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One Enzyme (one + enzyme)
Selected AbstractsInvestigation of the Substrate Specificity of Lacticin 481 Synthetase by Using Nonproteinogenic Amino AcidsCHEMBIOCHEM, Issue 5 2009Matthew R. Levengood Abstract One enzyme, many substrates. The substrate specificity of a lantibiotic biosynthetic enzyme, lacticin 481 synthetase, was probed by using synthetic prepeptides containing a variety of nonproteinogenic amino acids, including unnatural ,-amino acids, ,-amino acids, D -amino acids, and peptoids. Lantibiotics are peptide antimicrobial compounds that are characterized by the thioether-bridged amino acids lanthionine and methyllanthionine. For lacticin 481, these structures are installed in a two-step post-translational modification process by a bifunctional enzyme, lacticin 481 synthetase (LctM). LctM catalyzes the dehydration of Ser and Thr residues to generate dehydroalanine or dehydrobutyrine, respectively, and the subsequent intramolecular regio- and stereospecific Michael-type addition of cysteines onto the dehydroamino acids. In this study, semisynthetic substrates containing nonproteinogenic amino acids were prepared by expressed protein ligation and [3+2]-cycloaddition of azide and alkyne-functionalized peptides. LctM demonstrated broad substrate specificity toward substrates containing ,-amino acids, D -amino acids, and N -alkyl amino acids (peptoids) in certain regions of its peptide substrate. These findings showcase its promise for use in lantibiotic and peptide-engineering applications, whereby nonproteinogenic amino acids might impart improved stability or modulated biological activities. Furthermore, LctM permitted the incorporation of an alkyne-containing amino acid that can be utilized for the site-selective modification of mature lantibiotics and used in target identification. [source] A gene duplication led to specialized ,-aminobutyrate and ,-alanine aminotransferase in yeastFEBS JOURNAL, Issue 7 2007Gorm Andersen In humans, ,-alanine (BAL) and the neurotransmitter ,-aminobutyrate (GABA) are transaminated by a single aminotransferase enzyme. Apparently, yeast originally also had a single enzyme, but the corresponding gene was duplicated in the Saccharomyces kluyveri lineage. SkUGA1 encodes a homologue of Saccharomyces cerevisiae GABA aminotransferase, and SkPYD4 encodes an enzyme involved in both BAL and GABA transamination. SkPYD4 and SkUGA1 as well as S. cerevisiaeUGA1 and Schizosaccharomyces pombeUGA1 were subcloned, over-expressed and purified. One discontinuous and two continuous coupled assays were used to characterize the substrate specificity and kinetic parameters of the four enzymes. It was found that the cofactor pyridoxal 5,-phosphate is needed for enzymatic activity and ,-ketoglutarate, and not pyruvate, as the amino group acceptor. SkPyd4p preferentially uses BAL as the amino group donor (Vmax/Km = 0.78 U·mg,1·mm,1), but can also use GABA (Vmax/Km = 0.42 U·mg,1·mm,1), while SkUga1p only uses GABA (Vmax/Km = 4.01 U·mg,1·mm,1). SpUga1p and ScUga1p transaminate only GABA and not BAL. While mammals degrade BAL and GABA with only one enzyme, but in different tissues, S. kluyveri and related yeasts have two different genes/enzymes to apparently ,distinguish' between the two reactions in a single cell. It is likely that upon duplication ,200 million years ago, a specialized Uga1p evolved into a ,novel' transaminase enzyme with broader substrate specificity. [source] Inhibition kinetic and mechanism of polyphenol oxidase from various sources by diethyldithiocarbamic acidINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 7 2008Serap Do Summary Inhibition kinetics and mechanism of polyphenol oxidases (PPO) partially purified from various sources such as Thymbra spicata L. var. spicata and Ocimum basilicum L., and of mushroom PPO bought from Sigma by diethyldithiocarbamic acid have been described using catechol, 4-methylcatechol and pyrogallol as substrates. The inhibition type was competitive for O. basilicum L. PPO using catechol and 4-methylcatechol as substrates, for mushroom PPO using catechol, 4-methylcatechol and pyrogallol as substrates, and for T. spicata L. var. spicata PPO using 4-methylcatechol as a substrate; uncompetitive inhibition for T. spicata L. var. spicata PPO using pyrogallol as a substrate; and non-competitive inhibition for O. basilicum L. and T. spicata L. var. spicata PPO using pyrogallol and catechol as substrates, respectively. The inhibition effect of diethyldithiocarbamic acid on enzymatic browning varied greatly from one phenol to another and from one enzyme to another. Hence, no general rule can easily be established with regard to the type of inhibition observed. [source] Promiscuous Zinc-Dependent Acylase-Mediated One-Pot Synthesis of Monosaccharide-Containing Pyrimidine Derivatives in Organic MediumADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 11-12 2009Qi Wu Abstract A facile one-pot synthesis route to monosaccharide-containing pyrimidine derivatives was developed by combining the two