Enzymatic Characterization of the Isocitrate Dehydrogenase with Dual Coenzyme Specificity from the Marine Bacterium Umbonibacter marinipuiceus.

Isocitrate dehydrogenase (IDH) can be divided into NAD+-dependent and NADP+-dependent types based on the coenzyme specificity. It is worth noting that some IDHs exhibit dual coenzyme specificity characteristics. Herein, a dual coenzyme-dependent IDH from Umbonibacter Marinipuiceus (UmIDH) was expressed, purified, and identified in detail for the first time. SDS-PAGE and Gel filtration chromatography analyses showed that UmIDH is an 84.7 kDa homodimer in solution. The Km values for NAD+ and NADP+ are 1800.0 ± 64.4 µM and 1167.7 ± 113.0 µM in the presence of Mn2+, respectively. Meanwhile, the catalytic efficiency (kcat/Km) of UmIDH is only 2.3-fold greater for NADP+ than NAD+. The maximal activity for UmIDH occurred at pH 8.5 (with Mn2+) or pH 8.7 (with Mg2+) and at 35 °C (with Mn2+ or Mg2+). Heat inactivation assay revealed that UmIDH sustained 50% of maximal activity after incubation at 57 °C for 20 min with either Mn2+ or Mg2+. Moreover, three putative core coenzyme binding residues (R345, L346, and V352) of UmIDH were evaluated by site-directed mutagenesis. This recent work identified a unique dual coenzyme-dependent IDH and achieved the groundbreaking bidirectional modification of this specific IDH's coenzyme dependence for the first time. This provides not only a reference for the study of dual coenzyme-dependent IDH, but also a basis for the investigation of the coenzyme-specific evolutionary mechanisms of IDH.

From a dimer to a monomer: Construction of a chimeric monomeric isocitrate dehydrogenase.

Many isocitrate dehydrogenases (IDHs) are dimeric enzymes whose catalytic sites are located at the intersubunit interface, whereas monomeric IDHs form catalytic sites with single polypeptide chains. It was proposed that monomeric IDHs were evolved from dimeric ones by partial gene duplication and fusion, but the evolutionary process had not been reproduced in laboratory. To construct a chimeric monomeric IDH from homo-dimeric one, it is necessary to reconstitute an active center by a duplicated region; to properly link the duplicated region to the rest part; and to optimize the newly formed protein surface. In this study, a chimeric monomeric IDH was successfully constructed by using homo-dimeric Escherichia coli IDH as a start point by rational design and site-saturation mutagenesis. The ~67 kDa chimeric enzyme behaved as a monomer in solution, with a Km of 61 µM and a kcat of 15 s-1 for isocitrate in the presence of NADP+ and Mn2+ . Our result demonstrated that dimeric IDHs have a potential to evolve monomeric ones. The evolution of the IDH family was also discussed.

A broad-spectrum virus- and host-targeting peptide against respiratory viruses including influenza virus and SARS-CoV-2.

The 2019 novel respiratory virus (SARS-CoV-2) causes COVID-19 with rapid global socioeconomic disruptions and disease burden to healthcare. The COVID-19 and previous emerging virus outbreaks highlight the urgent need for broad-spectrum antivirals. Here, we show that a defensin-like peptide P9R exhibited potent antiviral activity against pH-dependent viruses that require endosomal acidification for virus infection, including the enveloped pandemic A(H1N1)pdm09 virus, avian influenza A(H7N9) virus, coronaviruses (SARS-CoV-2, MERS-CoV and SARS-CoV), and the non-enveloped rhinovirus. P9R can significantly protect mice from lethal challenge by A(H1N1)pdm09 virus and shows low possibility to cause drug-resistant virus. Mechanistic studies indicate that the antiviral activity of P9R depends on the direct binding to viruses and the inhibition of virus-host endosomal acidification, which provides a proof of concept that virus-binding alkaline peptides can broadly inhibit pH-dependent viruses. These results suggest that the dual-functional virus- and host-targeting P9R can be a promising candidate for combating pH-dependent respiratory viruses.

