Enzymatic characterization and functional implication of two structurally different isocitrate dehydrogenases from Xylella fastidiosa

Abstract

Isocitrate dehydrogenase (IDH) is a key enzyme at the critical junction between the tricarboxylic acid cycle and the glyoxylate cycle. Most bacteria have only one IDH, while a few contain two IDH isozymes. The coexistence of two different type IDHs in one organism was little known. Xylella fastidiosa is a nutritionally fastidious plant pathogen that contains two structurally different IDHs, an NAD -dependent homodimeric IDH (diXfIDH) and an NADP -dependent monomeric IDH (monoXfIDH). Kinetic characterization showed that diXfIDH displayed 206-fold preferences for NAD over NADP , while monoXfIDH showed 13,800-fold preferences for NADP over NAD . The putative coenzyme crucial amino acids (Asp-268, Ile-269, and Ala-275 in diXfIDH, and Lys-589, His-590, and Arg-601 in monoXfIDH) were studied by site-directed mutagenesis. The coenzyme specificities of the three diXfIDH mutants (D268K, D268K/I269Y, and D268K/I269Y/A275V) were switched successfully from NAD to NADP . Meanwhile, the mutant monoXfIDHs (H590L/R601L and K589T/H590L/R601L) greatly reduced the affinity for NADP , but failed to improve the ability to use NAD and had similar affinity to NADP and NAD . The biochemical properties of diXfIDH and monoXfIDH were investigated in detail. This study gives a further insight into the determinants of the coenzyme specificity in both monomeric and dimeric forms of IDHs.

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.…”

“…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.…”

Melatonin Attenuates Potato Late Blight by Disrupting Cell Growth, Stress Tolerance, Fungicide Susceptibility and Homeostasis of Gene Expression in Phytophthora infestans

“…Particularly, the genes encoding rate-limiting enzymes: pyruvate kinase, fructose-bisphosphate aldolase, isocitrate dehydrogenase [NADP] were down-regulated as well (Table 5 , Tables S6A,B ). The genetic mutation of these rate-limiting enzymes can reduce amino acid metabolism levels (Heneka et al, 2001 ; Malay et al, 2005 ; Gupta and Bamezai, 2010 ; Grace et al, 2015 ; Lv et al, 2017 ), indicating that the amino acid metabolism was suppressed after melatonin treatment. Collectively, the results and observations from the transcriptome suggest that amino acid metabolism of P. infestans is likely the main target of melatonin.…”

Biochemical Characterization and Crystal Structure of a Novel NAD+-Dependent Isocitrate Dehydrogenase from Phaeodactylum tricornutum

“…Early phylogenetic analysis and competition experiments have demonstrated that NAD + dependence is an ancestral trait, NADP + dependence by bacterial IDHs is an adaptive trait, and the alteration of the coenzyme specificity is an adaptive result [ 45 ]. For IDHs of the type I subfamily, the coenzyme specificity can be completely converted from NAD + to NADP + , such as Xylella fastidiosa NAD-IDH and Pyrococcus furiosus NAD-IDH [ 31 , 46 ], or from NADP + to NAD + , such as E. coli NADP-IDH [ 47 ]. Interestingly, the evolutionary mechanism may be common for all members of type II IDHs.…”