Molecular dynamics analysis of the structural and dynamic properties of the functionally enhanced hepta-variant of mouse 5-aminolevulinate synthase

Heme biosynthesis, a complex, multistage, and tightly controlled process, starts with 5-aminolevulinate (ALA) production, which, in metazoa and certain bacteria, is a reaction catalyzed by 5-aminolevulinate synthase (ALAS), a pyridoxal 5′-phosphate (PLP)-dependent enzyme. Functional aberrations in ALAS are associated with several human diseases. ALAS can adopt open and closed conformations, with segmental rearrangements of a C-terminal, 16-amino acid loop and an α-helix regulating accessibility to the ALAS active site. Of the murine erythroid ALAS (mALAS2) forms previously engineered to assess the role of the flexible C-terminal loop versus mALAS2 function one stood out due to its impressive gain in catalytic power. To elucidate how the simultaneously introduced seven mutations of this activity-enhanced variant affected structural and dynamic properties of mALAS2, we conducted extensive molecular dynamics simulation analysis of the dimeric forms of wild-type mALAS2, hepta-variant and Rhodobacter capsulatus ALAS (aka R. capsulatus HemA). This analysis revealed that the seven simultaneous mutations in the C-terminal loop, which extends over the active site of the enzyme, caused the bacterial and murine proteins to adopt different conformations. Specifically, a new β-strand in the mutated ‘loop’ led to interaction with two preexisting β-strands and formation of an anti-parallel three-stranded β-sheet, which likely endowed the murine hepta-variant a more ‘stable’ open conformation than that of wild-type mALAS2, consistent with a kinetic mechanism involving a faster closed-to-open conformation transition and product release for the mutated than wild-type enzyme. Further, the dynamic behavior of the mALAS2 protomers was strikingly different in the two dimeric forms.     

Poly-3-hydroxybutyrate production by washed cells of Alcaligenes eutrophus; purification, characterisation and potential regulatory role of citrate synthase

Washed cells prepared from carbon-limited continuous cultures of Alcaligenes eutrophus synthesised poly-3-hydroxybutyrate (PHB) rapidly when supplied with glucose, dl-lactate or l-lactate. Unlike growing cultures, washed cells excreted significant amounts of pyruvate. The combined rates of PHB production (qPHB) and pyruvate excretion (qPyr) were linearly related to the rate of carbon substrate utilisation (qS), showing that washed cells behaved similarly to growing cultures when corrected for the absence of non-PHB biomass production. The addition of formate (as a potential source of NADH and/or ATP) significantly stimulated both qPHB and qPyr, but slightly decreased qS and substantially decreased the flux of carbon through the tricarboxylic acid cycle (qTCA). Citrate synthase activity of broken cells was inhibited by physiological concentrations of NADH, but not of ATP, in a manner that was not reversible by AMP. Citrate synthase was purified and shown to be a “large” form of the enzyme (M _r 227,000), comprising a single type of subunit (M _r 47,000) as found in several other gram-negative aerobes. The potential role of citrate synthase in the regulation of PHB production via its ability to control carbon flux into the tricarboxylic acid cycle is discussed. Received: 14 March 1997 / Accepted: 9 July 1997     

Pisatin metabolism in pea (Pisum sativum L.) cell suspension cultures

Cell suspension cultures were established from germinating pea (Pisum sativum L.) seeds. This cell culture, which accumulated pisatin, consisted mostly of single cells containing a few cell aggregates. The cells responded to treatment with a yeast glucan preparation with transient accumulation of pisatin in both cells and culture media. Addition of pisatin to cell cultures resulted in increased synthesis of pisatin. Phenylalanine ammonia-lyase, chalcone synthase and isoflavone reductase activities were present in untreated cells. Upon treatment with an elicitor preparation the activities of the first two enzymes showed a rapid, transient increase up to 20 hours after treatment. Isoflavone reductase showed a major and minor peak at 16 and 36 h, respectively, after elicitor treatment. The time course of the enzyme activity and pisatin accumulation is consistent with an elicitor-mediated response.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - IFR isoflavone reductase - 2iP 6-(dimethylallylamino)-purine - MS Murashige & Skoog basal salt medium - PAL phenylalanine ammonia-lyase - PMSF phenylmethylsulfonyl fluoride - POPOP 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene - PPO 2,5-diphenyloxazole     

Transfer of a grapevine stilbene synthase gene to rice (Oryza sativa L.)

