Analysis of the proteome of intracellular Shigella flexneri reveals pathways important for intracellular growth.

Global proteomic analysis was performed with Shigella flexneri strain 2457T in association with three distinct growth environments: S. flexneri growing in broth (in vitro), S. flexneri growing within epithelial cell cytoplasm (intracellular), and S. flexneri that were cultured with, but did not invade, Henle cells (extracellular). Compared to in vitro and extracellular bacteria, intracellular bacteria had increased levels of proteins required for invasion and cell-to-cell spread, including Ipa, Mxi, and Ics proteins. Changes in metabolic pathways in response to the intracellular environment also were evident. There was an increase in glycogen biosynthesis enzymes, altered expression of sugar transporters, and a reduced amount of the carbon storage regulator CsrA. Mixed acid fermentation enzymes were highly expressed intracellularly, while tricarboxylic acid (TCA) cycle oxidoreductive enzymes and most electron transport chain proteins, except CydAB, were markedly decreased. This suggested that fermentation and the CydAB system primarily sustain energy generation intracellularly. Elevated levels of PntAB, which is responsible for NADPH regeneration, suggested a shortage of reducing factors for ATP synthesis. These metabolic changes likely reflect changes in available carbon sources, oxygen levels, and iron availability. Intracellular bacteria showed strong evidence of iron starvation. Iron acquisition systems (Iut, Sit, FhuA, and Feo) and the iron starvation, stress-associated Fe-S cluster assembly (Suf) protein were markedly increased in abundance. Mutational analysis confirmed that the mixed-acid fermentation pathway was required for wild-type intracellular growth and spread of S. flexneri. Thus, iron stress and changes in carbon metabolism may be key factors in the S. flexneri transition from the extra- to the intracellular milieu.

Activating transhydrogenase and NAD kinase in combination for improving isobutanol production.

Isobutanol is an excellent alternative biofuel. Fermentative production of isobutanol had been realized in several microorganisms by combining branched-chain amino acids synthetic pathway and Ehrlich pathway. In contrast to using plasmid overexpression and inducible promoters, genetically stable Escherichia coli strains for isobutanol production were constructed in this work by integrating essential genes into chromosome. A chromosome-based markerless gene modulation method was then developed for fine-tuning gene expression with multiple regulatory parts to improve isobutanol production. There was also a cofactor imbalance problem for anaerobic isobutanol synthesis. NADPH is the reducing equivalent required for isobutanol production, while the common reducing equivalent under anaerobic condition is NADH. Two strategies were used to modulate expression of transhydrogenase (pntAB) and NAD kinase (yfjB) genes to increase NADPH supply for improving isobutanol production. Plasmid overexpression of pntAB and yfjB genes either individually or in combination had little effect on isobutanol production. In contrast, modulating pntAB and yfjB gene expression in chromosome with multiple regulatory parts identified optimal modulators under aerobic and anaerobic conditions, respectively, and improved isobutanol production. Modulating pntAB gene alone led to 20% and 8% increase of anaerobic isobutanol titer and yield. Although modulating yfjB gene alone had nearly no effect, modulating pntAB and yfjB genes in combination led to 50% and 30% increase of isobutanol titer and yield in comparison with modulating pntAB gene alone. It was also found that increasing pntAB gene expression alone had a threshold for improving anaerobic isobutanol production, while activating NAD kinase could break through this threshold, leading to a yield of 0.92mol/mol. Our results suggested that transhydrogenase and NAD kinase had a synergistic effect on increasing NADPH supply and improving anaerobic isobutanol production. This strategy will be useful for improving production of target compounds using NADPH as reducing equivalent within their synthetic pathways. In addition, combined activation of PntAB and YfjB led to 28% and 22% increase of aerobic isobutanol titer and yield, resulting in production of 10.8g/L isobutanol in 24h with a yield of 0.62mol/mol.

Interactions of the NADP(H)-binding domain III of proton-translocating transhydrogenase from escherichia coli with NADP(H) and the NAD(H)-binding domain I studied by NMR and site-directed mutagenesis.

