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Bioinformatics and functional analysis define four distinct groups of AlkB DNA-dioxygenases in bacteria.

ABSTRACT: The iron(II)- and 2-oxoglutarate (2OG)-dependent dioxygenase AlkB from Escherichia coli (EcAlkB) repairs alkylation damage in DNA by direct reversal. EcAlkB substrates include methylated bases, such as 1-methyladenine (m(1)A) and 3-methylcytosine (m(3)C), as well as certain bulkier lesions, for example the exocyclic adduct 1,N(6)-ethenoadenine (epsilonA). EcAlkB is the only bacterial AlkB protein characterized to date, and we here present an extensive bioinformatics and functional analysis of bacterial AlkB proteins. Based on sequence phylogeny, we show that these proteins can be subdivided into four groups: denoted 1A, 1B, 2A and 2B; each characterized by the presence of specific conserved amino acid residues in the putative nucleotide-recognizing domain. A scattered distribution of AlkB proteins from the four different groups across the bacterial kingdom indicates a substantial degree of horizontal transfer of AlkB genes. DNA repair activity was associated with all tested recombinant AlkB proteins. Notably, both a group 2B protein from Xanthomonas campestris and a group 2A protein from Rhizobium etli repaired etheno adducts, but had negligible activity on methylated bases. Our data indicate that the majority, if not all, of the bacterial AlkB proteins are DNA repair enzymes, and that some of these proteins do not primarily target methylated bases.

SUBMITTER: van den Born E

PROVIDER: S-EPMC2790896 | biostudies-other | 2009 Nov

REPOSITORIES: biostudies-other

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Bioinformatics and functional analysis define four distinct groups of AlkB DNA-dioxygenases in bacteria.

van den Born Erwin E Bekkelund Anders A Moen Marivi N MN Omelchenko Marina V MV Klungland Arne A Falnes Pål Ø PØ

Nucleic acids research 20091101 21

The iron(II)- and 2-oxoglutarate (2OG)-dependent dioxygenase AlkB from Escherichia coli (EcAlkB) repairs alkylation damage in DNA by direct reversal. EcAlkB substrates include methylated bases, such as 1-methyladenine (m(1)A) and 3-methylcytosine (m(3)C), as well as certain bulkier lesions, for example the exocyclic adduct 1,N(6)-ethenoadenine (epsilonA). EcAlkB is the only bacterial AlkB protein characterized to date, and we here present an extensive bioinformatics and functional analysis of ba ...[more]

PMID: 19786499

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Project description:Ring cleavage dioxygenases catalyze the critical ring-opening step in the catabolism of aromatic compounds. The archetypal filamentous fungus Aspergillus niger previously has been reported to be able to utilize a range of monocyclic aromatic compounds as sole sources of carbon and energy. The genome of A. niger has been sequenced, and deduced amino acid sequences from a large number of gene models show various levels of similarity to bacterial intradiol ring cleavage dioxygenases, but no corresponding enzyme has been purified and characterized. Here, the cloning, heterologous expression, purification, and biochemical characterization of four nonheme iron(III)-containing intradiol dioxygenases (NRRL3_02644, NRRL3_04787, NRRL3_05330, and NRRL3_01405) from A. niger are reported. Purified enzymes were tested for their ability to cleave model catecholate substrates, and their apparent kinetic parameters were determined. Comparisons of k cat /Km values show that NRRL3_02644 and NRRL3_05330 are specific for hydroxyquinol (1,2,4-trihydroxybenzene), and phylogenetic analysis shows that these two enzymes are related to bacterial hydroxyquinol 1,2-dioxygenases. A high-activity catechol 1,2-dioxygenase (NRRL3_04787), which is phylogenetically related to other characterized and putative fungal catechol 1,2-dioxygenases, was also identified. The fourth enzyme (NRRL3_01405) appears to be a novel homodimeric Fe(III)-containing protocatechuate 3,4-dioxygenase that is phylogenetically distantly related to heterodimeric bacterial protocatechuate 3,4-dioxygenases. These investigations provide experimental evidence for the molecular function of these proteins and open the way to further investigations of the physiological roles for these enzymes in fungal metabolism of aromatic compounds.IMPORTANCE Aromatic ring opening using molecular oxygen is one of the critical steps in the degradation of aromatic compounds by microorganisms. While enzymes catalyzing this step have been well-studied in bacteria, their counterparts from fungi are poorly characterized despite the abundance of genes annotated as ring cleavage dioxygenases in fungal genomes. Aspergillus niger degrades a variety of aromatic compounds, and its genome harbors 5 genes encoding putative intracellular intradiol dioxygenases. The ability to predict the substrate specificities of the encoded enzymes from sequence data are limited. Here, we report the characterization of four purified intradiol ring cleavage dioxygenases from A. niger, revealing two hydroxyquinol-specific dioxygenases, a catechol dioxygenase, and a unique homodimeric protocatechuate dioxygenase. Their characteristics, as well as their phylogenetic relationships to predicted ring cleavage dioxygenases from other fungal species, provide insights into their molecular functions in aromatic compound metabolism by this fungus and other fungi.

Dataset Information

Bioinformatics and functional analysis define four distinct groups of AlkB DNA-dioxygenases in bacteria.

Publications

Bioinformatics and functional analysis define four distinct groups of AlkB DNA-dioxygenases in bacteria.

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