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Buchanan, B. B.

Publications and source records attributed to Buchanan, B. B..

10 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS1991

Thioredoxin and evolution

Comparisons of primary structure have revealed significant homology between the m type thioredoxins of chloroplasts and the thioredoxins from a variety of bacteria. Chloroplast thioredoxin f, by comparison, remains an enigma: certain residues are invariant with those of the other thioredoxins, but a phylogenetic relationship to bacterial or m thioredoxins seems distant. Knowledge of the evolutionary history of thioredoxin f is, nevertheless, of interest because of its role in photosynthesis. Therefore, we have attempted to gain information on the evolutionary history of chloroplast thioredoxin f, as well as m. Our goal was first to establish the utility of thioredoxin as a phylogenetic marker, and, if found suitable, to deduce the evolutionary histories of the chloroplast thioredoxins. To this end, we have constructed phylogenetic (minimal replacement) trees using computer analysis. The results show that the thioredoxins of bacteria and animals fall into distinct phylogenetic groups - the bacterial group resembling that derived from earlier 16s RNA analysis and the animal group showing a cluster consistent with known relationships. The chloroplast thioredoxins show a novel type of phylogenetic arrangement: one m type aligns with its counterpart of eukaryotic algae, cyanobacteria and other bacteria, whereas the second type (f type) tracks with animal thioredoxin. The results give new insight into the evolution of photosynthesis.

Buchanan, B. B.↗
NASA NTRS1991

Identification of an NADP/thioredoxin system in Chlamydomonas reinhardtii

The protein components of the NADP/thioredoxin system, NADP-thioredoxin reductase (NTR) and thioredoxin h, have been purified and characterized from the green alga, Chlamydomonas reinhardtii. The analysis of this system confirms that photoautotrophic Chlamydomonas cells resemble leaves in having both an NADP- and ferrodoxin-linked thioredoxin redox system. Chlamydomonas thioredoxin h, which is smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than thioredoxin m from the same source, cross-reacted with antisera to thioredoxin h from spinach (Spinacia oleracea L.) and wheat germ (Triticum vulgaris L.) but not with antisera to m or f thioredoxins. In these properties, the thioredoxin h resembled a thioredoxin from Chlamydomonas, designated Ch1, whose sequence was reported recently (P. Decottignies et al., 1991, Eur. J. Biochem. 198, 505-512). The differential reactivity of thioredoxin h with antisera was used to demonstrate that thioredoxin h is enriched outside the chloroplast. The NTR was purified from Chlamydomonas using thioredoxin h from the same source. Similar to its counterpart from other organisms, Chlamydomonas NTR had a subunit size of approx. 36 kDa and was specific for NADPH. Chlamydomonas NTR effectively reduced thioredoxin h from the same source but showed little activity with the other thioredoxins tested, including spinach thioredoxin h and Escherichia coli thioredoxin. Comparison of the reduction of Chlamydomonas thioredoxins m and h by each of the endogenous thioredoxin reductases, NTR and ferredoxin-thioredoxin reductase, revealed a differential specificity of each enzyme for thioredoxin. Thus, NTR showed increased activity with thioredoxin h and ferredoxin-thioredoxin reductase with thioredoxins m and f.

Non-NASA Center↗
NASA NTRS1991

Ferredoxin and ferredoxin-NADP reductase from photosynthetic and nonphotosynthetic tissues of tomato

Ferredoxin and ferredoxin-NADP+ oxidoreductase (FNR) were purified from leaves, roots, and red and green pericarp of tomato (Lycopersicon esculentum, cv VFNT and cv Momotaro). Four different ferredoxins were identified on the basis of N-terminal amino acid sequence and charge. Ferredoxins I and II were the most prevalent forms in leaves and green pericarp, and ferredoxin III was the most prevalent in roots. Red pericarp of the VFNT cv yielded variable amounts of ferredoxins II and III plus a unique form, ferredoxin IV. Red pericarp of the Momotaro cv contained ferredoxins I, II, and IV. This represents the first demonstration of ferredoxin in a chromoplast-containing tissue. There were no major differences among the tomato ferredoxins in absorption spectrum or cytochrome c reduction activity. Two forms of FNR were present in tomato as judged by anion exchange chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. FNR II had a lower apparent relative molecular weight, a slightly altered absorption spectrum, and a lower specific activity for cytochrome c reduction than FNR I. FNR II could be a partially degraded form of FNR I. The FNRs from the different tissues of tomato plants all showed diaphorase activity, with FNR II being more active than FNR I. The presence of ferredoxin and FNR in heterotrophic tissues of tomato is consistent with the existence of a nonphotosynthetic ferredoxin/FNR redox pathway to support the function of ferredoxin-dependent enzymes.

