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The 2C-methyl-D-erythritol 4-phosphate (MEP) pathway is a carbon-efficient route for synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the building blocks of isoprenoids. However, practical application of a native or recombinant MEP pathway for the mass production of isoprenoids in Escherichia coli has been unsatisfactory. In this study, the entire recombinant MEP pathway was established with plasmids and used for the production of an isoprenoid, protoilludene. E. coli harboring the recombinant MEP pathway plasmid (ME) and a protoilludene synthesis pathway plasmid (AO) produced 10.4mg/L of protoilludene after 48h of culture. To determine the rate-limiting gene on plasmid ME, each constituent gene of the MEP pathway was additionally overexpressed on the plasmid AO. The additional overexpression of IPP isomerase (IDI) enhanced protoilludene production to 67.4mg/L. Overexpression of the Fpr and FldA protein complex, which could mediate electron transfer from NADPH to Fe-S cluster proteins such as IspG and IspH of the MEP pathway, increased protoilludene production to 318.8mg/L. Given that it is required for IspC as well as IspG/H, the MEP pathway has high demand for NADPH. To increase the supply of NADPH, a NADH kinase from Saccharomyces cerevisiae (tPos5p) that converts NADH to NADPH was introduced along with the deletion of a promiscuous NADPH-dependent aldehyde reductase (YjgB) that consumes NADPH. This resulted in a protoilludene production of 512.7mg/L. The results indicate that IDI, Fpr-FldA redox proteins, and NADPH regenerators are key engineering points for boosting the metabolic flux toward a recombinant MEP pathway.

The cofactor NADPH participates in a variety of anabolic reactions and its availability is considered to play a critical role in biotransformation processes. NADH kinase (Pos5) from Saccharomyces cerevisiae catalyzes the phosphorylation of NADH to generate NADPH. To investigate the effect of NADH kinase on poly-3-hydroxybutyrate (PHB) production, pos5 was co-expressed with PHB synthetic operon phbCAB in Escherichia coli. The recombinant strain carrying pos5 and phbCAB co-expression plasmid reached 5.96 g/L cell dry weight with 64.1% PHB accumulation in 72 h shake flask cultivation, while the control strain without pos5 yielded 3.93 g/L cell dry weight with 58.5% PHB content. PHB production titer was enhanced from 2.30 g/L to 3.82 g/L. Intracellular cofactor concentration analysis revealed that the ratio of NADP/NAD in pos5 overexpression strain was two times more compared with that of the control without pos5. The results showed that NADH kinase could be employed as an effective metabolic manipulation target to improve PHB synthesis.

Auranofin is a gold based drug in clinical use since 1985 for the treatment of rheumatoid arthritis. Beyond its antinflammatory properties, auranofin exhibits other attractive biological and pharmacological actions such as a potent in vitro cytotoxicity and relevant antimicrobial and antiparasitic effects that make it amenable for new therapeutic indications. For instance, auranofin is currently tested as an anticancer agent in four independent clinical trials; yet, its mode of action is highly controversial. With the present study, we explore the effects of auranofin in Saccharomyces cerevisiae and its likely mechanism. Notably, auranofin is reported to induce remarkable yeast growth inhibition. Solid evidence is provided that growth inhibition is the consequence of a direct cytotoxic insult occurring at the mitochondrial level; a profound depression of cell respiration is indeed clearly documented as the main cause of cell death while induction of ROS plays only a secondary role. More in detail, the mitochondrial NADH kinase Pos5 is identified as a primary target for auranofin. The implications of these results are discussed in the frame of current mechanistic knowledge on the cellular effects of auranofin and of its role as a prospective anticancer drug.

