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The cysteine dioxygenase homologue from Pseudomonas aeruginosa is a 3-mercaptopropionate dioxygenase.

ABSTRACT: Thiol dioxygenation is the initial oxidation step that commits a thiol to important catabolic or biosynthetic pathways. The reaction is catalyzed by a family of specific non-heme mononuclear iron proteins each of which is reported to react efficiently with only one substrate. This family of enzymes includes cysteine dioxygenase, cysteamine dioxygenase, mercaptosuccinate dioxygenase, and 3-mercaptopropionate dioxygenase. Using sequence alignment to infer cysteine dioxygenase activity, a cysteine dioxygenase homologue from Pseudomonas aeruginosa (p3MDO) has been identified. Mass spectrometry of P. aeruginosa under standard growth conditions showed that p3MDO is expressed in low levels, suggesting that this metabolic pathway is available to the organism. Purified recombinant p3MDO is able to oxidize both cysteine and 3-mercaptopropionic acid in vitro, with a marked preference for 3-mercaptopropionic acid. We therefore describe this enzyme as a 3-mercaptopropionate dioxygenase. Mössbauer spectroscopy suggests that substrate binding to the ferrous iron is through the thiol but indicates that each substrate could adopt different coordination geometries. Crystallographic comparison with mammalian cysteine dioxygenase shows that the overall active site geometry is conserved but suggests that the different substrate specificity can be related to replacement of an arginine by a glutamine in the active site.

SUBMITTER: Tchesnokov EP

PROVIDER: S-EPMC4591825 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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The cysteine dioxygenase homologue from Pseudomonas aeruginosa is a 3-mercaptopropionate dioxygenase.

Tchesnokov Egor P EP Fellner Matthias M Siakkou Eleni E Kleffmann Torsten T Martin Lois W LW Aloi Sekotilani S Lamont Iain L IL Wilbanks Sigurd M SM Jameson Guy N L GN

The Journal of biological chemistry 20150813 40

Thiol dioxygenation is the initial oxidation step that commits a thiol to important catabolic or biosynthetic pathways. The reaction is catalyzed by a family of specific non-heme mononuclear iron proteins each of which is reported to react efficiently with only one substrate. This family of enzymes includes cysteine dioxygenase, cysteamine dioxygenase, mercaptosuccinate dioxygenase, and 3-mercaptopropionate dioxygenase. Using sequence alignment to infer cysteine dioxygenase activity, a cysteine ...[more]

PMID: 26272617

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Project description:3,4-dihydroxyphenylacetate (DHPA) dioxygenase (DHPAO) from Pseudomonas aeruginosa (PaDHPAO) was overexpressed in Escherichia coli and purified to homogeneity. As the enzyme lost activity over time, a protocol to reactivate and conserve PaDHPAO activity has been developed. Addition of Fe(II), DTT and ascorbic acid or ROS scavenging enzymes (catalase or superoxide dismutase) was required to preserve enzyme stability. Metal content and activity analyses indicated that PaDHPAO uses Fe(II) as a metal cofactor. NMR analysis of the reaction product indicated that PaDHPAO catalyzes the 2,3-extradiol ring-cleavage of DHPA to form 5-carboxymethyl-2-hydroxymuconate semialdehyde (CHMS) which has a molar absorptivity of 32.23 mM-1cm-1 at 380 nm and pH 7.5. Steady-state kinetics under air-saturated conditions at 25°C and pH 7.5 showed a Km for DHPA of 58 ± 8 ?M and a kcat of 64 s-1, indicating that the turnover of PaDHPAO is relatively fast compared to other DHPAOs. The pH-rate profile of the PaDHPAO reaction shows a bell-shaped plot that exhibits a maximum activity at pH 7.5 with two pKa values of 6.5 ± 0.1 and 8.9 ± 0.1. Study of the effect of temperature on PaDHPAO activity indicated that the enzyme activity increases as temperature increases up to 55°C. The Arrhenius plot of ln(k'cat) versus the reciprocal of the absolute temperature shows two correlations with a transition temperature at 35°C. Two activation energy values (Ea) above and below the transition temperature were calculated as 42 and 14 kJ/mol, respectively. The data imply that the rate determining steps of the PaDHPAO reaction at temperatures above and below 35°C may be different. Sequence similarity network analysis indicated that PaDHPAO belongs to the enzyme clusters that are largely unexplored. As PaDHPAO has a high turnover number compared to most of the enzymes previously reported, understanding its biochemical and biophysical properties should be useful for future applications in biotechnology.

Project description:Pseudomonas aeruginosa encodes eight members of the Rid protein superfamily. PA5339, a member of the RidA subfamily, is required for full growth and motility of P. aeruginosa. Our understanding of RidA integration into the metabolic network of P. aeruginosa is at an early stage, with analyses largely guided by the well-established RidA paradigm in Salmonella enterica. A P. aeruginosa strain lacking RidA has a growth and motility defect in a minimal glucose medium, both of which are exacerbated by exogenous serine. All described ridA mutant phenotypes are rescued by supplementation with isoleucine, indicating the primary generator of the reactive metabolite 2-aminoacrylate (2AA) in ridA mutants is a threonine/serine dehydratase. However, the critical (i.e., phenotype determining) targets of 2AA leading to growth and motility defects in P. aeruginosa remained undefined. This study was initiated to probe the effects of 2AA stress on the metabolic network of P. aeruginosa by defining the target(s) of 2AA that contribute to physiological defects of a ridA mutant. Suppressor mutations that restored growth to a P. aeruginosa ridA mutant were isolated, including an allele of iscS (encoding cysteine desulfurase). Damage to IscS was identified as a significant cause of growth defects of P. aeruginosa during enamine stress. A suppressing allele encoded an IscS variant that was less sensitive to damage by 2AA, resulting in a novel mechanism of phenotypic suppression of a ridA mutant. IMPORTANCE 2-aminoacrylate (2AA) is a reactive metabolite formed as an intermediate in various enzymatic reactions. In the absence of RidA, this metabolite can persist in vivo where it attacks and inactivates specific PLP-dependent enzymes, causing metabolic defects and organism-specific phenotypes. This work identifies the cysteine desulfurase IscS as the critical target of 2AA in Pseudomonas aeruginosa. A single substitution in IscS decreased sensitivity to 2AA and suppressed growth phenotypes of a ridA mutant. Here, we provide the first report of suppression of a ridA mutant phenotype by altering the sensitivity of a target enzyme to 2AA.

Dataset Information

The cysteine dioxygenase homologue from Pseudomonas aeruginosa is a 3-mercaptopropionate dioxygenase.

Publications

The cysteine dioxygenase homologue from Pseudomonas aeruginosa is a 3-mercaptopropionate dioxygenase.

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