NASA Discipline Number 52-30↗
NASA NTRS1990

A reverse KREBS cycle in photosynthesis: consensus at last

The Krebs cycle (citric acid or tricarboxylic acid cycle), the final common pathway in aerobic metabolism for the oxidation of carbohydrates, fatty acids and amino acids, is known to be irreversible. It liberates CO2 and generates NADH whose aerobic oxidation yields ATP but it does not operate in reverse as a biosynthetic pathway for CO2 assimilation. In 1966, our laboratory described a cyclic pathway for CO2 assimilation (Evans, Buchanan and Arnon 1966) that was unusual in two respects: (i) it provided the first instance of an obligate photoautotroph that assimilated CO2 by a pathway different from Calvin's reductive pentose phosphate cycle (Calvin 1962) and (ii) in its overall effect the new cycle was a reversal of the Krebs cycle. Named the 'reductive carboxylic acid cycle' (sometimes also called the reductive tricarboxylic acid cycle) the new cycle appeared to be the sole CO2 assimilation pathway in Chlorobium thiosulfatophilum (Evans et al. 1966) (now known as Chlorobium limicola forma thiosulfatophilum). Chlorobium is a photosynthetic green sulfur bacterium that grows anaerobically in an inorganic medium with sulfide and thiosulfate as electron donors and CO2 as an obligatory carbon source. In the ensuing years, the new cycle was viewed with skepticism. Not only was it in conflict with the prevailing doctrine that the 'one important property ... shared by all (our emphasis) autotrophic species is the assimilation of CO2 via the Calvin cycle' (McFadden 1973) but also some of its experimental underpinnings were challenged. It is only now that in the words of one of its early skeptics (Tabita 1988) 'a long and tortuous controversy' has ended with general acceptance of the reductive carboxylic acid cycle as a photosynthetic CO2 assimilation pathway distinct from the pentose cycle. (Henceforth, to minimize repetitiveness, the reductive pentose phosphate cycle will often be referred to as the pentose cycle and the reductive carboxylic acid cycle as the carboxylic acid cycle.) Aside from photosynthetic pathways which are the focus of this article, CO2 assimilation is also known to sustain autotrophic growth via the acetyl-CoA pathway (Wood et al. 1986). Our aim here is to discuss (i) the findings that led our group to the discovery of the reductive carboxylic acid cycle, (ii) the nature and resolution of the controversy that followed, and (iii) the possible evolutionary implications of the cycle as an ancient mechanism for photosynthetic CO2 assimilation that preceded the pentose cycle and served as a precursor of the Krebs cycle in aerobic metabolism.

Non-NASA Center↗
NASA NTRS1990

The ferredoxin-thioredoxin system of a green alga, Chlamydomonas reinhardtii: identification and characterization of thioredoxins and ferredoxin-thioredoxin reductase components

The components of the ferredoxin-thioredoxin (FT) system of Chlamydomonas reinhardtii have been purified and characterized. The system resembled that of higher plants in consisting of a ferredoxin-thioredoxin reductase (FTR) and two types of thioredoxin, a single f and two m species, m1 and m2. The Chlamydomonas m and f thioredoxins were antigenically similar to their higher-plant counterparts, but not to one another. The m thioredoxins were recognized by antibodies to both higher plant m and bacterial thioredoxins, whereas the thioredoxin f was not. Chlamydomonas thioredoxin f reacted, although weakly, with the antibody to spinach thioredoxin f. The algal thioredoxin f differed from thioredoxins studied previously in behaving as a basic protein on ion-exchange columns. Purification revealed that the algal thioredoxins had molecular masses (Mrs) typical of thioredoxins from other sources, m1 and m2 being 10700 and f 11500. Chlamydomonas FTR had two dissimilar subunits, a feature common to all FTRs studied thus far. One, the 13-kDa ("similar") subunit, resembled its counterpart from other sources in both size and antigenicity. The other, 10-kDa ("variable") subunit was not recognized by antibodies to any FTR tested. When combined with spinach, (Spinacia oleracea L.) thylakoid membranes, the components of the FT system functioned in the light activation of the standard target enzymes from chloroplasts, corn (Zea mays L.) NADP-malate dehydrogenase (EC 1.1.1.82) and spinach fructose 1,6-bisphosphatase (EC 3.1.3.11) as well as the chloroplast-type fructose 1,6-bisphosphatase from Chlamydomonas. Activity was greatest if ferredoxin and other components of the FT system were from Chlamydomonas. The capacity of the Chlamydomonas FT system to activate autologous FBPase indicates that light regulates the photosynthetic carbon metabolism of green algae as in other oxygenic photosynthetic organisms.

