Characterization of a superoxide dismutase gene from the archaebacterium Methanobacterium thermoautotrophicum

Takao, Masashi; Oikawa, Atsushi; Yasui, Akira

doi:10.1016/0003-9861(90)90633-a

Cited by 50 publications

(44 citation statements)

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“…Standard errors in metal determination and activity measurements were less than 5 and 10%, respectively. has been ascribed to the oxidation of an essential tryptophan residue in the vicinity of the iron binding site (4,42,46), although the actual identity of the reactive amino acid is still a matter of debate (7,27,37,45). A recombinant SOD Rc reconstituted with Mn was virtually insensitive to H 2 O 2 , whereas Fe incorporation led to a holoenzyme that was inhibited 60 to 70% by a similar treatment (data not shown).…”

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confidence: 94%

“…The two types of dismutases have often been distinguished by the presence of some key discriminating amino acids (27) and by their differential sensitivities to H 2 O 2 inhibition (4). However, these criteria are sometimes misleading (14,37), and the identity of the metal cofactor needs to be confirmed by direct analysis of the purified holoproteins. This precaution is particularly relevant since a small group of these enzymes, termed cambialistic, are able to display significant activity with either of the two metals bound at the same active site (23).…”

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confidence: 99%

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The Single Superoxide Dismutase of Rhodobacter capsulatus Is a Cambialistic, Manganese-Containing Enzyme

et al. 2003

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The phototrophic bacterium Rhodobacter capsulatus contains a single, oxygen-responsive superoxide dismutase (SOD Rc ) homologous to iron-containing superoxide dismutase enzymes. Recombinant SOD Rc , however, displayed higher activity after refolding with Mn 2؉ , especially when the pH of the assay mixture was raised. SOD Rc isolated from Rhodobacter cells also preferentially contains manganese, but metal discrimination depends on the culture conditions, with iron fractions increasing from 7% in aerobic cultures up to 40% in photosynthetic cultures. Therefore, SOD Rc behaves as a Mn-containing dismutase with cambialistic properties.

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confidence: 94%

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confidence: 99%

The Single Superoxide Dismutase of Rhodobacter capsulatus Is a Cambialistic, Manganese-Containing Enzyme

et al. 2003

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“…This observation is not surprising in view of the high primary sequence and structural homology demonstrated by these two proteins (Asada et al 1977;Steinman and Hill 1973;Takao et al 1990;Grace, 1990).…”

Section: Spmentioning

confidence: 85%

Debaryomyces hansenii: A Model System for Marine Molecular Biology

Nadathur¹

1991

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For the use of the constructed plasmids Ura 3" mutants had to be gnerated.Attempts were thus made to generate these mutants by chemical mutagenesis. The (,a 3' phenotype was selected by the negative selection utilizing 5-fluoro orotic acid. Even 4 though a large number of mutants were generated by this technique they were very unstable. All the mutants that were isolated reverted to their original phenotype. Dr. L.Adler of the University of Goteborg has recently sent me D. hanseni strain J,.26 which he has successfully used in mutant generation. He has also agreed to generate auxtrophic as weil as salt intolerant mutants of his strain. Transformations of Debaryomyces have been attempted with plasmid pLG90(kindly supplied by Dr. L. G. Gritz, Applied Biotechnology Inc.). This plasmid contains a Saccharomyces ARS and bacterial hygromycin resistance under the control of the yeast CYC.1 promoter. The plasmid is functional in providing hygromycin resistance in both E.coli and Saccharomyces. Several transformation techniques were attempted and low frequencies of transformation of Debaryomyces to hygromycin resistance were obtained with the protoplasting technique. The transformants are now being analyzed for the presence of the plasmid. This result, in addition to fact that the ARS sequence fromDebaryomyces is very similar to that of other organisms such as Candida albicans, Phycomyces blakesleeanus and Saccharomyces indicates that the plasmid that we have constructed will be successful in transforming Debayomyces. The experiments will be performed as soon as stable mutants are generated.The SSUrRNA from Debaryomyces was amplified using polymerase chain reaction and cloned into the polylinker cloning site of the plasmid pUC19. This was then sequenced and the sequences were aligned to organisms across kingdoms based on 23 taxa.Debaryomyces clusters among the yeasts but appears more closely related to Candida albicans than the group represented by S. than Saccharomyces. It also appears from these analyses that the capacity of these organisms to tolerate high salt is independent of phylogenetic affiliations based on SSU rDNA analysis.The rationale behind the isolation of regulatory sequences of inducible genes from Debaryonyces is that it would help in the construction of expression vectors. This in turnLi would help in the expression of foreign genes in this organism. Non-ambiguously aligned positions were analyzed by computation of 50 bootstrap resampled DNA distance matrices using the maximum likelihood metric (DNADIST). Resolution of the matrices was accomplished by Fitch and Margoliash's algorithm (FITCH), where input order of species added to the topology under conauction was randomized ten time. A consensus tree across 500 independent mees was generated using CONSENSE. Using the same alignment, 50 rounds of bootstrap resampled maximum parsimony (DNABOOT) were also conducted. All programs mentioned are from the PHYLIP 3.3 package (Felsenstein, 1990).-A Results and DiscussionDNA samples after PCR were separated on a...

