Temperature and Light modulate the trans-delta3-hexadecenoic acid content of phosphatidylglycerol: light-harvesting complex II organization and non-photochemical quenching

The interaction of light and temperature in the modulation of the trans-delta3-hexadecenoic acid (trans-16:1) content of phosphatidylglycerol (PG) in winter rye (Secale cereale L.) was assessed and related to the organization of light-harvesting complex II (LHCII). Increasing the growth irradiance from 50 to 800 micromol m(-2) s(-1) at 20 degrees C resulted in a 1.8-fold increase in the trans-16:1 content in PG which favoured a greater preponderance of oligomeric LHCII, measured in vitro as the ratio of oligomer : monomer. Similar irradiance-dependent increases were observed during growth at 5 degrees C; however, 1.4-fold lower trans-16:1 contents and lower LHCII oligomer : monomer ratios were observed compared with growth at 20 degrees C and the same irradiance. These trends were also observed under natural field conditions. Thus, the accumulation of trans-16:1, as well as the organization of LHCII are modulated by both growth irradiance and growth temperature in an independent but additive manner. We also examined how changes in the supramolecular organization of LHCII affected the capacity for non-photochemical quenching (q(N)) and photoprotection via antenna quenching (q(O)). While q(O) was positively correlated with q(N), there was no correlation with either LHCII organization or xanthophyll cycle activity under the steady-state growth conditions examined.     

Biosynthesis of riboflavin: mechanism of formation of the ribitylamino linkage

Feeding experiments with Ashbya gossypii followed by NMR analysis of the resulting riboflavin showed incorporation of deuterium from D-[2-2H]ribose at C-2' and from D-[1-2H]ribose in the pro-R position at C-1' of the ribityl side chain. The results rule out an Amadori rearrangement mechanism for the reduction of the ribosylamino to the ribitylamino linkage and point to formation of a Schiff base that is reduced stereospecifically opposite to the face from which the oxygen has departed. As prerequisite for the analysis, the 1H NMR signals for the pro-R and pro-S hydrogens at C-1' of 6,7-dimethyl-8-ribityllumazine and riboflavin and its tetraacetate were assigned with the aid of synthetic stereospecifically deuteriated samples.     

Biosynthesis of D-alanyl-lipoteichoic acid: role of diglyceride kinase in the synthesis of phosphatidylglycerol for chain elongation.

Lipophilic and hydrophilic D-alanyl-lipoteichoic acids are elongated in Lactobacillus casei by the transfer of sn-glycerol 1-phosphate units from phosphatidylglycerol to the poly(glycerophosphate) moiety of the polymer. These sn-glycerol 1-phosphate units are added to the end of the poly(glycerophosphate) which is distal to the glycolipid anchor; 1,2-diglyceride results from this addition. The presence of a diglyceride kinase was suggested by the ATP-dependent phosphorylation of 1,2-diglyceride to phosphatidic acid. Inorganic phosphate was used to initiate the synthesis of lipophilic lipoteichoic acid (LTA) and the elongation of both lipophilic and hydrophilic LTA. Three observations suggest that phosphate and other anions play a role in the in vitro synthesis of LTA and its precursors. First, the conversion of 1,2-diglyceride to phosphatidic acid by diglyceride kinase was stimulated. Second, the synthesis of phosphatidylglycerol was increased. Third, the elongation of lipophilic and hydrophilic LTA was enhanced. These observations indicated that one effect of phosphate might be to enhance the utilization of 1,2-diglyceride for the synthesis of phosphatidic acid. This phospholipid is a precursor of phosphatidylglycerol, the donor of sn-glycerol 1-phosphate for elongation of LTA.     

Cellulose component of Acetobacter xylinum pellicle: morphology and its significance for the mechanism of microfibril formation

The cellulose component of native, minimally disturbed pellicles of Acetobacter sylinum has a three-dimensional, microfibrillar, interconnected, ‘brush-wood’ structure. This structure could not originate from a spinneret or extrusion mechanism of cellulose microfibril formation. It may be produced by a mechanism of spontaneous association and post factor crystallization of preformed, transient I → 4β glucans.     

FUdR induction of the X chromosome fragile site: evidence for the mechanism of folic acid and thymidine inhibition

Experiments designed to illuminate the mechanism by which folic acid and thymidine inhibit expression of the Xq28 fragile site in human lymphocytes are described. The fragile site is induced by 5-fluorodeoxyuridine (FUdR), a potent inhibitor of thymidylate synthetase, in the presence of otherwise inhibiting concentrations of folic acid but not in the presence of thymidine. These results indicate that the fragile site is expressed because of depletion of deoxythymidine monophosphate (dTMP) available for DNA synthesis.     

