Abstract
Many organisms possess a non-heme, mononuclear iron enzyme termed cysteine dioxygenase (CDO) that aids in homeostasis. This enzyme irreversibly adds molecular oxygen to the thiol group of the N-terminal cysteine residue of proteins in plants, acting as an oxygen sensor. Plant CDO targets are transcription factors which permit transcription of enzymes required for anaerobic metabolism. Under hypoxic conditions, the molecular oxygen co-substrate is not present at a high concentration therefore these transcription factors cannot be dioxidised and remain able to elicit an anoxic response. In normoxia, plant CDO dioxidises the N-terminal cysteine of these transcription factors, tagging them for degradation. This study evaluated recombinant expression and purification of CDO from a three plant species. CDO from Arabidopsis thaliana and Zea mays were then characterised both structurally and kinetically.
Plant CDO’s were expressed in Escherichia coli cells and purified using Strep¬-tag® affinity chromatography. Plant cysteine oxidase 1 (PCO1) and plant cysteine oxidase 2 (PCO2) isoforms from Arabidopsis thaliana co-purified with chaperones, and DNA. Two shortened variants of PCO1 were designed to abolish these interactions and improve homogeneity. A variant, ΔN, had residues 2-51 removed and produced contaminant-free product. When residues 2-52 and 247-293 were removed in the variant termed ΔNΔC, more co-purifying contaminants were present than with full-length PCO1. Plant CDO from Zea mays was expressed and purified both as a full length construct and a variant missing the first 32 residues. The full length Z. mays construct co-purified with a range of contaminants, and removal of the N-terminal did not improve protein homogeneity. A PCO2 isoform from Orzya sativa was attempted to be produced, resulting in no expression.
Homology models of PCO1 were produced to assess structural characteristics. Surface charge distribution, disulfide bonding and accessibility of the active site was explored in these models. Mass spectrometry (MS) showed that disulfide bonding was present in PCO1. PCO1, PCO2, ΔN, ΔNΔC and both maize constructs were subjected to a range of high-throughput crystallography screens. Promising conditions were optimised, but no diffracting crystals were produced. Metal ion binding to plant CDO was assessed using intact MS, and showed that the protein may also bind zinc in vitro. Nuclear magnetic resonance showed that plant CDO is not able to dioxidise free cysteine. Other colorimetric kinetic assays were performed to show that plant CDO is able to act on N-terminal cysteine as part of a di-, tri- or penta-peptide having the N-terminal of target molecules. Plant CDO has greater affinity for longer peptides.
DNA binding is predicted to be via an electrostatic interaction with N-terminal, which also appears to also contain a nuclear localisation signal. Nuclear localisation, followed by DNA binding could permit localisation to targets in vivo. This would allow plant CDO to quickly bind transcription factor targets as they are also DNA binding proteins. Disulfide bonding may also play a role in modulating protein activity. As disulfide bonding relates to cell oxidation state, this could permit conformational changes that allow the protein activity to be increased or decrease accordingly.