4WVZ

Crystal structure of artificial crosslinked thiol dioxygenase G95C variant from Pseudomonas aeruginosa

Summary for 4WVZ

• Entry DOI: 10.2210/pdb4wvz/pdb
• Related: 4TLF
• Descriptor: 3-mercaptopropionate dioxygenase, FE (II) ION (3 entities in total)
• Functional Keywords: thiol dioxygenase, cysteine dioxygenase, 3-mpa dioxygenase, 3-mercaptopropionic acid, non-heme mono-iron, cupin, beta barrel, crosslink, oxidoreductase
• Biological source: Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
• Total number of polymer chains: 4
• Total formula weight: 95518.04

Authors

Fellner, M.,Tchesnokov, E.P.,Jameson, G.N.L.,Wilbanks, S.M. (deposition date: 2014-11-09, release date: 2016-02-24, Last modification date: 2023-09-27)

Primary citation

Fellner, M.,Aloi, S.,Tchesnokov, E.P.,Wilbanks, S.M.,Jameson, G.N.
Substrate and pH-Dependent Kinetic Profile of 3-Mercaptopropionate Dioxygenase from Pseudomonas aeruginosa.
Biochemistry, 55:1362-1371, 2016

PubMed Abstract: 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.

PubMed: 26878277
DOI: 10.1021/acs.biochem.5b01203

Experimental method

X-RAY DIFFRACTION (2.09 Å)