a strong impact on its biodegradability. The most important... 

The i-poly(3HB) depolymerase of R. rubrum is the only i-poly(3HB) depolymerase that has been purified [174]. The enzyme consists of one polypeptide of 30-32 kDa and has a pH and temperature optimum of pH 9 and 55 °C, respectively. A specific activity of 4 mmol released 3-hydroxybutyrate/min x mg protein was determined (at 45 °C). The purified enzyme was inactive with denatured poly(3HB) and had no lipase-, protease-, or esterase activity with p-nitro-phenyl fatty acid esters (2-8 carbon atoms). Native poly(3HO) granules were not hydrolyzed by i-poly(3HB) depolymerase, indicating a high substrate specificity similar to extracellular poly(3HB) depolymerases. Recently, the DNA sequence of the i-poly(3HB) depolymerase of R. eutropha was published (AB07612). Surprisingly, the DNA-deduced amino acid sequence (47.3 kDa) did not contain a lipase box fingerprint. A more detailed investigation of the structure and function of bacterial i-poly(HA) depolymerases will be necessary in future. 

Only a few bacterial and viral sialidases have been purified to high purity or even to protein homogeneity."0 Complete purification of sia-lidase on a preparative scale from the culture filtrate ofC. perfringens was achieved111 by using poly(acrylamide) gel-electrophoresis as the final purification step (see Section VI,1). It is necessary that such purified sialidases be available, as the presence of proteases or other gly-cosidases in the enzyme preparations would lead to severe errors, not only in studies of substrate specificity, but also in cell biological and medical studies (see Sections VI and VII). 

Lipases are of remarkable practical interest since they have been used in numerous biocatalytic applications, such as kinetic resolution of alcohols and carboxyl esters (both in water and in non-aqueous media) [1], regioselective acylations of poly-hydroxylated compounds, and the preparation of enantiopure amino acids and amides [2, 3]. Moreover, lipases are stable in organic solvents, do not require cofactors, possess broad substrate specificity, and exhibit, in general, a high enantioselectivity. All these features have contributed to make hpases the class of enzyme with the highest number of biocatalytic applications carried out in neat organic solvents. 

For HSV at least three mechanisms have been described that generate resistance to AC V deficiency or loss of viral TK activity, alteration in substrate specificity of the virus-encoded TK, and alteration in the substrate specificity of the viral DNA polymerase (1,8). Most of the ACVr mutants that have been isolated in vitro and recovered from clinical specimens are TK-deficient (TK). However, resistant clinical mutants that have an altered TK or altered DNA polymerase activity have occasionally been described too. Although TK mutants are crossresistant with drugs that also depend on viral TK for their activation (i.e., GCV, penciclovir and brivudin (BVDU), they remain sensitive to agents, such as PFA, vidarabine (Ara-A), and the acyclic nucleoside phosphonate (ANP) analogs. PFA, a pyrophosphate analog, is a direct inhibitor of the viral DNA poly-merase in which it binds to the site involved in releasing the pyrophosphate product of DNA synthesis. Phosphorylation of Ara-A to Ara-A triphosphate is carried out by cellular enzymes phosphorylation of ANP derivatives to their mono- and diphosphoryl derivatives is also carried out by cellular enzymes. 

It has been suggested that carboxyl ester lipase has two different bile-salt-binding sites [34-36], one nonspecific for the bile salt structure and one specific for primary bile salts causing the di(poly)merization. An interesting question is whether the bile salts regulate substrate specificity not only by bile salt-enzyme interactions but also by forming the appropriate substrate surface structure [27]. 

Further development of the SLM-ionic liquid methodology has been coupled with lipase-catalyzed esterification and ester-hydrolysis reactions in the feed gas (interface 1) and receiving phase (interface 2), respectively, to facilitate selective transport of various organic acids with aryl groups (via their esters) from aqueous solutions by utilizing different substrate specificity of lipases in a SLM system (Fig. 5.6-11) [118]. In the enzymatic SLM systems a poly(propene) membrane with water-immiscible [RMIM][X] (R = butyl, hexyl, octyl and X = [PFe]" and [(CF3S02)2N] ) ionic liquid phases were used. 

