Structure of pyridine nucleotide transhydrogenase from Azotobacter vinelandii.

1. Pyridine nucleotide transhydrogenase of Azotobacter vinelandii purified by affinity chromatography consists of a mixture of polydisperse rods at neutral pH. No other structures are seen by electron microscopy. 2. At high pH (8.5--9.0) the rods depolymerize. Complete depolymerization can be achieved in 0.1 M Tris-Cl pH 9.0. The depolymerized enzyme has a molecular weight of 421000 (sedimentation equilibrium), its sedimentation coefficient s20, w = 15 S and its Stokes' radius Rs = 7 nm. Since gel electrophoresis in the presence of sodium dodecyl sulphate shows that transhydrogenase consists of a single polypeptide chain of molecular weight (54 +/- 2) X 10(3) it follows that the depolymerized enzyme has an octameric quaternary structure. We propose that this octamer serves as the functional monomeric unit ('unimer') from which the polymeric form of transhydrogenase is constructed. 3. Gel filtration and sucrose gradient centrifugation studies of cell-free extracts from A. vinelandii show the unimer to be the predominant active species.     

Repression of Escherichia coli pyridine nucleotide transhydrogenase by leucine.

Addition of 0.1% casein hydrolysate to a minimal growth medium decreased membrane-bound transhydrogenase activity in Escherichia coli by about 80%. Of the amino acids added individually to the growth medium, only leucine and, to a lesser extent, methionine and alanine were effective, alpha-Ketoisocaproate- and leucine-containing peptides repressed the activity, and leucine also repressed activity in adenyl cyclase-deficient and relaxed strains. Derepression of transhydrogenase followed the removal of leucine from the growth medium and was sensitive to rifampin and chloramphenicol. A phosphoglucoisomerase-deficient strain that was forced to use the hexose monophosphate shunt exclusively had normal levels of transhydrogenase, which was repressed by leucine. Transhydrogenase activity doubled in mutants lacking either of the shunt dehydrogenases but was still repressed by leucine. In strains constitutive for the leucine biosynthetic operon, transhydrogenase was repressed by leucine but in strains livR and lst R, with leucine transport resistant to leucine repression, transhydrogenase was not repressed by leucine. These data suggest that transhydrogenase may have a function in the transport of branched-chain amino acids. In a hisT strain (which has altered leucyl-tRNA), transhydrogeanse was at a repressed level without the addition of leucine, suggesting that leucyl-tRNA may be involved in the regulation.     

Energy-linked mitochondrial pyridine nucleotide transhydrogenase of adult Hymenolepis diminuta.

Employing "phosphorylating" submitochondrial particles as the source of pyridine nucleotide transhydrogenase, the occurrence of an energy-linked NADH----NADP+ transhydrogenation in the adult cestode Hymenolepis diminuta was demonstrated. The isolated particles displayed rotenone-sensitive NADH utilization and the reversible transhydrogenase, with the NADPH----NAD+ transhydrogenation being more prominent. Although not inhibiting the NADPH----NAD+ reaction, rotenone, but not oligomycin, inhibited the catalysis of NADH----NADP+ transhydrogenation. In the presence of rotenone, Mg2+ plus ATP stimulated by more than 3-fold NADH----NADP+ transhydrogenation. This stimulation was ATP specific and was abolished by EDTA or oligomycin. Succinate was essentially without effect on the NADH----NADP+ reaction. These data demonstrate the occurrence of an energy-linked transhydrogenation between NADH and NADP+ with energization resulting from either electron transport-dependent NADH oxidation or ATP utilization via the phosphorylating mechanism in accord with the preparation of "phosphorylating" particles. This is the first demonstration of an energy-linked transhydrogenation in the parasitic helminths and apparently in the invertebrates generally.     

Overexpression and biochemical characterization of soluble pyridine nucleotide transhydrogenase from Escherichia coli

The soluble pyridine nucleotide transhydrogenase (STH) is an energy-independent flavoprotein that directly catalyzes hydride transfer between NAD(H) and NADP(H) to maintain homeostasis of these two redox cofactors. The sth gene in Escherichia coli was cloned and expressed as a fused protein (EcSTH). The purified EcSTH displayed maximal activity at 35 °C, pH 7.5. Heat-inactivation studies showed that EcSTH retains 50% activity after 5 h at 50 °C. The enzyme was stable at 4 °C for 25 days. The apparent K(m) values of EcSTH were 68.29 μM for NADPH and 133.2 μM for thio-NAD(+) . The k(cat) /K(m) ratios showed that EcSTH had a 1.25-fold preference for NADPH over thio-NAD(+) . Product inhibition studies showed that EcSTH activity was strongly inhibited by excess NADPH, but not by thio-NAD(+) . EcSTH activity was enhanced by 2 mM adenine nucleotide and inhibited by divalent metal ions: Mn(2+) , Co(2+) , Zn(2+) , Ni(2+) and Cu(2+) . However, after preincubation for 30 min, most divalent metal ions had little effect on EcSTH activity, except Zn(2+) , Ni(2+) and Cu(2+) . The enzymatic analysis could provide the important basic knowledge for EcSTH utilizations.     

