The critical role of residues Phe120 and Val161 of (2 R,3 R)­2,3­butanediol dehydrogenase from Neisseria gonorrhoeae as probed by molecular docking and site-directed mutagenesis.

NAD+-dependent (2 R,3 R)­2,3­butanediol dehydrogenase (BDH) from Neisseria gonorrhoeae (NgBDH) is a representative member of the medium-chain dehydrogenase/reductase (MDR) superfamily. To date, little information is available on the substrate binding sites and catalytic residues of BDHs from this superfamily. In this work, according to molecular docking studies, we found that conserved residues Phe120 and Val161 form strong hydrophobic interactions with both (2 R,3 R)­2,3­butanediol (RR-BD) and meso-2,3­butanediol (meso-BD) and that mutations of these residues to alanine or threonine impair substrate binding. To further evaluate the roles of these two residues, Phe120 and Val161 were mutated to alanine or threonine. Kinetic analysis revealed that, relative to those of wild type, the apparent KM values of the Phe120Ala mutant for RR-BD and meso-BD increased 36- and 369-fold, respectively; the catalytic efficiencies of this mutant with RR-BD and meso-BD decreased approximately 586- and 3528-fold, respectively; and the apparent KM values of the Val161Ala mutant for RR-BD and meso-BD increased 4- and 37-fold, respectively, the catalytic efficiencies of this mutant with RR-BD and meso-BD decreased approximately 3- and 28-fold, respectively. Additionally, the Val161Thr mutant slightly decreased catalytic efficiencies (twofold with RR-BD; 7.3-fold with meso-BD) due to an increase in KM (sixfold for RR-BD; 24-fold for meso-BD) and a slight increase (2.8-fold with RR-BD; 3.3-fold with meso-BD) in kcat. These findings validate the critical roles of Phe120 and Val161 of NgBDH in substrate binding and catalysis. Overall, the current study provides a better understanding of the substrate binding and catalysis of BDHs within the MDR superfamily.

Expression, Purification, and Bioinformatic Prediction of Mycobacterium tuberculosis Rv0439c as a Potential NADP+-Retinol Dehydrogenase.

Although the genome of Mycobacterium tuberculosis (Mtb) H37Rv, the causative agent of tuberculosis, has been repeatedly annotated and updated, a range of proteins from this human pathogen have unknown functions. Mtb Rv0439c, a member of the short-chain dehydrogenase/reductases superfamily, has yet to be cloned and characterized, and its function remains unclear. In this work, we present for the first time the optimized expression and purification of this enzyme, as well as bioinformatic analysis to unveil its potential coenzyme and substrate. Optimized expression in Escherichia coli yielded soluble Rv0439c, while certain tag fusions resulted in insolubility. Sequence and docking analyses strongly suggested that Rv0439c has a clear preference for NADP+, with Arg53 being a key residue that confers coenzyme specificity. Furthermore, functional prediction using CLEAN and DEEPre servers suggested that this protein is a potential NADP+-retinol dehydrogenase (EC No. 1.1.1.300) in retinol metabolism, and this was supported by a BLASTp search and docking studies. Collectively, our findings provide a solid basis for future functional characterization and structural studies of Rv0439c, which will contribute to enhanced understanding of Mtb biology.

Structure-based virtual screening of ROCK1 inhibitors for the discovery of Enterovirus-A71 antivirals.

Human enterovirus A71 (EV-A71) is the major causative agent of hand, foot, and mouth disease (HFMD), which may lead to neurological sequelae and even death. Although EV-A71 seriously threatens public health, there remains no efficient drug for the treatment of EV-A71 infection. We previously demonstrated that ROCK1 is a novel host dependency factor for EV-A71 replication and can serve as a target for the development of anti-EV-A71 therapeutics. In this study, we identified a subset of inhibitors with potential anti-EV-A71 activity by virtual screening using ROCK1 as a target. Among the hits, Dasabuvir, an HCV polymerase inhibitor, was found to have the best antiviral activity which is consistent with the ranking scores in Autodock Vina and iGEMDOCK. We found that Dasabuvir efficiently suppressed EV-A71 replication in a dose-dependent manner. Moreover, Dasabuvir not only efficiently suppressed the replication of EV-A71 in RD cells, but also in multiple cell lines, including HEK-293T, Caco-2, HT-29, HepG2, and Huh7. Besides, Dasabuvir alleviated the release of proinflammatory cytokines caused by EV-A71 infection. Notably, Dasabuvir also exhibited antiviral activity of CVA10, indicating it may have broad-spectrum antiviral activity against species Enteroviruses A. Hence, our results further confirm that ROCK1 can be a potential drug target and suggest Dasabuvir could be a clinical candidate for the treatment of EV-A71 infection.