Characterization of the nicotinamide adenine dinucleotides (NAD+ and NADP+) binding sites of the monomeric isocitrate dehydrogenases from Campylobacter species.

Monomeric isocitrate dehydrogenases (IDHs) have once been proposed to be exclusively NADP+-specific. Intriguingly, we recently have reported an NAD+-specific monomeric IDH from Campylobacter sp. FOBRC14 (CaIDH). Moreover, bioinformatic analysis revealed at least three different coenzyme-binding motifs among Campylobacter IDHs. Besides the NAD+-binding motif in CaIDH (Leu584/Asp595/Ser644), a typical NADP+-binding motif was also identified in Campylobacter corcagiensis IDH (CcoIDH, His582/Arg593/Arg638). Meanwhile, a third putative NAD+-binding motif was found in Campylobacter concisus IDH (CcIDH, Leu580/Leu591/Ala640). In this study, CcIDH was overexpressed in Escherichia coli and purified to homogeneity. Gel filtration chromatography demonstrated that the recombinant CcIDH exists as a monomer in solution. Kinetic analysis showed that the Km value of CcIDH for NADP+ was over 49-fold higher than that for NAD+ and the catalytic efficiency (kcat/Km) of CcIDH is 115-fold greater for NAD+ than NADP+. Thus, CcIDH is indeed an NAD+-specific enzyme. However, the catalytic efficiency (kcat/Km = 0.886 µM-1 s-1) of CcIDH for NAD+ is much lower (<5%) when compared to that of the typical monomeric NADP-IDHs for NADP+. Then, the three core NAD+-binding sites were evaluated by site-directed mutagenesis. The mutant CcIDH (H580R591R640) showed a 51-fold higher Km value for NAD+ and 21-fold lower Km value for NADP+ as compared to that of the wild type enzyme, respectively. The overall specificity of the mutant CcIDH was 12-fold greater for NADP+ than that for NAD+. Thus, the coenzyme specificity of CcIDH was converted from NAD+ to NADP+. Isocitrate dependence of enzyme kinetics showed that although the mutant H580R591R640 preferred NADP+ as its coenzyme, its catalytic efficiency for isocitrate reduced to half of that for the wild-type CcIDH as using NAD+. The finding of both NAD+ and NADP+-binding sites in monomeric IDHs from Campylobacter species will provide us a chance to explore the evolution of the coenzyme specificity in monomeric IDH subfamily.

Heteroexpression and biochemical characterization of a glucose-6-phosphate dehydrogenase from oleaginous yeast Yarrowia lipolytica.

Yarrowia lipolytica, a nonpathogenic, nonconventional, aerobic and dimorphic yeast, is considered an oleaginous microorganism due to its excellent ability to accumulate large amounts of lipids. Glucose-6-phosphate dehydrogenase (G6PD) is one of two key enzymes involved in the lipid accumulation in this fungi, which catalyzes the oxidative dehydrogenation of glucose-6-phosphate to 6-phosphoglucono-δ-lactone with the reduction of NADP+ to NADPH. In this study, the full-length gene of G6PD from Y. lipolytica (YlG6PD) was cloned without intron and heterogeneously expressed in E. coli. Then, YlG6PD was purified and biochemically characterized in details. Kinetic analysis showed that YlG6PD was completely dependent on NADP+ and its apparent Km for NADP+ was 33.3 µM. The optimal pH was 8.5 and the maximum activity was around 47.5 °C. Heat-inactivation profiles revealed that it remained 50% of maximal activity after incubation at 48 °C for 20 min YlG6PD activity was competitively inhibited by NADPH with a Ki value of 56.04 µM. Most of the metal ions have no effect on activity, but Zn2+ was a strong inhibitor. Furthermore, the determinants in the coenzyme specificity of YlG6PD were investigated. Kinetic analysis showed that the single mutant R52D completely lost the ability to utilize NADP+ as its coenzyme, suggesting that Arg-52 plays a decisive role in NADP+ binding in YlG6PD. The identification of Y. lipolytica G6PD may provide useful scientific information for metabolic engineering of this yeast as a model for bio-oil production.