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Albino mutants of Streptomyces glaucescens tyrosinase.

ABSTRACT: Site-directed mutagenesis was used to determine the functional role of several residues of Streptomyces glaucescens tyrosinase. Replacement of His-37, -53, -193 or -215 by glutamine yields albino phenotypes, as determined by expression on melanin-indicator plates. The purified mutant proteins display no detectable oxy-enzyme and increased Cu lability at the binuclear active site. The carbonyl derivatives of H189Q and H193Q luminesce, with lambda max. displaced more than 25 nm to a longer wavelength compared with native tyrosinase. The remaining histidine mutants display no detectable luminescence. The results are consistent with these histidine residues (together with His-62 and His-189 reported earlier) acting as Cu ligands in the Streptomyces glaucescens enzyme. Conservative substitution of the invariant Asn-190 by glutamine also gives an albino phenotype, no detectable oxy-enzyme and labilization of active-site Cu. The luminescence spectrum of carbonyl-N190Q, however, closely resembles that of the native enzyme under conditions promoting double Cu occupancy of the catalytic site. A critical role for Asn-190 in active-site hydrogen-bonding interactions is proposed.

SUBMITTER: Jackman MP

PROVIDER: S-EPMC1149969 | biostudies-other | 1991 Mar

REPOSITORIES: biostudies-other

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Enantioselective sulfoxidation using <i>Streptomyces glaucescens</i> GLA.0.

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Chromosomal deletion and rearrangement in Streptomyces glaucescens.

Project description:The Streptomyces glaucescens genome frequently undergoes gross genomic rearrangement events which result in the deletion of extremely large segments of chromosomal DNA. The structure and origin of the DNA forming the novel junctions arising from five of these deletion events are described. Only one junction proved to be the result of a relatively simple event; the remainder were more complex, with one involving DNA which originated from at least five distinct loci. In three of the investigated cases, DNA sequences present in the junctions appeared to have resulted from the duplication of previously unique sequences, suggesting that duplication of chromosomal segments may be an important factor in genetic instability. The nucleotide sequences surrounding these junctions and their respective wild-type termini were determined.

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Transcript profiling of Arabidopsis albino msh1 mutants

2011-11-29 | GSE30281 | GEO

Streptomyces glaucescens strain:GLA.O

Project description:Streptomyces glaucescens GLA.O Genome sequencing

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Purification and characterization of a nutritionally controlled endodeoxyribonuclease from Streptomyces glaucescens.

Project description:Streptomyces glaucescens has a DNAase whose synthesis is under nutritional control. We have purified this enzyme to apparent homogeneity by phosphocellulose chromatography followed by heparin-agarose, Cibacron Blue F3-GA-Sepharose and Sephadex G-75 chromatography and MonoQ f.p.l.c. The enzyme had an apparent Mr of 39,600 and a pI of approx. 8.15. The Mr of the native enzyme estimated by gel chromatography was 49,000. The DNAase had a pH optimum of 7.5 and an absolute requirement for bivalent cations in the reaction buffer. It was inhibited by high salt concentrations, chelating agents or phosphate-containing compounds and was stimulated by dimethyl sulphoxide. The activity was greatly diminished unless dithiothreitol or 2-mercaptoethanol was included in the reaction mixture. Reagents such as Hg2+ or iodoacetate strongly inhibited the enzyme. The nuclease hydrolysed both double-stranded and single-stranded DNA, showing greater affinity for double-stranded DNA, and no detectable hydrolysis of RNA. The enzyme produced nicks in double-stranded DNA, generating 3'-hydroxy and 5'-phosphate termini, and degraded circular DNA.

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Albino mice with the point mutation at the tyrosinase locus show high cholesterol diet-induced NASH susceptibility.

Project description:Non-alcoholic fatty liver disease (NAFLD) constitutes a metabolic disorder with high worldwide prevalence and increasing incidence. The inflammatory progressive state, non-alcoholic steatohepatitis (NASH), leads to liver fibrosis and carcinogenesis. Here, we evaluated whether tyrosinase mutation underlies NASH pathophysiology. Tyrosinase point-mutated B6 (Cg)-Tyrc-2J/J mice (B6 albino) and C57BL/6J black mice (B6 black) were fed with high cholesterol diet (HCD) for 10 weeks. Normal diet-fed mice served as controls. HCD-fed B6 albino exhibited high NASH susceptibility compared to B6 black, a phenotype not previously reported. Liver injury occurred in approximately 50% of B6 albino from one post HCD feeding, with elevated serum alanine aminotransferase and aspartate aminotransferase levels. NASH was induced following 2 weeks in severe-phenotypic B6 albino (sB6), but B6 black exhibited no symptoms, even after 10 weeks. HCD-fed sB6 albino showed significantly higher mortality rate. Histological analysis of the liver revealed significant inflammatory cell and lipid infiltration and severe fibrosis. Serum lipoprotein analysis revealed significantly higher chylomicron and very low-density lipoprotein levels in sB6 albino. Moreover, significantly higher small intestinal lipid absorption and lower fecal lipid excretion occurred together with elevated intestinal NPC1L1 expression. As the tyrosinase point mutation represents the only genetic difference between B6 albino and B6 black, our work will facilitate the identification of susceptible genetic factors for NASH development and expand the understanding of NASH pathophysiology.

