Correlating crosslink formation with enzymatic activity in cysteine dioxygenase

https://doi.org/10.1016/j.bbapap.2011.07.019Get rights and content

Abstract

Cysteine dioxygenase (CDO) from rat and other mammals exhibits a covalent post-translational modification between the residues C93 and Y157 that is in close proximity to the active site, and whose presence enhances the enzyme's activity. Protein with and without C93-Y157 crosslink migrates as distinct bands in SDS-PAGE, allowing quantification of the relative ratios between the two forms by densitometry of the respective bands. Expression of recombinant rat wild type CDO in Escherichia coli typically produces 40–50% with the C93-Y157 crosslink. A strategy was developed to increase the ratio of the non-crosslinked form in an enzyme preparation of reasonable quantity and purity, allowing direct assessment of the activity of non-crosslinked CDO and mechanism of formation of the crosslink. The presence of ferrous iron and oxygen is a prerequisite for C93-Y157 crosslink formation. Absence of oxygen during protein expression increased the fraction of non-crosslinked CDO, while presence of the metal chelator EDTA had little effect. Metal affinity chromatography was used to enrich non-crosslinked content. Both the enzymatic rate of cysteine oxidation and the amount of cross-linking between C93 and Y157 increased significantly upon exposure of CDO to air/oxygen and substrate cysteine in the presence of iron in a hitherto unreported two-phase process. The instantaneous activity was proportional to the amount of crosslinked enzyme present, demonstrating that the non-crosslinked form has negligible enzymatic activity. The biphasic kinetics suggest the existence of an as yet uncharacterised intermediate in crosslink formation and enzyme activation.

Highlights

► Anaerobic expression of mainly non-crosslinked cysteine dioxygenase. ► Separation of different ratios crosslinked/non-crosslinked enzyme using IMAC. ► Mainly non-crosslinked enzyme shows low initial activity. ► Enzyme activity increases with crosslink formation.

Introduction

The mononuclear non-heme iron enzyme cysteine dioxygenase (CDO, EC 1.13.11.20) is a unique and highly conserved protein of the cupin superfamily expressed in both mammals [1], [2], [3] and bacteria [4]. CDO catalyses the addition of dioxygen to the thiol group of cysteine, forming cysteine sulfinate (CSA). This reaction is the first step in cysteine catabolism, which mobilises cysteine carbon for gluconeogenesis or oxidative metabolism and taurine synthesis, and supplies inorganic sulphur for sulfation reactions. Tight regulation of cysteine levels has been shown to be important for health. High levels of cysteine are neuroexcitory [5] and have been associated with various diseases including rheumatoid arthritis [6] and several neurological diseases, such as Alzheimer's and Parkinson's diseases [7], [8], [9].

The crystal structures of human [3] and rodent [1], [2] CDO show a ferrous ion in the active site coordinated by three histidines and in close proximity to an unusual covalent cysteinyl-tyrosine crosslink between C93 and Y157 (Fig. 1). Although the cysteine and tyrosine which form the crosslink are completely conserved in eukaryotes, they are found in only a minority of bacterial CDO sequences. In rodents, both crosslinked and non-crosslinked CDO are expressed in vitro and in vivo and both are present in preparations used for biochemical analysis. Intriguingly, only the crosslinked form appears to crystallise. In a similar manner to the enzyme galactose oxidase [10], [11] that also forms this post translational modification, the two forms of CDO can be separated by SDS-PAGE into two distinctive bands, with the faster migrating band being the crosslinked form. Crosslink formation in galactose oxidase has been carefully investigated and shown to depend upon copper, though there is debate as to whether dioxygen is required [10]. CDO, on the other hand, has not been fully investigated and the mechanism of maturation is not entirely understood. Although the crosslink has been shown to form under high cysteine and iron concentrations [12], we have proposed that this may be non-physiological [13].

