Substrate Specificity in Thiol Dioxygenases.

Thiol dioxygenases make up a class of ferrous iron-dependent enzymes that oxidize thiols to their corresponding sulfinates. X-ray diffraction structures of cysteine-bound cysteine dioxygenase show how cysteine is coordinated via its thiolate and amine to the iron and oriented correctly for O atom transfer. There are currently no structures with 3-mercaptopropionic acid or mercaptosuccinic acid bound to their respective enzymes, 3-mercaptopropionate dioxygenase or mercaptosuccinate dioxygenase. Sequence alignments and comparisons of known structures have led us to postulate key structural features that define substrate specificity. Here, we compare the rates and reactivities of variants of Rattus norvegicus cysteine dioxygenase and 3-mercaptopropionate dioxygenases from Pseudomonas aureginosa and Ralstonia eutropha (JMP134) and show how binary variants of three structural features correlate with substrate specificity and reactivity. They are (1) the presence or absence of a cis-peptide bond between residues Ser158 and Pro159, (2) an Arg or Gln at position 60, and (3) a Cys or Arg at position 164 (all RnCDO numbering). Different permutations of these features allow sulfination of l-cysteine, 3-mercaptopropionic acid, and ( R)-mercaptosuccinic acid to be promoted or impeded.

Substrate and pH-Dependent Kinetic Profile of 3-Mercaptopropionate Dioxygenase from Pseudomonas aeruginosa.

Thiol dioxygenases catalyze the synthesis of sulfinic acids in a range of organisms from bacteria to mammals. A thiol dioxygenase from the bacterium Pseudomonas aeruginosa oxidizes both 3-mercaptopropionic acid and cysteine, with a ∼70 fold preference for 3-mercaptopropionic acid over all pHs. This substrate reactivity is widened compared to other thiol dioxygenases and was exploited in this investigation of the residues important for activity. A simple model incorporating two protonation events was used to fit profiles of the Michaelis-Menten parameters determined at different pH values for both substrates. The pKs determined using plots of k(cat)/Km differ at low pH, but not in a way easily attributable to protonation of the substrate alone and share a common value at higher pH. Plots of k(cat) versus pH are also quite different at low pH showing the monoprotonated ES complexes with 3-mercaptopropionic acid and cysteine have different pKs. At higher pH, k(cat) decreases sigmoidally with a similar pK regardless of substrate. Loss of reactivity at high pH is attributed to deprotonation of tyrosine 159 and its influence on dioxygen binding. A mechanism is proposed by which deprotonation of tyrosine 159 both blocks oxygen binding and concomitantly promotes cystine formation. Finally, the role of tyrosine 159 was further probed by production of a G95C variant that is able to form a cysteine-tyrosine crosslink homologous to that found in mammalian cysteine dioxygenases. Activity of this variant is severely impaired. Crystallography shows that when un-crosslinked, the cysteine thiol excludes tyrosine 159 from its native position, while kinetic analysis shows that the thioether bond impairs reactivity of the crosslinked form.

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

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.