Influence of cysteine 164 on active site structure in rat cysteine dioxygenase

Abstract

Cysteine dioxygenase (CDO) is a non-heme mononuclear iron enzyme with unique structural features, namely an intramolecular thioether crosslink between cysteine 93 and tyrosine 157, and a disulfide between substrate L-cysteine and cysteine 164 in the entrance channel to the active site. We investigated how these posttranslational modifications affect catalysis through a kinetic, crystallographic and computational study. The enzyme kinetics of a C164S variant are identical to WT indicating that disulfide formation at C164 does not significantly impair access to the active site at physiological pH. However, at high pH the cysteine-tyrosine crosslink formation is enhanced in C164S. This supports the view that disulfide formation at position 164 can limit access to the active site. The C164S yielded crystal structures of unusual clarity in both resting state and with cysteine bound. Both show that the iron in the cysteine bound complex is a mixture of penta-and hexa-coordinate with a water molecule taking up the final site (60% occupancy), which is where dioxygen is believed to coordinate during turnover. The serine also displays stronger hydrogen bond interactions to a water bound to the amine of the substrate cysteine. However, the interactions between cysteine and iron appear unchanged. DFT calculations support this and show that WT and C164S have similar binding energies for the water molecule in the final site. This variant therefore provides evidence that WT also exists in this equilibrium between penta-and hexa-coordinate forms and presence of the sixth ligand does not strongly affect dioxygen binding.

Structure-Based Insights into the Role of the Cys–Tyr Crosslink and Inhibitor Recognition by Mammalian Cysteine Dioxygenase

“…The strong density is well modeled as a chloride ion, which is present at 15 mM in the crystallization buffer, is known to bind to iron, 41; 42; 43; 44 and was also recently similarly modeled in a crystal structure of a CDO Cys164Ser mutant. 45 The fully occupied water/hydroxide ligand is opposite to His140, and both the chloride and water appear stabilized by reasonably well-aligned bifurcated hydrogen-bonds from the Tyr157 hydroxyl (Figure 2A). …”

How Are Substrate Binding and Catalysis Affected by Mutating Glu127 and Arg161 in Prolyl-4-hydroxylase? A QM/MM and MD Study

“…Very recently, we used QM/MM modeling to predict spectroscopic fingerprints of short-lived catalytic cycle intermediates and used this on cysteine dioxygenase enzymes. Calculated UV-Vis absorption spectra and Mössbauer and EPR parameters enabled the experimental characterization of a short-lived oxygen-bound intermediate (Fellner et al, 2016 ; Tchesnokov et al, 2016 ).…”

A Comparative Review on the Catalytic Mechanism of Nonheme Iron Hydroxylases and Halogenases

“…Most of these enzymes are associated with cysteinate binding and catalysis. For instance, cysteine dioxygenase binds cysteinate and converts it into cysteine sulfinic acid on a NHFeHy active site with three facial His residues [129][130][131][132][133]. Another NHFeHy with a 3-His ligand coordination is the sulfoxide synthase enzyme EgtB that is involved in the biosynthesis of ergotheinine [134].…”