NAD kinase catalyzes the phosphorylation of NAD(+) to synthesize NADP(+), whereas NADH kinase catalyzes conversion of NADH to NADPH. The mitochondrial protein Pos5 of Saccharomyces cerevisiae shows much higher NADH kinase than NAD kinase activity and is therefore referred to as NADH kinase. To clarify the structural determinant underlying the high NADH kinase activity of Pos5 and its selectivity for NADH over NAD(+), we determined the tertiary structure of Pos5 complexed with NADH at a resolution of 2.0 Å. Detailed analysis, including a comparison of the tertiary structure of Pos5 with the structures of human and bacterial NAD kinases, revealed that Arg-293 of Pos5, corresponding to His-351 of human NAD kinase, confers a positive charge on the surface of NADH-binding site, whereas the corresponding His residue does not. Accordingly, conversion of the Arg-293 into a His residue reduced the ratio of NADH kinase activity to NAD kinase activity from 8.6 to 2.1. Conversely, simultaneous changes of Ala-330 and His-351 of human NAD kinase into Ser and Arg residues significantly increased the ratio of NADH kinase activity to NAD kinase activity from 0.043 to 1.39; human Ala-330 corresponds to Pos5 Ser-272, which interacts with the side chain of Arg-293. Arg-293 and Ser-272 were highly conserved in Pos5 homologs (putative NADH kinases), but not in putative NAD kinases. Thus, Arg-293 of Pos5 is a major determinant of NADH selectivity. Moreover, Ser-272 appears to assist Arg-293 in achieving the appropriate conformation.

In Saccharomyces cerevisiae, the mitochondrial inner membrane readily allows transport of cytosolic NAD(+), but not NADPH, to the matrix. Pos5p is the only known NADH kinase in the mitochondrial matrix. The enzyme phosphorylates NADH to NADPH and is the major source of NADPH in the matrix. The importance of mitochondrial NADPH for cellular physiology is underscored by the phenotypes of the Δpos5 mutant, characterized by oxidative stress sensitivity and iron-sulfur (Fe-S) cluster deficiency. Fe-S clusters are essential cofactors of proteins such as aconitase [4Fe-4S] and ferredoxin [2Fe-2S] in mitochondria. Intact mitochondria isolated from wild-type yeast can synthesize these clusters and insert them into the corresponding apoproteins. Here, we show that this process of Fe-S cluster biogenesis in wild-type mitochondria is greatly stimulated and kinetically favored by the addition of NAD(+) or NADH in a dose-dependent manner, probably via transport into mitochondria and subsequent conversion into NADPH. Unlike wild-type mitochondria, Δpos5 mitochondria cannot efficiently synthesize Fe-S clusters on endogenous aconitase or imported ferredoxin, although cluster biogenesis in isolated Δpos5 mitochondria is restored to a significant extent by a small amount of imported Pos5p. Interestingly, Fe-S cluster biogenesis in wild-type mitochondria is further enhanced by overexpression of Pos5p. The effects of Pos5p on Fe-S cluster generation in mitochondria indicate that one or more steps in the biosynthetic process require NADPH. The role of mitochondrial NADPH in Fe-S cluster biogenesis appears to be distinct from its function in anti-oxidant defense.

Yeast cells lacking the mitochondrial NADH kinase encoded by POS5 display increased sensitivity to hydrogen peroxide, a slow-growth phenotype, reduced mitochondrial function and increased levels of mitochondrial protein oxidation and mtDNA mutations. Here we examined gene expression in pos5 Δ cells, comparing these data to those from cells containing deletions of superoxide dismutase-encoding genes SOD1 or SOD2 . Surprisingly, stress–response genes were down-regulated in pos5 Δ, sod1 Δ and sod2 Δ cells, implying that cells infer stress levels from mitochondrial activity rather than sensing reactive oxygen species directly. Additionally, pos5 Δ, but not sod1 or sod2 , cells displayed an anaerobic expression profile, indicating a defect in oxygen sensing that is specific to pos5 , and is not a general stress–response. Finally, the pos5 Δ expression profile is quite similar to the hap1 Δ expression profile previously reported, which may indicate a shared mechanism.

The complete genome sequence of the filamentous fungi Aspergillus nidulans has paved the way for fundamental research on this industrially important species. To the best of our knowledge, this is the first time a gene encoding for ATP-dependent NADH kinase (ATP:NADH 2'-phosphotransferase, EC 2.7.1.86) has been identified. The enzyme has a predicted molecular weight of 49 kDa. We characterised the role of this NADH kinase by genomic integration of the putative gene AN8837.2 under a strong constitutive promoter. The physiological effects of overexpressed NADH kinase in combination with different aeration rates were studied in well-controlled glucose batch fermentations. Metabolite profiling and metabolic network analysis with [1-(13)C] glucose were used for characterisation of the strains, and the results demonstrated that NADH kinase activity has paramount influence on growth physiology. Biomass yield on glucose and the maximum specific growth rate increased from 0.47 g/g and 0.22 h(-1) (wild type) to 0.54 g/g and 0.26 h(-1) (NADH kinase overexpressed), respectively. The results suggest that overexpression of NADH kinase improves the growth efficiency of the cell by increasing the access to NADPH. Our findings indicate that A. nidulans is not optimised for growth in nutrient-rich conditions typically found in laboratory and industrial fermentors. This conclusion may impact the design of new strains capable of generating reducing power in the form of NADPH, which is crucial for efficient production of many industrially important metabolites and enzymes.