Chen GQ, Zhang G, Park SJ, Lee SJ (2001) Industrial production of poly(hydroxybutyrate-co-hydroxyhexanoate). Appl Microbiol Biotechnol 57 50-55 Chen JY, Liu T, Zheng Z, Chen JC, Chen GQ (2004) PolyhydroxyaUcanoate synthases PhaCl rmd PhaC2 from Pseudomonas stutzeri 1317 had different substrate specificities. FEMS Microbiol Lett 234 231-237... 

Arias S, Sandoval A, Arcos M, Canedo LM, Maestro B, Sanz JM, Naharro G, Luengo JM (2(X)8) Poly-3-hydroxyalkanoate synthases from Pseudomonas putida U substrate specificity and ultrastructural studies. Microb Biotechnol 1 170-176 Arkin AH, Hazer B (2002) Chemical modification of chlorinated microbitil polyesters. Biomacromolecules 3 1327-1335... 

There was a breakthrough when it was found that one mutant of PHA synthase was capable of incorporating lactic acid (LA) from its CoA form, lactyl-CoA (LA-CoA), into the polymer chain [67]. PHAs containing 2HA monomers, lactic acid (LA), glycolate (GL), and 2HB can be synthesized by engineered microbes in which the broad substrate specificities of PHA synthase and propionyl-CoA transferase (PCX) are critical factors for the incorporation of the monomers into the polymer chain. LA-based polymers, such as P[LA-co-3HB], have the properties of pliability and stretchiness which are distinctly different from those of the rigid poly(lactic acid) and P(3HB) homopolymers. 

SDS-polyacrylamide gel electrophoresis. The substrate specificity was rather loose with the protein portion ADP-ribosyl histones HI and H2B, peptide fragments of H2B, and nonhistone proteins (a mixture) served as substrates. In contrast, the specificity was very tight with the mono(ADP-ribosyl) portion and the carboxyl ester bond poly- or oligo(ADP-ribosyl) histones were hardly split, and ADP-ribose histone adducts formed chemically through Schiff base reduction [3] or ADP-ribosyl arginine bond formed by avian erythrocyte ADP-ribosyltransferase [14] did not serve as substrate. 

Ribosomal protein S6 from Escherichia coli undergoes a unique post-translation modification where up to six glutamic acid residues are ligated to the C-terminus. [82-84] RimK, also a member of carboxylate-amine/thiol Ugase superfamily, mediated this post-translahonal modification. [84] In vitro analysis of RimK s)rnthesis resulted in 46-mer (maximum length) of a-poly (L-Glu) at pH 9.0, 30 °C. The maximum chain length was pH dependent. Furthermore, RimK demonstrated strict substrate specificity for Glu. [72]... 

It is weU established that the enzyme that catalyses the polymerisation of the (7()-3-hydroxyacyl CoA into poly-(/ )-3-hydroxyacyl CoA is the PHA synthase (Peoples and Sinskey, 1989). The PHA synthase enzyme can be classified into four different classes based on their substrate specificity and subunit composition as shown in Table 8.1. 

To date, at least five biotinylation sites have been identified in histones H3 [lysine (K)-4, K9, K18 and probably K23] and H4 (K8, K12 and probably K16) (Camporeale et al. 2004 Kobza et al. 2005 Kobza et al. 2008). K9 and K13 in histone H2A might also be biotinylated (Chew et al. 2006), but the abundance of these two marks appears to be very low (Stanley et al. 2001). Studies with synthetic HLCS substrates provide unambiguous evidence that biotinylation of histones by HLCS is a substrate-specific process (Hassan et al. 2009a). Histone biotinylation is a comparably rare event (<0.1% of histones are biotinylated), but the abundance of an epigenetic mark is not necessarily a marker for its importance. For example, serine-14 phosphorylation in histone H2B and histone poly(ADP-ribosylation) are detectable only after induction of apoptosis... 

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