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Novel Hypomorphic Alleles of the Mouse Tyrosinase Gene Induced by CRISPR-Cas9 Nucleases Cause Non-Albino Pigmentation Phenotypes.

Project description:Tyrosinase is a key enzyme in melanin biosynthesis. Mutations in the gene encoding tyrosinase (Tyr) cause oculocutaneous albinism (OCA1) in humans. Alleles of the Tyr gene have been useful in studying pigment biology and coat color formation. Over 100 different Tyr alleles have been reported in mice, of which ?24% are spontaneous mutations, ?60% are radiation-induced, and the remaining alleles were obtained by chemical mutagenesis and gene targeting. Therefore, most mutations were random and could not be predicted a priori. Using the CRISPR-Cas9 system, we targeted two distinct regions of exon 1 to induce pigmentation changes and used an in vivo visual phenotype along with heteroduplex mobility assays (HMA) as readouts of CRISPR-Cas9 activity. Most of the mutant alleles result in complete loss of tyrosinase activity leading to an albino phenotype. In this study, we describe two novel in-frame deletion alleles of Tyr, dhoosara (Sanskrit for gray) and chandana (Sanskrit for sandalwood). These alleles are hypomorphic and show lighter pigmentation phenotypes of the body and eyes. This study demonstrates the utility of CRISPR-Cas9 system in generating domain-specific in-frame deletions and helps gain further insights into structure-function of Tyr gene.

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Transcript profiling of Arabidopsis albino msh1 mutants

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2011-11-29 | E-GEOD-30281 | biostudies-arrayexpress

Characterization of Temperature-Dependent Kinetics of Oculocutaneous Albinism-Causing Mutants of Tyrosinase.

Project description:Human tyrosinase (Tyr) is a glycoenzyme that catalyzes the first and rate-limiting step in melanin production, and its gene (TYR) is mutated in many cases of oculocutaneous albinism type 1 (OCA1). The mechanisms by which individual mutations contribute to the diverse pigmentation phenotype in patients with OCA1 have only began to be examined and remain to be delineated. Here, we analyze the temperature-dependent kinetics of wild-type Tyr (WT) and two OCA1B mutant variants (R422Q and P406L) using Michaelis-Menten and Van't Hoff analyses. Recombinant truncated human Tyr proteins (residues 19-469) were produced in the whole insect Trichoplusia Ni larvae. Proteins were purified by a combination of affinity and size-exclusion chromatography. The temperature dependence of diphenol oxidase protein activities and kinetic parameters were measured by dopachrome absorption. Using the same experimental conditions, computational simulations were performed to assess the temperature-dependent association of L-DOPA and Tyr. Our results revealed, for the first time, that the association of L-DOPA with R422Q and P406L followed by dopachrome formation is a complex reaction supported by enthalpy and entropy forces. We show that the WT has a higher turnover number as compared with both R422Q and P406L. Elucidating the kinetics and thermodynamics of mutant variants of Tyr in OCA1B helps to understand the mechanisms by which they lower Tyr catalytic activity and to discover novel therapies for patients.

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Bioproduction, characterization, anticancer and antioxidant activities of extracellular melanin pigment produced by newly isolated microbial cell factories Streptomyces glaucescens NEAE-H.

Project description:In this present study, a newly isolated strain, Streptomyces sp. NEAE-H, capable of producing high amount of black extracellular melanin pigment on peptone-yeast extract iron agar and identified as Streptomyces glaucescens NEAE-H. Plackett-Burman statistical design was conducted for initial screening of 17 independent (assigned) variables for their significances on melanin pigment production by Streptomyces glaucescens NEAE-H. The most significant factors affecting melanin production are incubation period, protease-peptone and ferric ammonium citrate. The levels of these significant variables and their interaction effects were optimized by using face-centered central composite design. The maximum melanin production (31.650??g/0.1?ml) and tyrosinase activity (6089.10?U/ml) were achieved in the central point runs under the conditions of incubation period (6 days), protease-peptone (5?g/L) and ferric ammonium citrate (0.5?g/L). Melanin pigment was recovered by acid-treatment. Higher absorption of the purified melanin pigment was observed in the UV region at 250?nm. It appeared to have defined small spheres by scanning electron microscopy imaging. The maximum melanin yield was 350?mg dry wt/L of production medium. In vitro anticancer activity of melanin pigment was assayed against skin cancer cell line using MTT assay. The IC50 value was 16.34?±?1.31??g/ml for melanin and 8.8?±?0.5??g/ml for standard 5-fluorouracil.

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