title: Cloning and functional analysis of the genes coding for 4-aminobenzenesulfonate 3,4-dioxygenase from Hydrogenophaga sp. PBC.
abstract: The gene coding for the oxygenase component, sadA, of 4-aminobenzenesulfonate (4-ABS) 3,4-dioxygenase in Hydrogenophaga sp. PBC was previously identified via transposon mutagenesis. Expression of wild-type sadA in trans restored the ability of the sadA mutant to grow on 4-ABS. The inclusion of sadB and sadD, coding for a putative glutamine-synthetase-like protein and a plant-type ferredoxin, respectively, further improved the efficiency of 4-ABS degradation. Transcription analysis using the gfp promoter probe plasmid showed that sadABD was expressed during growth on 4-ABS and 4-sulfocatechol. Heterologous expression of sadABD in Escherichia coli led to the biotransformation of 4-ABS to a metabolite which shared a similar retention time and UV/vis profile with 4-sulfocatechol. The putative reductase gene sadC was isolated via degenerate PCR and expression of sadC and sadABD in E. coli led to maximal 4-ABS biotransformation. In E. coli, the deletion of sadB completely eliminated dioxygenase activity while the deletion of sadC or sadD led to a decrease in dioxygenase activity. Phylogenetic analysis of SadB showed that it is closely related to the glutamine-synthetase-like proteins involved in the aniline degradation pathway. This is the first discovery, to our knowledge, of the functional genetic components for 4-ABS aromatic ring hydroxylation in the bacterial domain.
authors.affiliation: Department of Biological Sciences, Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, Johor 81310, Malaysia.
authors.forename: Han Ming
authors.initials: HM
authors.lastname: Gan
authors.affiliation: Department of Biological Sciences, Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, Johor 81310, Malaysia.
authors.forename: Shafinaz
authors.initials: S
authors.lastname: Shahir
authors.affiliation: Department of Industrial Biotechnology, Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, Johor 81310, Malaysia.
authors.forename: Adibah
authors.initials: A
authors.lastname: Yahya
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D042621:Comamonadaceae / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D049308:Dioxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D013425:Sulfanilic Acids / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D013425:Sulfanilic Acids; C100016:4-sulfanilic acid; D049308:Dioxygenases