A gene derived from grapevine (Vitis vinifera) coding for stilbene synthase has been transferred into protoplasts of the commercially important japonica rice cultivar Nipponbare using PEG-mediated direct gene transfer. Transgenic plants were regenerated from calli selected on kanamycin. Southern blot analysis of genomic DNA isolated from regenerants and progeny plants demonstrated that the stilbene synthase gene is stably integrated in the genome of transgenic rice plants and inherited in the offspring. The transient formation of stilbene-synthase-specific mRNA shortly after inoculation with the fungus of the rice blast Pyricularia oryzae has demonstrated that the grapevine stilbene synthase promoter is also active in monocotyledonous plants. Preliminary results indicate an enhanced resistance of transgenic rice to P. oryzae. Received: 1 July 1996 / Revision received: 5 November 1996 / Accepted: 30 November 1996     

Biosynthesis of 5-aminolevulinic Acid via the Shemin Pathway in a Green Sugar Beet Callus

5-Aminolevulinic acid synthase (ALAS) has been detected in a normal (auxin- and cytokinin-dependent) green sugar beet callus under light and under darkness. ALAS activity was lower when the callus was grown under light. The supply of precursors of the Shemin pathway (glycine and succinate) to dark-grown callus enhanced considerably the capacity of the 5-aminolevulinic acid (ALA) formation. Glutamate, -aminobutyrate or -ketoglutarate also increased ALA accumulation. Such an accumulation was also obtained after inhibition of polyamine synthesis. The results show that glutamate or its derivatives might feed the Shemin pathway in conditions preventing glutamate to be used through the Beale pathway.     

With chlorophyll pigments from prolamellar bodies to light-harvesting complexes

The biosynthetic chain leading from 5-aminolevulinic acid to chlorophyll is localised to the plastid. Many of the enzymes are nuclear-encoded. NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33) is one such enzyme which is encoded by two different genes and can exist in an A and a B form. Its import into the plastid seems to be facilitated when protochlorophyllide is present in the chloroplast envelope. Within the plastid the reductase is assembled to thylakoids or prolamellar bodies. The specific properties of the reductase together with the specific properties of the lipids present in the etioplast inner membranes promote the formation of the three-dimensional regular network of the prolamellar bodies. The reductase forms a ternary complex with protochlorophyllide and NADPH that gives rise to different spectral forms of protochlorophyllide. Light transforms protochlorophyllide into chlorophyllide and this photoreaction induces a conformational change in the reductase protein which leads to a process of disaggregation of enzyme, pigment aggregates and membranes, which can be followed spectroscopically and with electron microscopy. The newly formed chlorophyllide is esterified by a membrane-bound nuclear-encoded chlorophyll synthase and the chlorophyll molecule is then associated with proteins into active pigment protein complexes in the photosynthetic machinery.     

Control of plant disease by perturbation of fungal polyamine metabolism

The diamine putrescine and the polyamines spermidine and spermine are ubiquitous in nature and are essential for cell proliferation. Since polyamine biosynthesis in plants can start from either ornithine or arginine, while fungal polyamine biosynthesis appears to utilise only the ornithine route, it was suggested that specific inhibition of fungal polyamine biosynthesis should be lethal. Indeed, inhibitors of polyamine biosynthesis, e.g. the ornithine decarboxylase inhibitor α-difluoromethylornithine, have been shown to inhibit fungal growth in vitro and to control fungal infections on a variety of plants under glasshouse and field conditions. It is now known that polyamine analogues can perturb polyamine metabolism leading to powerful antiproliferative effects in cancer cells. This paper reviews the results of a research programme focused on the synthesis and evaluation of putrescine analogues as novel fungicides. A number of aliphatic, alicyclic and cyclic diamines have been shown to possess considerable fungicidal activity, but although many of these compounds perturb polyamine metabolism in fungal cells, such changes are not considered sufficient to account for the observed antifungal effects. More recent work on spermidine analogues is also described.     

Elicitor-mediated induction of anthraquinone biosynthesis and regulation of isopentenyl diphosphate isomerase and farnesyl diphosphate synthase activities in cell suspension cultures of Cinchona robusta How.

Treatment of a Cinchona robusta How. cell suspension culture with a homogenate of Phytophthora cinnamomi resulted in cessation of growth and a rapid induction of the biosynthesis of anthraquinone-type phytoalexins. The strongest induction of anthraquinone biosynthesis was obtained when the elicitor was added in the early growth phase of the growth cycle. The accumulation of anthraquinones was accompanied by a tri-phasic response in the activity of isopentenyl diphosphate (IPP) isomerase (EC 5.3.3.2): phase I was characterised by a rapid induction of activity, reaching a maximum at 12 h after elicitation. During phase II, IPP isomerase rapidly decreased to levels below those found in untreated cells. At phase III, IPP isomerase activity increased again, reaching a second maximum at about 72 h after elicitation. During phase I, the activity of farnesyl diphosphate synthase (EC 2.5.1.10) was found to be suppressed. Extraction and assay conditions were optimised for IPP isomerase. The presence of Mn²⁺ in the incubation buffer resulted in a marked increase in the activity of the enzymes obtained from cells in phase I. The induction of IPP isomerase in combination with a concomitant inhibition of farnesyl diphosphate synthase might result in an efficient channeling of C₅-precursors into phytoalexin biosynthesis. Received: 23 August 1996 / Accepted: 20 March 1997