Biomedical subjects

Guy T Hanke

Publications and source records attributed to Guy T Hanke.

4 recordsLinked to original sources

PubMed2007

The effect of Glc6P uptake and its subsequent oxidation within pea root plastids on nitrite reduction and glutamate synthesis.

In roots, nitrate assimilation is dependent upon a supply of reductant that is initially generated by oxidative metabolism including the pentose phosphate pathway (OPPP). The uptake of nitrite into the plastids and its subsequent reduction by nitrite reductase (NiR) and glutamate synthase (GOGAT) are potentially important control points that may affect nitrate assimilation. To support the operation of the OPPP there is a need for glucose 6-phosphate (Glc6P) to be imported into the plastids by the glucose phosphate translocator (GPT). Competitive inhibitors of Glc6P uptake had little impact on the rate of Glc6P-dependent nitrite reduction. Nitrite uptake into plastids, using (13)N labelled nitrite, was shown to be by passive diffusion. Flux through the OPPP during nitrite reduction and glutamate synthesis in purified plastids was followed by monitoring the release of (14)CO(2) from [1-(14)C]-Glc6P. The results suggest that the flux through the OPPP is maximal when NiR operates at maximal capacity and could not respond further to the increased demand for reductant caused by the concurrent operation of NiR and GOGAT. Simultaneous nitrite reduction and glutamate synthesis resulted in decreased rates of both enzymatic reactions. The enzyme activity of glucose 6-phosphate dehydrogenase (G6PDH), the enzyme supporting the first step of the OPPP, was induced by external nitrate supply. The maximum catalytic activity of G6PDH was determined to be more than sufficient to support the reductant requirements of both NiR and GOGAT. These data are discussed in terms of competition between NiR and GOGAT for the provision of reductant generated by the OPPP.

Electrons↗

PubMed2006

Higher order structure contributes to specific differences in redox potential and electron transfer efficiency of root and leaf ferredoxins.

Plant type ferredoxin (Fd) is a small [2Fe-2S] cluster containing electron-transfer protein with a highly negative redox potential. Higher plants contain different iso-protein types of Fd in roots and leaves, reflecting the difference in redox cascades between these two tissues. We have combined subdomains of leaf and root Fds in recombinant chimeras, to examine structural effects and the relationship between groups of residues on redox potential, electron transfer, and protein-protein interactions. All chimeras had redox potentials that were intermediate to the wild type leaf and root Fds. Surprisingly, the largest differences resulted from exchange of the N-terminus, the region farthest from the redox center. Homology modeling and energy minimization calculations suggest that the N-terminal chimeras may indirectly influence redox potentials by structurally perturbing the active site. Measurements of electron transport and protein interaction indicate that synergistic interaction between the C- and N-terminal of root Fd bestows a specific high affinity for accepting electrons in the root type electron cascade, and that there is discrimination against photosynthetic electron donation to root Fd based on the C-terminus of the molecule. Taken together, the experimental and computational studies support a model in which higher order structure contributes to iso-protein specific interaction and electron-transfer properties.

Amino Acid Sequence↗

PubMed2005

Three maize leaf ferredoxin:NADPH oxidoreductases vary in subchloroplast location, expression, and interaction with ferredoxin.

In higher plants, ferredoxin (Fd):NADPH oxidoreductase (FNR) catalyzes reduction of NADP+ in the final step of linear photosynthetic electron transport and is also implicated in cyclic electron flow. We have identified three leaf FNR isoenzymes (LFNR1, LFNR2, and LFNR3) in maize (Zea mays) chloroplasts at approximately equivalent concentrations. Fractionation of chloroplasts showed that, while LFNR3 is an exclusively soluble enzyme, LFNR1 is only found at the thylakoid membrane and LFNR2 has a dual location. LFNR1 and LFNR2 were found to associate with the cytochrome b6f complex following its partial purification. We cloned LFNR3 and produced all three isoenzymes as stable, soluble proteins. Measurement of Fd reduction ability showed no significant differences between these recombinant enzymes. Column chromatography revealed variation between the interaction mechanisms of LFNR1 and LFNR2 with Fd, as detected by differential dependence on specific intermolecular salt bridges and variable sensitivity of interactions to changes in pH. A comparison of LFNR transcripts in leaves of plants grown on variable nitrogen regimes revealed that LFNR1 and LFNR2 transcripts are relatively more abundant under conditions of high demand for NADPH. These results are discussed in terms of the functional differentiation of maize LFNR isoenzymes.

Amino Acid Sequence↗

PubMed2004

Fd : FNR Electron Transfer Complexes: Evolutionary Refinement of Structural Interactions.

During the evolution of higher-plant root and leaf-type-specific Fd : FNR complexes from an original cyanobacterial type progenitor, rearrangement of molecular interaction has altered the relative orientation of prosthetic groups and there have been changes in complex induced conformational change. Selection has presumably worked on mutation of residues responsible for interaction between the two proteins, favoring optimized electron flow in a specific direction, and efficient dissociation following specific oxidation of leaf Fd and reduction of root Fd. Major changes appear to be: loss in both leaf and root complexes of a cyanobacterial mechanism that ensures Fd dissociation from the complex following change in Fd redox state, development of a structural rearrangement of Fd on binding to leaf FNR that results in a negative shift in Fd redox potential favorable to photosynthetic electron flow, creation of a vacant space in the root Fd:FNR complex that may allow access to the redox centers of other enzymes to ensure efficient channeling of heterotrophic reductant into bioassimilation. Further structural analysis is essential to establish how root type FNR distinguishes between Fd isoforms, and discover how residues not directly involved in intermolecular interactions may affect complex formation.

Journal Article↗