The Sec2 translocase of the chloroplast inner envelope contains a unique and dedicated SECE2 component

Biogenesis of chloroplasts involves a series of protein trafficking events. Nuclear‐encoded proteins are imported into the organelle, and then trafficked to various chloroplast locations by systems that are directly homologous to bacterial systems. Although the thylakoid‐based systems have been studied extensively, much less is known about the systems that reside and function in the inner envelope membrane. One such system, the Sec2 system, is homologous to both the thylakoid‐based Sec1 system and bacterial Sec systems, and may mediate both integration and translocation across the inner envelope. At a minimum, this system is expected to include three components, but only two, SCY2 and SECA2, have been identified in Arabidopsis. Bioinformatics and protein modeling were used to identify the protein encoded by At4g38490 as a candidate for the missing component (SECE2). Cellular localization, biochemistry, protein interaction assays in yeast, and co‐immunoprecipitation experiments were used to establish that this protein is an integral membrane protein of the inner envelope, and specifically interacts with the SCY2 component in vivo. Sequence analyses indicated that SECE2 proteins are found in a variety of plants, and differ from the thylakoid SECE1 proteins in a stroma‐exposed helical domain, which may contribute to their specificity. Finally, a genetic analysis indicated that SECE2 plays an essential role in plant growth and development.     

An hypothesis and evidence concerning the genetic components controlling tumor formation in Nicotiana

Summary The evidence discussed in this paper demonstrates unuqual genetic contribution of N. debneyi-tabacum and N. longiflora to the development of tumors in hybrids between them. Tumor formation depends upon the presence of a specific longiflora chromosome fragment in an otherwise debneyi-tabacum background and consequently is transmitted as a dominant trait. Tumor expression remains relatively constant among those segregants which carry the complete complements of N. debneyi-tabacum or N. tabacum along with the longiflora chromosome, but tumors fail to develop on plants with a few debneyi chromosomes on a diploid longiflora background. These results suggest that gene(s) on a single longiflora chromosome fragment are sufficient, whereas from N. debneyi or N. tabacum a large number of genes distributed over many chromosomes are required for tumor formation. An hypothesis concerning genetic components controlling tumor initiation (I) and expression (ee) is proposed, supported by these observations, and by previous studies both genetic and physiological, on another tumorous hybrid between N. glauca and N. langsdorffii. (I) and (ee), representing unequal contributions from two evolutionarily diverse species, must both be present in the hybrid for tumors to develop. Evidence is presented to indicate that N. longiflora and N. langsdorffii, belonging to the section Alatae, represent species carrying (I) and that N. debneyi, N. tabacum and N. glauca, belonging to different sections of the genus Nicotiana, are (ee) carriers. It is predicted that genetic analyses will reveal that the genes for tumor initiation (I) will be carried invariably by species of the section Alatae, or the so-called plus group of Näf, and genes modifying expression (ee) by species from other sections but belonging to the so-called minus group. Specific characterization of (I) and (ee) in biochemical terms is under investigation.     

阿卡波糖及其结构类似杂质生物合成与调控的研究进展

阿卡波糖是一种用于Ⅱ型糖尿病治疗的糖苷酶抑制剂,工业上采用游动放线菌Actinoplanes sp.生产.作为一种次级代谢产物,阿卡波糖生物合成复杂,游动放线菌发酵液中除阿卡波糖外还会积累大量结构类似杂质组分.由于缺乏对阿卡波糖及其杂质合成和调控机制的系统了解,难以通过调控来阻断或降低杂质合成,从而导致纯化难度大.近年...     

