1115-65-7Relevant articles and documents
Steady-state substrate specificity and O2-coupling efficiency of mouse cysteine dioxygenase
Li, Wei,Pierce, Brad S.
, p. 49 - 56 (2015)
Cysteine dioxygenase (CDO) is a non-heme mononuclear iron enzyme that catalyzes the oxygen-dependent oxidation of l-cysteine (Cys) to produce l-cysteine sulfinic acid (CSA). Sequence alignment of mammalian CDO with recently discovered thiol dioxygenase enzymes suggests that the mononuclear iron site within all enzymes in this class share a common 3-His first coordination sphere. This implies a similar mechanistic paradigm among thiol dioxygenase enzymes. Although steady-state studies were first reported for mammalian CDO over 45 years ago, detailed analysis of the specificity for alternative thiol-bearing substrates and their oxidative coupling efficiencies have not been reported for this enzyme. Assuming a similar mechanistic theme among this class of enzymes, characterization of the CDO substrate specificity may provide valuable insight into substrate-active site intermolecular during thiol oxidation. In this work, the substrate-specificity for wild-type Mus musculus CDO was investigated using NMR spectroscopy and LC-MS for a variety of thiol-bearing substrates. Tandem mass spectrometry was used to confirm dioxygenase activity for each non-native substrate investigated. Steady-state Michaelis-Menten parameters for sulfinic acid product formation and O2-consumption were compared to establish the coupling efficiency for each reaction. In light of these results, the minimal substrate requirements for CDO catalysis and O2-activation are discussed.
Bioinformatic and biochemical characterizations of C-S bond formation and cleavage enzymes in the fungus neurospora crassa ergothioneine biosynthetic pathway
Hu, Wen,Song, Heng,Her, Ampon Sae,Bak, Daniel W.,Naowarojna, Nathchar,Elliott, Sean J.,Qin, Li,Chen, Xiaoping,Liu, Pinghua
, p. 5382 - 5385 (2014)
Ergothioneine is a histidine thiol derivative. Its mycobacterial biosynthetic pathway has five steps (EgtA-E catalysis) with two novel reactions: a mononuclear nonheme iron enzyme (EgtB) catalyzed oxidative C-S bond formation and a PLP-mediated C-S lyase (EgtE) reaction. Our bioinformatic and biochemical analyses indicate that the fungus Neurospora crassa has a more concise ergothioneine biosynthetic pathway because its nonheme iron enzyme, Egt1, makes use of cysteine instead of γ-Glu-Cys as the substrate. Such a change of substrate preference eliminates the competition between ergothioneine and glutathione biosyntheses. In addition, we have identi fied the N. crassa C-S lyase (NCU11365) and reconstituted its activity in vitro, which makes the future ergothioneine production through metabolic engineering feasible. (Chemical Equation Presented).
Shifting redox states of the iron center partitions CDO between crosslink formation or cysteine oxidation
Njeri, Catherine W.,Ellis, Holly R.
, p. 61 - 69 (2014)
Cysteine dioxygenase (CDO) is a mononuclear iron-dependent enzyme that catalyzes the oxidation of l-cysteine to l-cysteine sulfinic acid. The mammalian CDO enzymes contain a thioether crosslink between Cys93 and Tyr157, and purified recombinant CDO exists as a mixture of the crosslinked and non crosslinked isoforms. The current study presents a method of expressing homogenously non crosslinked CDO using a cell permeative metal chelator in order to provide a comprehensive investigation of the non crosslinked and crosslinked isoforms. Electron paramagnetic resonance analysis of purified non crosslinked CDO revealed that the iron was in the EPR silent Fe(II) form. Activity of non crosslinked CDO monitoring dioxygen utilization showed a distinct lag phase, which correlated with crosslink formation. Generation of homogenously crosslinked CDO resulted in an ～5-fold higher kcat/Km value compared to the enzyme with a heterogenous mixture of crosslinked and non crosslinked CDO isoforms. EPR analysis of homogenously crosslinked CDO revealed that this isoform exists in the Fe(III) form. These studies present a new perspective on the redox properties of the active site iron and demonstrate that a redox switch commits CDO towards either formation of the Cys93-Tyr157 crosslink or oxidation of the cysteine substrate.
Substrate and pH-Dependent Kinetic Profile of 3-Mercaptopropionate Dioxygenase from Pseudomonas aeruginosa
Fellner, Matthias,Aloi, Sekotilani,Tchesnokov, Egor P.,Wilbanks, Sigurd M.,Jameson, Guy N.L.
, p. 1362 - 1371 (2016)
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 kcat/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 kcat 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, kcat 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.