Using recombinant rat CDO with point mutations at C93 or Y157 position, Dominy et al. [12] showed that the crosslink is not a requirement for enzyme activity, but it increases turnover. Replacement of cysteine (C93A and C93S) reduced activity five-fold, while replacement of tyrosine (Y157F) reduced activity ten-fold. Likewise, Ye et al. [3] showed similar results for human CDO, with C93S and Y157F mutants being two- and twenty-fold less active, respectively. Computational investigations [14], [15] show that, although the crosslink may stabilise the distal oxygen of the initial iron(II) superoxo species, it does not appear to be required for reaction.

Dominy et al. [12] were able to increase the crosslinked fraction in wild type (WT) rat CDO by incubating the protein (μM concentration) with an excess of ferrous iron (0.3 mM) and cysteine (10 mM) in the presence of oxygen. It was therefore concluded that the presence of cysteine and iron are both required to form the post-translational modification. It was not clear, however, whether both forms were equally active and whether crosslink formation caused an increase in activity or was just a consequence of turnover. Furthermore, under these supra-physiological levels of cysteine and iron we subsequently showed that disulfides are formed between exogenous cysteine and both C93 and C164 [13]. C164 is situated at the entrance to the active site and although a disulfide formed at that residue would appear to block access, mutation of that residue (C164S) to impair disulfide formation in fact slightly decreases activity [3], suggesting that activation of the enzyme could involve modification of C164.

The aim of the present study was to further investigate conditions under which the crosslink is formed. In order to investigate the kinetic behaviour of non-crosslinked CDO it was necessary to obtain sufficient amounts thereof and two approaches towards its production were used. The first approach was to vary expression conditions of CDO in order to shift the usual approximate 1:1 proportion of crosslinked/non-crosslinked protein to a proportion in favour of the non-crosslinked form. The second approach was to separate CDO fractions with different crosslinked/non-crosslinked proportions during purification in order to obtain a fraction of mainly non-crosslinked protein.

Section snippets

Materials and methods

Isopropyl-β-d-thiogalactopyranoside (IPTG) was purchased from Gold Biotechnology, USA. Hydrochloric acid was purchased from Thermo Fisher, USA. Sulphuric acid 98% was purchased from BDH Laboratory Supplies, England. EDTA free Complete Protease Inhibitor Tablets were purchased from Roche Diagnostics GmbH, Germany. l-ascorbic acid 99% LR grade was purchased from J.T Baker, USA. l-cysteine non-animal source, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,4″-disulfonic acid sodium salt (ferrozine),

Aerobic and anaerobic expressions of mainly non-crosslinked WT and C164S CDO

WT and C164S CDO were expressed under a number of conditions, limiting the availability of either iron or oxygen. The presence of iron was modulated by the use of defined minimal media containing 30 μM Fe(II) and by the addition or lack of 1 mM EDTA. The final optical density at 600 nm (OD600) for each culture was not significantly different, showing that addition of EDTA at these concentrations did not impede cell growth (data not shown). The presence of the chelating agent EDTA did not notably

Obtaining mainly non-crosslinked CDO

The presence of both ferrous iron and oxygen has been reported to be a prerequisite for C93-Y157 crosslink formation in CDO [2], at least in vitro. We therefore assessed whether the non-crosslinked proportion could be increased by expressing CDO (a) in presence of the chelating agent EDTA, or (b) under anaerobic conditions.

To test method (a) WT and C164S CDO was expressed in E. coli in the presence and absence of 1 mM EDTA in the growth media. EDTA has been used previously by other researchers

Conclusion

In this work we describe how it is possible to obtain mainly non-crosslinked CDO for activity assays, either through anaerobic expression in E. coli, or during purification of the fusion protein as late eluting fraction on an IMAC column.

Presence of oxygen during protein expression had a notable influence on the expressed proportion of crosslinked to non-crosslinked form, which underlined the importance of oxygen in the formation of the C93-Y157 thioether bond. This reaction, however, appeared

Acknowledgments

The authors wish to thank Dr. Martha Stipanuk (Cornell University, USA) for the kind gift of the plasmid rCDO/pET32a. The research was funded by the Marsden Fund (UOO0923), Lottery Health (New Zealand), the University of Otago Research Committee (New Zealand) and the Department of Chemistry (University of Otago, New Zealand).

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