Expression, purification, and biochemical characterization of an NAD+-dependent homoserine dehydrogenase from the symbiotic Polynucleobacter necessarius subsp. necessarius.

Homoserine dehydrogenase (HSD), encoded by the hom gene, is a key enzyme in the aspartate pathway, which reversibly catalyzes the conversion of l-aspartate ß-semialdehyde to l-homoserine (l-Hse), using either NAD(H) or NADP(H) as a coenzyme. In this work, we presented the first characterization of the HSD from the symbiotic Polynucleobacter necessaries subsp. necessarius (PnHSD) produced in Escherichia coli. Sequence analysis showed that PnHSD is an ACT domain-containing monofunctional HSD with 436 amnio acid residues. SDS-PAGE and Western blot demonstrated that PnHSD could be overexpressed in E. coli BL21(DE3) cell as a soluble form by using SUMO fusion technique. It could be purified to apparent homogeneity for biochemical characterization. Size-exclusion chromatography revealed that the purified PnHSD has a native molecular mass of ∼160 kDa, indicating a homotetrameric structure. The oxidation activity of PnHSD was studied in this work. Kinetic analysis revealed that PnHSD displayed an up to 1460-fold preference for NAD+ over NADP+, in contrast to its homologs. The purified PnHSD displayed maximal activity at 35 °C and pH 11. Similar to its NAD+-dependent homolog, neither NaCl and KCl activation nor L-Thr inhibition on the enzymatic activity of PnHSD was observed. These results will contribute to a better understanding of the coenzyme specificity of the HSD family and the aspartate pathway of P. necessarius.

Biochemical characterization and redesign of the coenzyme specificity of a novel monofunctional NAD+-dependent homoserine dehydrogenase from the human pathogen Neisseria gonorrhoeae.

Gonorrhoea, caused by Neisseria gonorrhoeae, is a major global public health concern. Homoserine dehydrogenase (HSD), a key enzyme in the aspartate pathway, is a promising metabolic target against pathogenic infections. In this study, a monofunctional HSD from N. gonorrhoeae (NgHSD) was overexpressed in Escherichia coli and purified to >95% homogeneity for biochemical characterization. Unlike the classic dimeric structure, the purified recombinant NgHSD exists as a tetramer in solution. We determined the enzymatic activity of recombinant NgHSD for l-homoserine oxidation, which revealed that this enzyme was NAD+ dependent, with an approximate 479-fold (kcat/Km) preference for NAD+ over NADP+, and that optimal activity for l-homoserine oxidation occurred at pH 10.5 and 40 °C. At 800 mM, neither NaCl nor KCl increased the activity of NgHSD, in contrast to the behavior of several reported NAD+-independent homologs. Moreover, threonine did not markedly inhibit the oxidation activity of NgHSD. To gain insight into the cofactor specificity, site-directed mutagenesis was used to alter coenzyme specificity. The double mutant L45R/S46R, showing the highest affinity for NADP+, caused a shift in coenzyme preference from NAD+ to NADP+ by a factor of ~974, with a catalytic efficiency comparable with naturally occurring NAD+-independent homologs. Collectively, our results should allow the exploration of drugs targeting NgHSD to treat gonococcal infections and contribute to the prediction of the coenzyme specificity of novel HSDs.

Crystal structures of NAD+-linked isocitrate dehydrogenase from the green alga Ostreococcus tauri and its evolutionary relationship with eukaryotic NADP+-linked homologs.

NAD+-linked isocitrate dehydrogenases (NAD-IDHs) catalyze the oxidative decarboxylation of isocitrate into α-ketoglutarate. Previously, we identified a novel phylogenetic clade including NAD-IDHs from several algae in the type II subfamily, represented by homodimeric NAD-IDH from Ostreococcus tauri (OtIDH). However, due to its lack of a crystalline structure, the molecular mechanisms of the ligand binding and catalysis of OtIDH are little known. Here, we elucidate four high-resolution crystal structures of OtIDH in a ligand-free and various ligand-bound forms that capture at least three states in the catalytic cycle: open, semi-closed, and fully closed. Our results indicate that OtIDH shows several novel interactions with NAD+, unlike type I NAD-IDHs, as well as a strictly conserved substrate binding mode that is similar to other homologs. The central roles of Lys283' in dual coenzyme recognition and Lys234 in catalysis were also revealed. In addition, the crystal structures obtained here also allow us to understand the catalytic mechanism. As expected, structural comparisons reveal that OtIDH has a very high structural similarity to eukaryotic NADP+-linked IDHs (NADP-IDHs) within the type II subfamily rather than with the previously reported NAD-IDHs within the type I subfamily. It has also been demonstrated that OtIDH exhibits substantial conformation changes upon ligand binding, similar to eukaryotic NADP-IDHs. These results unambiguously support our hypothesis that OtIDH and OtIDH-like homologs are possible evolutionary ancestors of eukaryotic NADP-IDHs in type II subfamily.