Topological analysis of the pyridine nucleotide transhydrogenase of Escherichia coli using proteolytic enzymes

The pyridine nucleotide transhydrogenase of Escherichia coli has an alpha 2 beta 2 structure (alpha: Mr, 54,000; beta: Mr, 48,700). Hydropathy analysis of the amino acid sequences suggested that the 10 kDa C-terminal portion of the alpha subunit and the N-terminal 20-25 kDa region of the beta subunit are composed of transmembranous alpha-helices. The topology of these subunits in the membrane was investigated using proteolytic enzymes. Trypsin digestion of everted cytoplasmic membrane vesicles released a 43 kDa polypeptide from the alpha subunit. The beta subunit was not susceptible to trypsin digestion. However, it was digested by proteinase K in everted vesicles. Both alpha and beta subunits were not attacked by trypsin and proteinase K in right-side out membrane vesicles. The beta subunit in the solubilized enzyme was only susceptible to digestion by trypsin if the substrates NADP(H) were present. NAD(H) did not affect digestion of the beta subunit. Digestion of the beta subunit of the membrane-bound enzyme by trypsin was not induced by NADP(H) unless the membranes had been previously stripped of extrinsic proteins by detergent. It is concluded that binding of NADP(H) induces a conformational change in the transhydrogenase. The location of the trypsin cleavage sites in the sequences of the alpha and beta subunits were determined by N- and C-terminal sequencing. A model is proposed in which the N-terminal 43 kDa region of the alpha subunit and the C-terminal 30 kDa region of the beta subunit are exposed on the cytoplasmic side of the inner membrane of E. coli. Binding sites for pyridine nucleotide coenzymes in these regions were suggested by affinity chromatography on NAD-agarose columns.     

Genetic mapping of a mutation affecting pyridine nucleotide transhydrogenase in Escherichia coli.

A mutation, pnt-1, causing loss of pyridine nucleotide transhydrogenase activity in Escherichia coli, was mapped by assaying for the enzyme in extracts of recombinant strains produced by conjugation, F-duction, and P1 transduction. The site of this mutation was near min 35, counterclockwise from man, and it co-transduced 59% with man. The mutation was associated with loss from the cell membrane fraction of energy-independent and adenosine 5'-triphosphate-dependent transhydrogenase activities, but reduced nicotinamide adenine dinucleotide dehydrogenase activity was not affected. Strains were constructed which lack phosphoglucoisomerase (pgi-2) and which carry either pnt+ or pnt-1. Although such strains, when grown on glucose, are expected to produce a large excess of reduced nicotinamide adenine dinucleotide phosphate, the growth rate was not affected by the pnt-1 allele.     

Cofactor regeneration by a soluble pyridine nucleotide transhydrogenase for biological production of hydromorphone

We have applied the soluble pyridine nucleotide transhydrogenase of Pseudomonas fluorescens to a cell-free system for the regeneration of the nicotinamide cofactors NAD and NADP in the biological production of the important semisynthetic opiate drug hydromorphone. The original recombinant whole-cell system suffered from cofactor depletion resulting from the action of an NADP(+)-dependent morphine dehydrogenase and an NADH-dependent morphinone reductase. By applying a soluble pyridine nucleotide transhydrogenase, which can transfer reducing equivalents between NAD and NADP, we demonstrate with a cell-free system that efficient cofactor cycling in the presence of catalytic amounts of cofactors occurs, resulting in high yields of hydromorphone. The ratio of morphine dehydrogenase, morphinone reductase, and soluble pyridine nucleotide transhydrogenase is critical for diminishing the production of the unwanted by-product dihydromorphine and for optimum hydromorphone yields. Application of the soluble pyridine nucleotide transhydrogenase to the whole-cell system resulted in an improved biocatalyst with an extended lifetime. These results demonstrate the usefulness of the soluble pyridine nucleotide transhydrogenase and its wider application as a tool in metabolic engineering and biocatalysis.     

Improved cell growth and biosynthesis of glycolic acid by overexpression of membrane-bound pyridine nucleotide transhydrogenase

Journal of Industrial Microbiology & Biotechnology - The non-conventional d-xylose metabolism called the Dahms pathway which only requires the expression of at least three enzymes to produce...     

Isolation and partial characterization of a mutant of Escherichia coli lacking pyridine nucleotide transhydrogenase.

A mutant of Escherichia coli lacking pyridine nucleotide transhydrogenase (EC 1.6.1.1) was isolated by assaying activity in clones of cells mutagenized with N-methyl-N′-nitro-N-nitrosoguanidine. The mutant is missing both energy-independent and energy-dependent transhydrogenase, but has normal NADH dehydrogenase and ATPase activities. Compared to the parental strain, the mutant has normal growth rates with glucose, glycerol, or succinate aerobically and with glucose or glycerol plus fumarate anaerobically. The aerobic growth yield with limiting glucose concentrations is also normal. These growth properties indicate that the enzyme is not an essential source of NADPH or ATP in vivo.     

Pyridine nucleotide transhydrogenase from Chlorella

Summary A pyridine nucleotide transhydrogenase which could facilitate the transfer of hydrogen between chloroplastic and non-chloroplastic pools of pyridine nucleotides, was found to be present in Chlorella pyrenoidosa.