Two isocitrate dehydrogenases from a plant pathogen Xanthomonas campestris pv. campestris 8004. Bioinformatic analysis, enzymatic characterization, and implication in virulence

Abstract

Isocitrate dehydrogenase (IDH) is a key enzyme in the tricarboxylate (TCA) cycle, which may play an important role in the virulence of pathogenic bacteria. Here, two structurally different IDHs from a plant pathogen Xanthomonas campestris pv. campestris 8004 (XccIDH1 and XccIDH2) were characterized in detail. The recombinant XccIDH1 forms homodimer in solution, while the recombinant XccIDH2 is a typical monomer. Phylogenetic analysis showed that XccIDH1 belongs to the type I IDH subfamily and XccIDH2 groups into the monomeric IDH clade. Kinetic characterization demonstrated that XccIDH1's specificity towards NAD(+) was 110-fold greater than NADP(+) , while XccIDH2's specificity towards NADP(+) was 353-fold greater than NAD(+) . The putative coenzyme discriminating amino acids (Asp268, Ile269 and Ala275 for XccIDH1, and Lys589, His590 and Arg601 for XccIDH2) were studied by site-directed mutagenesis. The coenzyme specificities of the two mutants, mXccIDH1 and mXccIDH2, were completely reversed from NAD(+) to NADP(+) , and NADP(+) to NAD(+) , respectively. Furthermore, Ser80 of XccIDH1, and Lys256 and Tyr421 of XccIDH2, were the determinants for the substrate binding. The detailed biochemical properties, such as optimal pH and temperature, thermostability, and metal ion effects, of XccIDH1 and XccIDH2 were further investigated. The possibility of taking the two IDHs into consideration as the targets for drug development to control the plant diseases caused by Xcc 8004 were described and discussed thoroughly.

Biochemical and Phylogenetic Characterization of a Novel NADP+-Specific Isocitrate Dehydrogenase From the Marine Microalga Phaeodactylum tricornutum

“…Interestingly, even though the H 604 L/R 615 D/R 664 S mutant enzyme could utilize NAD + as the coenzyme, the catalytic efficiency was fairly low. Similar results were obtained in other studies that sought to convert the coenzyme dependence of IDH from NADP + to NAD + , such as for M. methylutens IDH (Wang et al, 2020), X. fastidiosa IDH (Lv et al, 2018), and X. campestris IDH (Lv et al, 2016) (Supplementary Table S2). These results indicated that additional amino acid residues are involved in the binding of NAD + by monomeric IDHs, and further imply that the mechanisms involved in NAD + catalysis are more complex than those involved in NADP + catalysis in the type III IDH subfamily.…”

“…Additionally, the K m values of PtIDH2 for NADP + were 37.37 ± 4.02 μM with Mn 2+ and 18.37 ± 2.94 μM with Mg 2+ , and the corresponding catalytic efficiency (k cat /K m ) values were 3.22 and 2.36 μM −1 s −1 (Supplementary Figure S2). These k cat /K m values were similar to those for the bacterial monomeric NADP-IDHs from Xanthomonas campestris (6.0 μM −1 s −1 with Mn 2+ ) and Streptomyces avermitilis (11.7 μM −1 s −1 with Mn 2+ ) (Wang et al, 2011;Lv et al, 2016), but higher than those for the homodimeric NADP-IDH (contains two monomeric IDH-like subunits) from A. baumannii (0.39 μM −1 s −1 with Mn 2+ ) (Wang et al, 2018) and lower than those for the monomeric IDHs from A. vinelandii (15.0 μM −1 s −1 with Mn 2+ ) and Xylella fastidiosa (96.5 μM −1 s −1 with Mn 2+ ) (Watanabe et al, 2005;Lv et al, 2018).…”

“…Kinetic studies revealed that PtIDH2 is a completely NADP + -specific IDH. Moreover, the K m of PtIDH2 for NADP + (37.4 μM) was slightly higher than those of typical monomeric NADP-IDHs such as X. campestris IDH (17.5 μM) ( Lv et al, 2016 ), A. vinelandii IDH (5.8 μM) ( Tsubouchi and Takada, 2019 ), S. avermitilis IDH (5.0 μM) ( Wang et al, 2011 ), C. glutamicum IDH (4.0 μM) ( Chen and Yang, 2000 ), and X. fastidiosa IDH (1.0 μM) ( Lv et al, 2018 ), but lower than that of the homodimeric IDH from A. baumannii (94.0 μM) ( Wang et al, 2018 ) ( Supplementary Table S2 ). This indicates that the affinity of PtIDH2 for NADP + is lower than that of monomeric NADP-IDHs, but higher than that of the homodimeric NADP-IDHs of the type III subfamily.…”

Crystal Structure of the Isocitrate Dehydrogenase 2 from Acinetobacter baumannii (AbIDH2) Reveals a Novel Dimeric Structure with Two Monomeric-IDH-Like Subunits

“…Furthermore, mAbIDH2 was totally abolished by incubation for 20 min at 45 °C while AbIDH2 still sustained 60% of the activity ( Figure 3 ). The results agree well with previous reports detailing that the monomeric IDHs were not stable above 25 °C [ 11 , 22 , 23 ]. It is not clear why the IDH2 from A. baumannii could bear a much higher temperature than that needed for normal growth.…”

Biochemical and molecular characterization of the isocitrate dehydrogenase with dual coenzyme specificity from the obligate methylotroph Methylobacillus Flagellatus

“…psychrerythraea and X . campestris have one homodimeric type I IDH and one monomeric IDH with different biochemical properties [8–10]. Most prokaryotic IDHs that have been investigated are NADP + -dependent and homodimeric [11–17].…”