Purification and Characterization of (2R,3R)-2,3-Butanediol Dehydrogenase of the Human Pathogen Neisseria gonorrhoeae FA1090 Produced in Escherichia coli.

2,3-Butanediol dehydrogenase (BDH), also known as acetoin/diacetyl reductase, is a pivotal enzyme for the formation of 2,3-butanediol (2,3-BD), a chiral compound with potential roles in the virulence of certain pathogens. Here, a NAD(H)-dependent (2R,3R)-BDH from Neisseria gonorrhoeae FA1090 (NgBDH), the causative agent of gonorrhoea, was functionally characterized. Sequence analysis indicated that it belongs to zinc-containing medium-chain dehydrogenase/reductase family. The recombinant NgBDH migrated as a single band with a size of around 45 kDa on SDS-PAGE and could be confirmed by Western blotting and mass spectrometry. For the oxidation of either (2R,3R)-2,3-BD or meso-2,3-BD, the enzyme exhibited a broad pH optimum between pH 9.5 to 11.5. For the reduction of (3R/3S)-acetoin, the pH optimum was around 6.5. The enzyme could catalyze the stereospecific oxidation of (2R,3R)-2,3-BD (Km = 0.16 mM, kcat/Km = 673 s-1 · mM-1) and meso-BD (Km = 0.72 mM, kcat/Km = 165 s-1 · mM-1). Moreover, it could also reduce (3R/3S)-acetoin with a Km of 0.14 mM and a kcat/Km of 885 s-1 · mM-1. The results presented here contribute to understand the 2,3-BD metabolism in N. gonorrhoeae and pave the way for studying the influence of 2,3-BD metabolism on the virulence of this pathogen in the future.

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

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.

A unique homodimeric NAD⁺-linked isocitrate dehydrogenase from the smallest autotrophic eukaryote Ostreococcus tauri.

In eukaryotes, NAD(+)-dependent isocitrate dehydrogenase (IDH) is strictly mitochondrial and is a key enzyme in the Krebs cycle. To date, all known NAD(+)-specific IDHs (NAD-IDHs) in the mitochondria are believed to be heteromeric in solution. Here, a unique homodimeric NAD-IDH from Ostreococcus tauri (OtIDH), the smallest autotrophic picoeukaryote, was unveiled. Active OtIDH has a molecular weight of ∼93 kDa with each subunit of 46.7 kDa. In the presence of Mn(2+) and Mg(2+), OtIDH displayed 42-fold and 51-fold preference for NAD(+) over NADP(+), respectively. Interestingly, OtIDH exhibited a sigmoidal kinetic behavior in response to isocitrate unlike other homodimeric homologs, and a remarkably high affinity for isocitrate (S0.5 < 10 µM) unlike other hetero-oligomeric homologs. Furthermore, its coenzyme specificity can be completely converted from NAD(+) (ancient trait) to NADP(+) (adaptive trait) by rational mutagenesis based on the evolutionary trace. Mutants D344R and D344R/M345H displayed a 15-fold and 72-fold preference for NADP(+) over NAD(+), respectively, indicating that D344 and M345 are the determinants of NAD(+) specificity. These findings also suggest that OtIDH may be an ancestral form of type II IDHs (all reported members are NADP(+)-linked enzymes) and may have evolved into NADP(+)-dependent IDH for adaptation to the increased demand of NADPH under carbon starvation.

Expression and characterization of a novel isocitrate dehydrogenase from Streptomyces diastaticus No. 7 strain M1033.

Isocitrate dehydrogenase (IDH) is one of the key enzymes in tricarboxylic acid cycle, widely distributed in Archaea, Bacteria and Eukarya. Here, we report for the first time the cloning, expression and characterization of a monomeric NADP(+)-dependent IDH from Streptomyces diastaticus No. 7 strain M1033 (SdIDH). Molecular mass of SdIDH was about 80 kDa and showed high amino acid sequence identity with known monomeric IDHs. Maximal activity of SdIDH was observed at pH 8.0 (Mn(2+)) and 9.0 (Mg(2+)), and the optimal temperature was 40 °C (Mn(2+)) and 37 °C (Mg(2+)). Heat-inactivation studies showed that SdIDH remained about 50 % activity after 20 min of incubation at 47 °C. SdIDH displayed a 19,000 and 32,000-fold (k (cat)/K (m)) preference for NADP(+) over NAD(+) with Mn(2+) and Mg(2+), respectively. Our work implicate that SdIDH is a divalent metal ion-dependent monomeric IDH with remarkably high coenzyme preference for NADP(+). This work may provide fundamental information for further investigation on the catalytic mechanism of monomeric IDH and give a clue to disclose the real cause of IDH monomerization.