types of catalytic activities of one enzyme in an organic medium, i.e., the Michael addition/acylation activities of zinc-dependent D -aminoacylase (DA) from Escherichia coli. First, the stepwise approach was investigated. DA showed higher activity towards the Michael addition than acylation in this reaction system. The enzymatic Michael additions of pyrimidines to vinyl acrylate proceeded very rapidly and the initial reaction rates for the Michael addition of pyrimidines to vinyl acrylate were 7.2,16.5,mM,min,1. The catalytic specificity of aminoacylases toward Michael addition was demonstrated by the combination of different control experiments. Then, the two steps could be performed in one pot and a single aminoacylase catalyzed one-pot biotransformation was constructed. Using this strategy, a series of saccharide-pyrimidine complexes with potentially biological and pharmacological applications was prepared efficiently. This high Michael addition activity of zinc-dependent aminoacylases and the novel single aminoacylase-catalyzed one-pot synthesis combining two catalytic activities in vitro is of practical significance in expanding the application of enzymes and in the evolution of new biocatalysts. [source] Photoreactivation in Paramecium tetraurelia under Conditions of Various Degrees of Ozone Layer Depletion,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2005Akihisa Takahashi ABSTRACT Photoreactivation (PR) is an efficient survival mechanism that helps protect cells against the harmful effects of solar-ultraviolet (UV) radiation. The PR mechanism involves photolyase, just one enzyme, and can repair DNA damage, such as cyclobutane-pyrimidine dimers (CPD) induced by near-UV/blue light, a component of sunlight. Although the balance of near-UV/blue light and far-UV light reaching the Earth's surface could be altered by the atmospheric ozone layer's depletion, experiments simulating this environmental change and its possible effects on life have not yet been performed. To quantify the strength of UVB in sunlight reaching the Earth's surface, we measured the number of CPD generated in plasmid DNA after UVB irradiation or exposure to sunlight. To simulate the increase of solar-UV radiation resulting from the ozone layer depletion, Paramecium tetraurelia was exposed to UVB and/or sunlight in clear summer weather. PR recovery after exposure to sunlight was complete at a low dose rate of 0.2 J/m2·s, but was less efficient when the dose rate was increased by a factor of 2.5 to 0.5 J/m2·s. It is suggested that solar-UV radiation would not influence the cell growth of P. tetraurelia for the reason of high PR activity even when the ozone concentration was decreased 30% from the present levels. [source] Evidence for two-step regulation of pheromone biosynthesis by the pheromone biosynthesis-activating neuropeptide in the moth Heliothis virescens,ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2007H. Eltahlawy Abstract The control of pheromone biosynthesis by the neuropeptide PBAN was investigated in the moth Heliothis virescens. When decapitated females were injected with [2- 14C] acetate, females co-injected with PBAN produced significantly greater quantities of radiolabeled fatty acids in their pheromone gland than females co-injected with saline. This indicates that PBAN controls an enzyme involved in the synthesis of fatty acids, probably acetyl CoA carboxylase. Decapitated females injected with PBAN showed a rapid increase in native pheromone, and a slower increase in the pheromone precursor, (Z)-11-hexadecenoate. Total native palmitate and stearate (both pheromone intermediates) showed a significant decrease after PBAN injection, before their titers were later restored to initial levels. In contrast, the acyl-CoA thioesters of these two saturated fatty acids increased during the period when their total titers decreased. When a mixture of labeled palmitic and heptadecanoic (an acid that cannot be converted to pheromone) acids was applied to the gland, PBAN-injected females produced greater quantities of labeled pheromone and precursor than did saline-injected ones. The two acids showed similar time-course patterns, with no difference in total titers of each of the respective acids between saline- and PBAN-injected females. When labeled heptadecanoic acid was applied to the gland alone, there was no difference in titers of either total heptadecanoate or of heptadecanoyl-CoA between PBAN- and saline-injected females, suggesting that PBAN does not directly control the storage or liberation of fatty acids in the gland, at least for this fatty acid. Overall, these data indicate that PBAN also controls a later step involved in pheromone biosynthesis, perhaps the reduction of acyl-CoA moieties. The control by PBAN of two enzymes, near the beginning and end of the pheromone biosynthetic process, would seem to allow for more efficient utilization of fatty acids and pheromone than control of only one enzyme. Arch. Insect Biochem. Physiol. 64:120,130, 2007. Published 2007 Wiley-Liss, Inc. [source] | ||||||||||