NASA Discipline Exobiology↗
NASA NTRS1989

Activation of a chloroplast type of fructose bisphosphatase from Chlamydomonas reinhardtii by light-mediated agents

A chloroplast type of fructose-1,6-bisphosphatase, a central regulatory enzyme of photosynthetic carbon metabolism, has been partially purified from Chlamydomonas reinhardtii. Unlike its counterpart from spinach chloroplasts, the algal FBPase showed a strict requirement for a dithiol reductant irrespective of Mg2+ concentration. The enzymes from the two sources resembled each other immunologically, in subunit molecular mass and response to pH. In the presence of dithiothreitol, the pH optimum for both the algal and spinach enzymes shifted from 8.5 to a more physiologic value of 8.0 as the Mg2+ concentration was increased from 1 to 16 mM. At 1 mM Mg2+, a concentration estimated to be close to physiological, the Chlamydomonas FBPase was active only in the presence of reduced thioredoxin and was most active with Chlamydomonas thioredoxin f. Under these conditions, the enzyme showed a pH optimum of 8.0. The data suggest that the Chlamydomonas enzyme resembles its spinach counterpart in most respects, but it has a stricter requirement for reduction and less strict reductant specificity. A comparison of the properties of the FBPases from Chlamydomonas and spinach will be helpful for elucidating the mechanism of the reductive activation of this enzyme.

NASA Discipline Exobiology↗
NASA NTRS1987

Thioredoxin and NADP-thioredoxin reductase from cultured carrot cells

Dark-grown carrot (Daucus carota L.) tissue cultures were found to contain both protein components of the NADP/thioredoxin system--NADP-thioredoxin reductase and the thioredoxin characteristic of heterotrophic systems, thioredoxin h. Thioredoxin h was purified to apparent homogeneity and, like typical bacterial counterparts, was a 12-kdalton (kDa) acidic protein capable of activating chloroplast NADP-malate dehydrogenase (EC 1.1.1.82) more effectively than fructose-1,6-bisphosphatase (EC 3.1.3.11). NADP-thioredoxin reductase (EC 1.6.4.5) was partially purified and found to be an arsenite-sensitive enzyme composed of two 34-kDa subunits. Carrot NADP-thioredoxin reductase resembled more closely its counterpart from bacteria rather than animal cells in acceptor (thioredoxin) specificity. Upon greening of the cells, the content of NADP-thioredoxin-reductase activity, and, to a lesser extent, thioredoxin h decreased. The results confirm the presence of a heterotrophic-type thioredoxin system in plant cells and raise the question of its physiological function.

Non-NASA Center↗
NASA NTRS1987

Ion-exchange chromatography separates activities synthesizing and degrading fructose 2,6-bisphosphate from C3 and C4 leaves but not from rat liver

Fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase were separated on the basis of charge from leaves of C3 (spinach, lettuce, and pea) and C4 (sorghum and amaranthus) plants but not from rat liver--a tissue known to contain a bifunctional enzyme with both activities. [2-32P]Fructose 2,6-bisphosphate binding experiments also suggest that the major forms of these activities reside on different proteins in leaves.

NASA Discipline Exobiology↗
NASA NTRS1986

Thioredoxins in evolutionarily primitive organisms

Thioredoxins are low molecular weight redox proteins, alternating between the S-S (oxidized) and SH (reduced) states, that function in a number of biochemical processes, including DNA synthesis, DNA replication, and enzyme regulation. Until recently, reduced ferredoxin was known to serve as the source of reducing power for the reduction of thioredoxins only in oxygenic photosynthetic cells. In all other organisms, the source of hydrogen (electrons) for thioredoxin reduction was considered to be NADPH. It was found that Clostridium pasteurianum, an anaerobic organism normally living in the soil unexposed to light, resembles photosynthetic cells in using ferredoxin for the reduction of thioredoxin. The results reveal the existence of a pathway in which ferredoxin, provides the reducing power for the reduction of thioredoxin via the flavoprotein enzyme, ferredoxinthioredoxin reductase. In related studies, it was found that Chromatium vinosum, an anaerobic photosynthetic purple sulfur bacterium, resembles evolutionarily more advanced micro-organisms in having an NADP-thioredoxin system composed of a single thioredoxin which is reduced by NADPH via NADP-thioredoxin reductase. The adoption of the NADP-thioredoxin system by Chromatium seems appropriate in view of evidence tha the organi sm utilizes ATP-driven reverse electron transport. Finally, results of research directed towards the identification of target enzymes of the ferredoxin/thioredoxin system in a cyanobacterium (Nostoc muscorum), show that thioredoxin-linked photosynthetic enzymes of cyanobateria are similar to those of chloroplasts. It now seems that the ferredoxin/thioredoxin system functions in regulating CO2 assimilation via the reductive pentose phosphate cycle in oxygenic but not anoxygenic photosynthetic cells.

Buchanan, B. B.↗