ATP-NADH kinase phosphorylates NADH to produce NADPH at the expense of ATP. The present study describes Fusarium oxysporum NADH kinase (ATP:NADH 2′-phosphotransferase, EC 2.7.1.86), a novel fungal enzyme capable of synthesizing NADPH using NADH as the preferred diphosphonicotinamide (diphosphopyridine) nucleotide donor. NADH kinase was highly purified (∼66-fold) and the enzyme was found to be a homodimeric with a subunit of M r 72,000. Isoelectric focusing in the pH range of 3.0–9.5 of the purified NADH kinase yielded a p I value of about 5.6. The K m values of NADH kinase for NADH and ATP were found to be 0.13 and 2.59 mM, respectively. Prediction of the secondary structure of the protein was performed in the PSIPRED server while modelling the three-dimensional (3D) structure was accomplished by the use of the HH 3D-structure prediction server.

Disruption of the Saccharomyces cerevisiae mitochondrial NADH kinase POS5 increases the mitochondrial mutation rate 50-fold. Whereas most multicellular eukaryotic genomes have one NADH kinase gene, the yeast genome contains three distinct genes encoding NAD/H kinase activity. To determine if all three genes are essential for viability we constructed combinations of gene knockouts. We show that only the pos5Deltautr1Delta combination is synthetically lethal, demonstrating an essential overlapping function, and showing that NAD/H kinase activity is essential for eukaryotic viability. The single human NAD/H kinase gene can rescue the lethality of the double knockout in yeast, demonstrating that the single human gene can fill the various functions provided by the three yeast genes. The human NAD/H kinase gene harbors very common sequence variants, but all of these equally complement the synthetic lethality in yeast, illustrating that each of these are functionally wild-type. To understand the molecular mechanism of the mitochondrial genome instability of pos5 mutation we performed gene expression analysis on the pos5Delta. The pos5Delta resulted in an increase in expression of most of the iron transport genes including key genes involved in iron-sulfur cluster assembly. Decreased expression occurred in many genes involved in the electron transport chain. We show that the pos5Delta expression pattern is similar to the frataxin homolog knockout (yfh1Delta), the yeast model for Friedreich's ataxia. These combined data show that the POS5 NAD/H kinase is an important protein required for a variety of essential cellular pathways and that deficient iron-sulfur cluster assembly may play a critical role in the mitochondrial mutator phenotype observed in the pos5Delta.

We have reacted calmodulins containing cysteines substituted at positions 3 and 146 or 5 and 146 with bismaleimidohexane (BMH) to generate intramolecularly cross-linked proteins termed BMHCM or BMHCM1, respectively. Reactions were also performed with N-ethylmaleimide (NEM) in place of BMH to generate corresponding S-ethylsuccinimidylated proteins termed NEMCM or NEMCM1. The abilities of these proteins to activate plant NAD kinase, erythrocyte Ca(2+)-ATPase and bovine brain calcineurin activities were assessed. The BMH- or NEM-reacted proteins activate calcineurin activity as does control calmodulin. Kact values for Ca(2+)-ATPase activation by BMHCM and BMHCM1 are increased 10-fold relative to the control value, with no corresponding change in Vmax values. Activation of this enzyme by NEMCM or NEMCM1 is not different from the control. In NAD kinase activation experiments BMHCM and BMHCM1 are associated with a 10 to 20-fold increase in Kact values and a 60-75% reduction in Vmax values relative to the control. NEMCM1 is not associated with any apparent changes in NAD kinase activation, however, NEMCM is associated with a 10-fold increase in the Kact value. NEM-reacted calmodulin containing a cysteine only at position 3 is not associated with an increased Kact value, implying that this change is due to interactions between S-(ethylsuccinimido)cysteines at positions 3 and 146. In conclusion, cross-linking and associated distortions in the structure of calmodulin appear to have little or no effect on activation of calcineurin enzyme activity. However, bending in the central helix and/or steric restrictions associated with cross-linking increase significantly the Kact value for Ca(2+)-ATPase and NAD kinase activation, and dramatically reduce maximal activation of NAD kinase activity.