Using the purified NADP(H)-binding domain of proton-translocating Escherichia coli transhydrogenase (ecIII) overexpressed in (15)N- and (2)H-labeled medium, together with the purified NAD(H)-binding domain from E. coli (ecI), the interface between ecIII and ecI, the NADP(H)-binding site and the influence on the interface by NAD(P)(H) was investigated in solution by NMR chemical shift mapping. Mapping of the NADP(H)-binding site showed that the NADP(H) substrate is bound to ecIII in an extended conformation at the C-terminal end of the parallel beta-sheet. The distribution of chemical shift perturbations in the NADP(H)-binding site, and the nature of the interaction between ecI and ecIII, indicated that the nicotinamide moiety of NADP(H) is located near the loop comprising residues P346-G353, in agreement with the recently determined crystal structures of bovine [Prasad, G. S., et al. (1999) Nat. Struct. Biol. 6, 1126-1131] and human heart [White, A. W., et al. (2000) Structure 8, 1-12] transhydrogenases. Further chemical shift perturbation analysis also identified regions comprising residues G389-I406 and G430-V434 at the C-terminal end of ecIII's beta-sheet as part of the ecI-ecIII interface, which were regulated by the redox state of the NAD(P)(H) substrates. To investigate the role of these loop regions in the interaction with domain I, the single cysteine mutants T393C, R425C, G430C, and A432C were generated in ecIII and the transhydrogenase activities of the resulting mutant proteins characterized using the NAD(H)-binding domain I from Rhodospirillum rubrum (rrI). All mutants except R425C showed altered NADP(H) binding and domain interaction properties. In contrast, the R425C mutant showed almost exclusively changes in the NADP(H)-binding properties, without changing the affinity for rrI. Finally, by combining the above conclusions with information obtained by a further characterization of previously constructed mutants, the implications of the findings were considered in a mechanistic context.

NMR characterization of the NADP(H)-binding domain of Escherichia coli transhydrogenase: sequential assignment and global fold.

The soluble NADP(H)-binding domain of Escherichia coli transhydrogenase (186 amino acids, 20.4 kDa, rotational correlation time 14 ns) was characterized using NMR techniques. The global fold is similar to that of a classical dinucleotide-binding fold with six parallel beta-strands in a central sheet surrounded by helices and irregular structures, but is lacking both alphaD and alphaE. The substrate is bound in an extended conformation at the C-terminal end of the parallel beta-sheet and our data support the notion of a redox dependent structural rearrangement.

A catalytically active complex formed from the recombinant dI protein of Rhodospirillum rubrum transhydrogenase, and the recombinant dIII protein of the human enzyme.

Transhydrogenase is a proton pump. It has three components: dI and dIII protrude from the membrane and contain the binding sites for NAD(H) and NADP(H), respectively, and dII spans the membrane. We have expressed dIII from Homo sapiens transhydrogenase (hsdIII) in Escherichia coli. The purified protein was associated with stoichiometric amounts of NADP(H) bound to the catalytic site. The NADP+ and NADPH were released only slowly from the protein, supporting the suggestion that nucleotide-binding by dIII is regulated by the membrane-spanning dII. HsdIII formed a catalytically active complex with recombinant dI from Rhodospirillum rubrum (rrdI), even in the absence of dII. The rates of forward and reverse transhydrogenation catalysed by this complex are probably limited by slow release from dIII of NADPH and NADP+, respectively. The hybrid complex also catalysed high rates of 'cyclic' transhydrogenation, indicating that hydride transfer, and exchange of nucleotides with dI, are rapid. Stopped-flow experiments revealed a rapid, monoexponential, single-turnover burst of reverse transhydrogenation in pre-steady-state. The apparent first-order rate constant of the burst increased with the concentration of rrdI. A deuterium isotope effect (kH/kD approximately 2 at 27 degrees C) was observed when [4B-1H]NADPH was replaced with [4B-2H]NADPH. The characteristics of the burst of transhydrogenation with rrdI:hsdIII differed from those previously reported for rrdI:rrdIII (J.D. Venning et al., Eur. J. Biochem. 257 (1998) 202-209), but the differences are readily explained by a greater dissociation constant of the hybrid complex. The steady-state rate of reverse transhydrogenation by the rrdI:hsdIII complex was almost independent of pH, but there was a single apparent pKa ( approximately 9.1) associated with the cyclic reaction. The reactions of the dI:dIII complex probably proceed independently of those protonation/deprotonation reactions which, in the complete enzyme, are associated with H+ translocation.

The udhA gene of Escherichia coli encodes a soluble pyridine nucleotide transhydrogenase.

The udhA gene of Escherichia coli was cloned and expressed in E. coli and found to encode an enzyme with soluble pyridine nucleotide transhydrogenase activity. The N-terminal end of the enzyme contains the fingerprint motif of a dinucleotide binding domain, not present in published E. coli genome sequences due to a sequencing error. E. coli is hereby the first organism reported to possess both a soluble and a membrane-bound pyridine nucleotide transhydrogenase.

Cloning, analysis and inactivation of the ndhK gene encoding a subunit of NADH quinone oxidoreductase from Anabaena PCC 7120.

