Dominique Job

The crystal structure of plant acetohydroxy acid isomeroreductase complexed with NADPH, two magnesium ions and a herbicidal transition state analog determined at 1.65 A resolution.

Acetohydroxy acid isomeroreductase catalyzes the conversion of acetohydroxy acids into dihydroxy valerates. This reaction is the second in the synthetic pathway of the essential branched side chain amino acids valine and isoleucine. Because this pathway is absent from animals, the enzymes involved in it are good targets for a systematic search for herbicides. The crystal structure of acetohydroxy acid isomeroreductase complexed with cofactor NADPH, Mg2+ ions and a competitive inhibitor with herbicidal activity, N‐hydroxy‐N‐isopropyloxamate, was solved to 1.65 Å resolution and refined to an R factor of 18.7% and an R free of 22.9%. The asymmetric unit shows two functional dimers related by non‐crystallographic symmetry. The active site, nested at the interface between the NADPH‐binding domain and the all‐helical C‐terminus domain, shows a situation analogous to the transition state. It contains two Mg2+ ions interacting with the inhibitor molecule and bridged by the carboxylate moiety of an aspartate residue. The inhibitor‐binding site is well adjusted to it, with a hydrophobic pocket and a polar region. Only 24 amino acids are conserved among known acetohydroxy acid isomeroreductase sequences and all of these are located around the active site. Finally, a 140 amino acid region, present in plants but absent from other species, was found to make up most of the dimerization domain.

The solubilization of the basic subunit of sugarbeet seed 11-S globulin during priming and early germination

AbstractThe basic B-subunit of the seed storage protein 11-Sglobulin (an 11-S-legumin type protein) is the majorpolypeptide in soluble protein extracts from primedsugarbeet {Beta vulgaris L.) seeds. In contrast, only asmall amount of this protein is present in correspondingextracts from untreated dry mature seeds. Here, and asfor all 11-S globulins describe B-chaid so far isn, thelinked to other polypeptide(s), corresponding mostpresumably to an acidic A-chain, through the formationof disulphide bridge(s). Polyacrylamide gel electrophoreticanalyses of total and soluble protein extracts fromuntreated and primed seeds strongly indicate that thispriming-induced solubilizatio B-chain of resulte thne dfrom an endoproteolytic attack on the A-chain.Microscopical immunolocalization showed a uniformdistribution of the 11-S globuli B-chain oven r the proteinbodies of the embryonic cells from the untreated seeds.For the primed seeds, however, the B-subunit of 11-Sglobulin diffused out of the protein bodies and invadedthe cytosolic compartment. This phenomenon occurredindependently of the manner of priming, being observedwith hydroprimed and osmoprimed seeds, as well as withsugarbeet seeds that had been primed by a prehydrationtreatment. Quantitative analyses of the amounts ofsoluble 11-S globuli B-chain havn e enabled the primingof sugarbeet seeds to be optimized.Keywords: germination, 11-S globulin mobilization,immunocytochemistry, seed priming, storage proteins,sugarbeet.CorrespondenceAbbreviations: DTT = dithiothreitol; PAGE = polyacrylamidegel electrophoresis; PEG = polyethylene glycol; SDS =sodium dodecyl sulphate.

The use of an ELISA to quantitate the extent of 11S globulin mobilization in untreated and primed sugar beet seed lots

Abstract Seed priming (controlled imbibition) is a widely used technique for improving crop establishment, because it allows a reduction of the time to radicle emergence following seed imbibition and synchronization of individual seeds within seed lots with respect to germination timing. The major problem encountered in seed priming is the control of seed imbibition to a level permitting pre-germinative processes to proceed but that blocks radicle emergence. If not, the consequence of drying back the seeds to initial moisture content for storage purposes could be a total loss of the treated batch. This is because, as long as radicle growth has not begun, seeds may be re-dried without any permanent deleterious effects upon subsequent germination or growth. Recently, we reported the discovery of a molecular marker of sugar beet seed priming, corresponding to the basic B-subunit of the seed storage protein 11S globulin. An ELISA based upon this molecular marker has been used to analyse how different sugar beet seed lots respond to a priming treatment. The results demonstrate that this ELISA allows us to readily distinguish between the primed seeds and the corresponding untreated seeds.

The Role of Protein Biotinylation in the Development and Germination of Seeds

Biotin levels of pea seeds were investigated in order to characterize the temporal expression of the vitamin and the pattern of protein biotinylation that can be used to define developmental stages of seed formation and germination. These studies disclosed that metabolically active and quiescent tissues can be distinguished by their biotin content. In young developing seeds, as well as in germinating seeds, there is an excess of free versus bound biotin. In young seeds biotin is bound to the housekeeping biotin enzymes. A different pattern is observed in late stages of seed formation and in mature dry seeds. Here, protein-bound biotin is in excess with respect to free biotin. This is accounted for by the accumulation of a biotinylated polypeptide called SBP65 that behaves as a putative sink for the free vitamin, representing more than 90% of the total protein-bound biotin in mature seeds. Because the biotinylation domain of SBP65 differs markedly from that of presently known biotin enzymes, this protein defines a previously unrecognized class of biotinylated proteins. Furthermore, SBP65 shares many features in common with the LEA (Late Embryogenic Abundant) group of proteins. These results suggest that metabolic control of seed maturation and germination may occur through modulation in the level of an essential cofactor such as biotin. They also document an as yet undescribed role for some LEA proteins.

Biochemical and molecular bases of the carbon flux regulation between threonine and methionine in plants

Methionine and threonine are two essential amino acids belonging to the aspartate-derived family of amino acids. In plants, they both originate from the same precursor, O-phosphohomoserine (OPH) (Fig. 1). This contrasts with the situation seen in bacteria where branching between the two pathways occurs upstream of OPH, at the level of homoserine (Saint-Girons et al, 1998). Despite intensive studies (for reviews, see Giovanelli et al., 1980; Leustek, 1996; Azevedo et al., 1997; Hell, 1997; Ravanel, 1997; Ravanel et al., 1998b), the specific mechanisms that are responsible for homeostatic regulation of methionine and threonine in plants are not fully understood.

Cysteine Biosynthesis in Higher Plants

Higher plants and many microoganisms reduce sulfate to sulfide for the synthesis of cysteine, the sulfur precursor for the formation of glutathione and methionine. In plants, this reaction sequence from sulfate to sulfide is called assimilatory sulfate reduction and involves 1) activation of sulfate to adenosine phosphosulfate (APS) by ATP-sulfurylase, 2) reduction of the activated sulfate to free sulfite by APS reductase, 3) reduction of sulfite to sulfide by the ferredoxin-dependent sulfite reductase (1). The final steps of cysteine biosynthesis involve the combination of sulfide with activated serine. Serine acetyltransferase (SAT), catalyses the acetylation of L-serine in the presence of acetylCoA, yielding the carbon precursor of cysteine, O-acetylserine (Eqn 1). O-acetylserine (thiol) lyase (OASTL), converts O-acetylserine to L-cysteine in the presence of sulfide (Eqn 2).

Cystathionine gamma-synthase from Arabidopsis thaliana: purification and biochemical characterization of the recombinant enzyme overexpressed in Escherichia coli.

L-Serine+acetyl-CoA\to O-Acetylserine+CoAsh

Spinach Chloroplast O‐Acetylserine (thiol)‐Lyase Exhibits two Catalytically Non‐Equivalent Pyridoxal‐5′‐Phosphate‐Containing Active Sites

(1)