Substrate Specificity in Thiol Dioxygenases

Abstract

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.

Structures of Arabidopsis thaliana oxygen-sensing plant cysteine oxidases 4 and 5 enable targeted manipulation of their activity

“…The AtPCOs are unique among the three types of thiol dioxygenases characterized to date (CDOs, 3MDOs [mercaptopropionate dioxygenases], and MSDOs [mercaptosuccinate dioxygenases]) and represent a new class. In the CDOs, 3MDOs and MSDOs, substrate specificity and reactivity are influenced by the presence and position of an Arg residue in the active site (25,26). In the CDOs, Arg60 forms a strong salt bridge with the carboxylate group of the active site metal bound L-cysteine, while in other thiol dioxygenases, there is a Gln at position 60 and an Arg residue elsewhere in the active site which contributes to substrate carboxylate binding (e.g., Arg168 in Pseudomonas aeruginosa 3MDO) (26).…”

“…In the CDOs, 3MDOs and MSDOs, substrate specificity and reactivity are influenced by the presence and position of an Arg residue in the active site (25,26). In the CDOs, Arg60 forms a strong salt bridge with the carboxylate group of the active site metal bound L-cysteine, while in other thiol dioxygenases, there is a Gln at position 60 and an Arg residue elsewhere in the active site which contributes to substrate carboxylate binding (e.g., Arg168 in Pseudomonas aeruginosa 3MDO) (26). The AtPCOs do not possess an Arg residue in the active site, which is unsurprising since the cysteinyl substrates of AtPCOs have a peptide bond in place of the charged carboxylate group of free cysteine.…”

Second‐Coordination Sphere Effects on Selectivity and Specificity of Heme and Nonheme Iron Enzymes

“…Another example of a nearby Arg residue in the active site that positions oxidant and substrate through a salt bridge is in the nonheme iron dioxygenase CDO . Interestingly, the cysteine dioxygenase does not use α‐ketoglutarate as a co‐substrate but reacts dioxygen directly with the substrate .…”

“…Thus, the work of Jameson et al. compared the structurally and functionally similar enzymes CDO and mercaptopropionate dioxygenase (MDO) . Both of these are nonheme iron enzymes that bind the metal through a 3‐His coordination environment (see Figure ).…”

“…Furthermore, the CDO structure has a kink in the peptide chain through a cis ‐Ser 158 ‐Pro 159 linkage that redirects the chain, which is missing in MDO. It has been hypothesized that the substrate in MDO will bind and form a salt bridge with Arg 168 and hence is positioned differently from cysteine in CDO . Indeed, site‐directed mutations of R60Q and R60Q/C164R of CDO showed a considerable drop in activity with respect to cysteine, which gives evidence of the importance of positioning the substrate in the binding pocket.…”