The function of the type-1 pyridine nucleotide dehydrogenase (NDH-1) in the cyanobacterium Anabaena PCC 7120 was investigated. Immunological analysis with antibodies raised against NdhK from Synechocystis PCC 6803, a subunit of NDH-1, showed that NdhK in Anabaena PCC 7120 is only present on the plasma membrane, which confirms the results of previous studies [Howitt, C.A., Smith, G.D. & Day, D. A. (1993) Biochim. Biophys. Acta 114], 313-320]. Southern analysis with probes from the operon encoding ndhC-K-J from Synechocystis PCC 6803 showed that this operon is also conserved in Anabaena PCC 7120. Part of the operon was amplified using PCR with degenerate primers designed against two sequences encoding regions of NdhC and NdhJ that are conserved between cyanobacteria and chloroplasts. The nucleotide sequence of ndhK encodes a protein of 245 amino acids with a predicted molecular mass of 27.5 kDa. The coding regions of ndhC and ndhK overlap by 7 bp, as found in the chloroplasts of liverwort, maize, and rice. This is markedly different from the case in Synechocystis PCC 6803 where a 71-bp non-coding, intergenic spacer region lies between ndhC and ndhK. The ndhK clone was interrupted by the insertion of a kanamycin-resistance gene and used to transform Anabaena PCC 7120.20 unsegregated transformants were produced, all of which died during attempts to segregate them. This indicates that under the selection conditions used, ndhK is an essential gene in Anabaena PCC 7120.

Identification of an aspartic acid residue in the beta subunit which is essential for catalysis and proton pumping by transhydrogenase from Escherichia coli.

Based on the alignment of 7 unknown amino acid sequences, including the recently determined sequences for the mouse and human enzymes, a highly conserved acidic domain was identified which in the Escherichia coli enzyme is located close to the C-terminal end of the predicted NADP(H)-binding site of the beta subunit. The effect of replacing the four conserved acidic residues, betaE361, betaE374, betaD383 and betaD392, in this domain on catalytic and proton-pumping activity was tested by site-directed mutagenesis. In addition, betaE371, which is not conserved but located in the same domain, was also mutated. Of these residues, betaAsp 392 proved to be the only residue which is essential for both activities. However, two betaAsp 392 mutants were still partly active in catalyzing the cyclic reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADPH, suggesting that the mutations did not cause a global change but rather a subtle local change influencing the dissociation of NADP(H). It is proposed that betaAsp 392 together with th previously identified betaHis91 form part of a proton wire in transhydrogenase.

The cDNA sequence of proton-pumping nicotinamide nucleotide transhydrogenase from man and mouse.

cDNA clones for the human and mouse nicotinamide nucleotide transhydrogenases have been isolated and their sequences have been determined. Multiple alignments show that the functional proteins are encoded by single mRNAs. The deduced amino acid sequences are approximately 95% identical for the previously known bovine, and the human and mouse proteins. The major variable region is located in the presequence. It is proposed that all mammalian transhydrogenases have a similar structure.

Cell-free translation of mitochondrial nicotinamide nucleotide transhydrogenase.

Mammalian nicotinamide nucleotide transhydrogenase is translated as a 5000 daltons larger molecular weight precursor in a cell-free system programmed with rat liver polysomes. The mature rat liver enzyme had the same molecular weight as the purified beef heart enzyme, 115 000 daltons. The precursor was not processed in vitro by liver mitochondria or by a rat liver mitochondrial matrix fraction, nor did it appear to bind to mitochondria. In contrast, pre-FeS protein of the cytochrome bc1 complex was processed in the same samples by both mitochondria and matrix, suggesting an important difference in the processing mechanisms or in the efficiency of processing of the two precursors.

Biosynthesis of rat liver transhydrogenase in vivo and in vitro.

The biosynthesis of pyridine dinucleotide transhydrogenase, a homodimeric inner mitochondrial membrane redox-linked proton pump, has been studied in isolated rat hepatocytes. Newly synthesized transhydrogenase, having an apparent molecular weight identical to the enzyme of isolated liver mitochondria, was selectively immunoprecipitated from detergent extracts of isolated hepatocytes which were labeled with [35S]methionine. That the enzyme is a nuclear gene product is indicated since 1) synthesis was inhibited by cycloheximide, but not by chloramphenicol and 2) no synthesis could be demonstrated in hepatocyte ghosts which are competent only in mitochondrial translation. In addition to the mature form of the enzyme, a species about 2000 daltons larger was also immunoprecipitated from pulse-labeled cells. The half-life of the larger form during a subsequent chase at 37 degrees C was about 2 min, whereas the mature form was not degraded. The relationship between the two forms of the enzyme was established by in vitro studies. A protein approximately 2000 daltons larger than mature transhydrogenase was immunoisolated from a rabbit reticulocyte lysate system programmed with sucrose gradient fractionated rat liver mRNA. This protein was converted to a species having the same size as mature enzyme after incubation with either intact rat liver mitochondria or a soluble matrix fraction derived from mitoplasts. These studies indicate that transhydrogenase is synthesized in the cytoplasm as a higher molecular weight precursor which is post-translationally processed to the mature protein by a soluble matrix protease during or after membrane insertion.