Functional Identification Of Two Isocitrate Dehydrogenases From Pathogenic Leptospira Interrogans Serovar Lai Strain56601

Isocitrate dehydrogenase (IDH) is a key enzyme in the tricarboxylic acidcycle (TCA) and ubiquitously distributed throughout Archaea, Bacteria andEukarya. Based on the phylogenetic analysis, IDH can be classified into twotypes, Type I IDH, and Type II IDH. According to their coenzyme specificity,IDH can be further divided into two groups: NAD+-specific IDH (NAD-IDH)and NADP+-specific IDH (NADP-IDH). Both of NAD-IDH and NADP-IDHare found in Type I. However, all reported members in Type II to date areNADP-IDH.Leptospira interrogans serovar Lai str.56601contains two different IDHgenes that belong to the Type I and Type II IDH family and termed as LiIDH-Iand LiIDH-II, respectively. Enzymatic properties of both IDHs have not beenreported yet. In this study, we reported, for the first time, the cloning,overexpression and detailed characterization of two IDHs from L. interrogansserovar Lai strain56601.Functional identification of LiIDH-I. The purified LiIDH-I gave a single53.8kDa band in SDS-PAGE. Gel filtration chromatography was thenperformed to determine the oligomerization status. The native molecular massof LiIDH-I either with or without glycerol in solution was estimated to be294kDa (stable and active) and127kDa (unstable and inactive), respectively,suggesting that the active form of LiIDH-I is a homohexamer.LiIDH-I was completely divalent cation-dependent as other typicaldimeric IDH. Mn2+was found to be the most effective cation for LiIDH-Iactivity, and Mg2+took the second place. However, its activity was stronglyinhibited by Cu2+, Zn2+, Ni2+and Ca2+. The optimal pH of LiIDH-I was8.0 (Mg2+) and7.0(Mn2+), and the maximum activity was found at55°C (Mg2+)and25°C (Mn2+), respectively. Heat inactivation studies showed that LiIDH-Iretained50%activity after20min of incubation at50°C. The Kmfor NAD+ofLiIDH (84.68μM with Mg2+and149μM with Mn2+) is slightly lower thanthose for the dimeric NAD-IDHs from Streptococcus suis (233μM with Mg2+and174μM with Mn2+) and Zymomonas mobilis IDH (245μM with Mg2+and312μM with Mn2+), but is higher than NAD-IDHs from Acidithiobacillusthiooxidans (180μM with Mg2+) and Pyrococcus furiosus (68.3μM with Mg2+).The kinetic studies showed that in the presence of Mg2+or Mn2+, the catalyticefficiency (kcat/Km) of LiIDH-I were97-fold and58-fold greater specificity forNAD+than NADP+, respectively. Therefore, the recombinant LiIDH-I can useboth coenzymes, but has a remarkable preference toward NAD+.Functional identification of LiIDH-II. The purified LiIDH-II gave asingle44.7kDa band in SDS-PAGE. The molecular weight of LiIDH-II wasestimated to be87kDa by filtration chromatography, suggesting LiIDH-II is atypical homodimer. The optimum temperature for LiIDH-II activity was foundat60°C, and the optimum pH was7.0(Mn2+) and8.0(Mg2+). Heat inactivationstudies showed that the heat treatment for20min at50°C caused a50%loss ofenzyme activity. LiIDH-II was completely divalent cation dependent as othertypical dimeric IDH. Mg2+was found to be the most effective cation, althoughits role could partially replace by Mn2+and Co2+(63%by Mn2+,52%by Co2+).The Kmfor NADP+of LiIDH-II (21.10μM with Mg2+and37.94μM with Mn2+)is similar to those of dimeric NADP-IDHs from Thermotoga maritime (55.2μM with Mg2+) and Escherichia coli (17μM with Mg2+), but higher than thoseof monomeric NADP-IDHs, such as Streptomyces avermitilis (4.9μM withMn2+), Streptomyces lividans (2.42μM with Mg2+) and Azotobacter vinelandii(5.8μM with Mg2+). Therefore, monomeric IDHs exhibited significantpreference for NADP+over dimeric IDHs. Due to the decreased cofactoraffinity, the dimeric NADP-IDHs have much lower catalytic efficiency such asLiIDH-II (1.21μM-1s-1) and E. coli IDH (4.7μM-1s-1) with Mg2+, compared with the monomeric NADP-IDHs such as S. avermitilis IDH (11.7μM-1s-1) andA. vinelandii IDH (15.9μM-1s-1). In the presence of Mg2+and Mn2+, thepreference of LiIDH-II were6,269-fold and1,000-fold greater for NADP+thanNAD+, respectively.Additionally, in the presence of Mg2+and Mn2+, the Kmfor isocitrate ofLiIDH-I was76.63μM and85.15μM, and the Kmfor isocitrate of LiIDH-IIwas7.33μM and2.84μM, respectively. Thus, the affinity to isocitrate ofLiIDH-I is much higher than that of LiIDH-I.The structural, catalytic and regulatory characteristics of NADP-IDHshave been extensively studied. However, the catalytic mechanism ofNAD-IDHs is not yet well known as NADP-IDHs. The current study onLiIDH-I would provide more useful information for the enzymologyexploration of NAD-IDHs and the further study on the three-dimentionalstructure and catalysis mechanism of them.Although many molecular and cellular studies have been carried out onleptospires, little is known about pathogenesis mechanisms. The study onLiIDH-II of L. interrogans would enlarge the basic information for Type IINADP-IDHs in prokaryotes and further understanding on the three-dimentionalstructure and catalysis mechanism. Also, it may be possible that LiIDH-II couldbecome a candidate target protein for the clinical diagnosis of the infection byL. interrogans.