MOLECULAR CHARACTERIZATION AND EXPRESSION OF GENTISATE 1,2-DIOXYGENASE FROM PSEUDOMONAS ALCALIGENES NCIB 9867

Abstract

Gentisate 1,2-dioxygenase (GDO, EC 1.13.11.4) utilizes Fe2+ as a cofactor in the aromatic ring cleavage reaction of gentisate, yielding maleylpyruvate as the ring fission product. Mutant gentisate 1,2 dioxygenases were generated by both random and site directed mutagenesis of the gene cloned from Pseudomonas alcaligenes NCIB 9867. Alignment of known GDO sequences indicated the presence of a conserved central core region containing a two-motif signature of the cupin superfamily, designated motifs 1 and 2. Mutations generated within this central core resulted in the complete loss of enzyme activity whereas mutations in the flanking regions yielded GDOs with enzyme activities that were reduced by up to 78%. Site directed mutagenesis was also performed on a pair of highly conserved HRH and HXH motifs found within motifs 1 and 2 respectively of this conserved region. Conversion of these His residues to Asp resulted in the complete loss of catalytic activity. Mutagenesis within the core region could have affected quaternary structure formation as well as cofactor binding resulting in the loss of GDO activity. Several enzyme evolution methods were employed to generate gentisate 1,2-dioxygenases with increased catalytic activities. Due to the low homology between the two donor templates, both gene shuffling and domain swapping did not yield enzymes with increased catalytic properties. A mutant enzyme with increased catalytic activities however, was generated by random mutagenesis. The mutant enzyme had an apparent Kcat/Km ratio of 83.20 x 10-2 min-1 μM-1, representing an approximately 13-fold increase in catalytic efficiency when compared with the wild type enzyme. GDO from Pseudomonas alcaligenes NCIB 9867 (P25X) was expressed using three different heterologous E. coli overexpression systems, namely the 6X His tag, GST tag and the His-Patch ThioFusion™ systems as well as the in vitro cell free expression RTS 500 system. Binding of a 6X His-GOO fusion protein to the Ni2+ affinity column was poor when the 6X His tag was fused to the N-terminal portion of the enzyme. However, fusing the 6X His tag to both the N- and Cterminus of GDO enabled the rapid purification of the enzyme to apparent homogeneity using a single step Ni2+ affinity column chromatography. Purification yields of 42% were obtained and the purified protein possessed a specific activity of 7.89 U mg-1 for gentisate. The enzyme also exhibited a similar Fe2+ requirement to that of the wild type enzyme. GDO activity was detected in crude extracts of clones harboring GST-GDO fusion constructs, but the purified GST-GDO fusion enzyme was functionally inactive possibly due to the heat sensitive nature of the enzyme as the purification of GST-fusion proteins were performed at room temperature. ThioHis-GDO fusion proteins were soluble and active in the crude extracts but exhibited poor binding efficiency to the ThioBond™ resin. GDO expressed in an in vitro system exhibited lower levels of activity compared to its in vivo counterpart. The data obtained suggest that rapid, one-step purification of large quantities of GDO could be carried out by the addition of 6X His tags on both the N-and C-terminals of GDO.