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“…A existência dessas enzimas era associada anteriormente apenas a organismos aeróbios (40), atualmente consideradas ubíquas, a presença dessas foi confirmada em anaeróbios como Mathanobacterium thermoautotrophicum (41). Adicionalmente, a baixa solubilidade do cobre em condições anaeróbias teria tornado inviável seu emprego como cofator enzimático nas primeiras formas de vida devido à sua baixa disponibilidade ambiental (44).…”

Section: Superóxido Dismutasesunclassified

“…Em humanos, o excesso de superóxido dismutase, que faz parte de uma das alterações causadas pela trissomia do cromossomo 21, condição da doença conhecida como síndrome de Down, resulta em acúmulo de peróxido de hidrogênio e neste caso o excesso de SOD pode ser prejudicial ao organismo (2). Apesar das diferenças clássicas encontradas nestas SODs, alguns organismos possuem enzimas tetraméricas ligadas a ferro com características de MnSODs, como resistência a íons azoteto e peróxido de hidrogênio (41). Embora essa substituição de metais possa ser obtida, as atividades dismutásicas resultantes são muito reduzidas quando comparadas com as proteínas na presença dos metais próprios (39,64 Se o MSA é composto por um número suficientemente grande e diverso de sequências individuais, admite-se que se um resíduo l não contribui para a estrutura e função da proteína sua freqüência no MSA deve ser próxima do valor médio calculado para todas as proteínas.…”

Section: Superóxido Dismutasesunclassified

Identificação dos determinantes estruturais de Fe/MnSODs necessários a especificidade por metal.

Fontolan¹

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Superoxide dismutases (SODs) are metalloenzymes that convert the superoxide anion in molecular oxygen (O2) and hydrogen peroxide (H2O2). The metal in the catalytic center of such enzymes is directly related to their catalysis mechanisms and tridimensional structures. Evolutionarily, FeSOD and MnSOD may have evolved from a common ancestor, because both proteins have homologous primary sequences and superposable crystallographic structures. However, at the catalytic level, both proteins diverged sufficiently to prevent interchange of their metallic centers, which would generate non-functional enzymes, indicating that these proteins have high metal specificity. The objective of this research project is to identify structural determinants of Fe/MnSODs necessary to metal specificity. We intend to use statistical coupling analysis (SCA) to select amino acid residues for site-directed mutagenesis in TrMnSOD e TbFeSODB2. Mutant genes will be constructed and their proteins expressed, purified and crystallized. The tridimensional structure of such mutants will be solved by X-ray crystallography and their enzymatic activities determined, as well as their electron paramagnetic resonance spectra. We hypothesize that SCA is useful to identify amino acid candidates for site-directed mutagenesis to design new SODs with intermediated Fe/Mn specificity, and even metal specificity interconversion, by studying the evolutionary history of these proteins.

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Characterization of a superoxide dismutase gene from the archaebacterium Methanobacterium thermoautotrophicum

Cited by 50 publications

References 28 publications

The Single Superoxide Dismutase of Rhodobacter capsulatus Is a Cambialistic, Manganese-Containing Enzyme

The Single Superoxide Dismutase of Rhodobacter capsulatus Is a Cambialistic, Manganese-Containing Enzyme

Debaryomyces hansenii: A Model System for Marine Molecular Biology

Identificação dos determinantes estruturais de Fe/MnSODs necessários a especificidade por metal.

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