Purification and characterization of 9-hexadecenoic acid cis-trans isomerase from Pseudomonas sp. strain E-3

A 9-hexadecenoic acid cis-trans isomerase (9-isomerase) that catalyzed the cis-to-trans isomerization of the double bond of free 9-cis-hexadecenoic acid [16:1(9c)] was purified to homogeneity from an extract of Pseudomonas sp. strain E-3 and characterized. Electrophoresis of the purified enzyme on both incompletely denaturing and denaturing polyacrylamide gels yielded a single band of a protein with a molecular mass of 80 kDa, suggesting that the isomerase is a monomeric protein of 80 kDa. The 9-isomerase, assayed with 16:1(9c) as a substrate, had a specific activity of 22.8 μmol h^–1 (mg protein)^–1 and a K _m of 117.6 mM. The optimal pH and temperature for catalysis were approximately pH 7–8 and 30° C, respectively. The 9-isomerase catalyzed the cis-to-trans conversion of a double bond at positions 9, 10, or 11, but not that of a double bond at position 6 or 7 of cis-mono-unsaturated fatty acids with carbon chain lengths of 14, 15, 16, and 17. Octadecenoic acids with a double bond at position 9 or 11 were not susceptible to isomerization. These results suggest that 9-isomerase has a strict specificity for both the position of the double bond and the chain length of the fatty acid. The enzyme catalyzed the cis-to-trans isomerization of fatty acids in a free form, and in the presence of a membrane fraction it was also able to isomerize 16:1(9c) esterified to phosphatidylethanolamine. The 9-isomerase was strongly inhibited by catecholic antioxidants such as α-tocopherol and nordihydroguaiaretic acid, but was not inhibited by 1,10-phenanthroline or EDTA or under anoxic conditions. Based on these results, the possible mechanism of catalysis by this enzyme is discussed. Received: 21 May 1997 / Accepted: 5 September 1997     

Biosynthesis of 3-O-sulfated heparan sulfate: unique substrate specificity of heparan sulfate 3-O-sulfotransferase isoform 5

Heparan sulfate 3-O-sulfotransferase transfers sulfate to the 3-OH position of a glucosamine to generate 3-O-sulfated heparan sulfate (HS), which is a rare component in HS from natural sources. We previously reported that 3-O- sulfotransferase isoform 5 (3-OST-5) generates both an antithrombin-binding site to exhibit anticoagulant activity and a binding site for herpes simplex virus 1 glycoprotein D to serve as an entry receptor for herpes simplex virus. In this study, we characterize the substrate specificity of 3-OST-5 using the purified enzyme. The enzyme was expressed in insect cells using the baculovirus expression approach and was purified by using heparin-Sepharose and 3',5'-ADP- agarose chromatographies. As expected, the purified enzyme generates both an antithrombin binding site and a glycoprotein D binding site. We isolated IdoUA-AnMan3S and IdoUA-AnMan3S6S from nitrous acid-degraded 3-OST-5-modified HS (pH 1.5), suggesting that 3-OST-5 enzyme sulfates the glucosamine residue that is linked to an iduronic acid residue at the nonreducing end. We also isolated a disaccharide with a structure of DeltaUA2S-GlcNS3S and a tetrasaccharide with a structure of DeltaUA2S-GlcNS-IdoUA2S-GlcNH23S6S from heparin lyases-digested 3-OST-5-modified HS. Our results suggest that 3-OST-5 enzyme sulfates both N-sulfated glucosamine and N-unsubstituted glucosamine residues. Taken together, the results indicate that 3-OST-5 has broader substrate specificity than those of 3-OST-1 and 3-OST-3. The unique substrate specificity of 3-OST-5 serves as an additional tool to study the mechanism for the biosynthesis of biologically active HS.     

A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation.

The inhibition of GSK3 is required for the stimulation of glycogen and protein synthesis by insulin and the specification of cell fate during development. Here, we demonstrate that the insulin-induced inhibition of GSK3 and its unique substrate specificity are explained by the existence of a phosphate binding site in which Arg-96 is critical. Thus, mutation of Arg-96 abolishes the phosphorylation of "primed" glycogen synthase as well as inhibition by PKB-mediated phosphorylation of Ser-9. Hence, the phosphorylated N terminus acts as a pseudosubstrate, occupying the same phosphate binding site used by primed substrates. Significantly, this mutation does not affect phosphorylation of "nonprimed" substrates in the Wnt-signaling pathway (Axin and beta-catenin), suggesting new approaches to design more selective GSK3 inhibitors for the treatment of diabetes.     

Complex structures of the abscisic acid receptor PYL3/RCAR13 reveal a unique regulatory mechanism

Abscisic acid (ABA) controls many physiological processes and mediates adaptive responses to abiotic stresses. The ABA signaling mechanisms for abscisic acid receptors PYR/PYL/RCAR (PYLs) were reported. However, it remains unclear whether the molecular mechanisms are suitable for other PYLs. Here, complex structures of PYL3 with (+)-ABA, pyrabactin and HAB1 are reported. An unexpected trans-homodimer intermediate observed in the crystal is confirmed in solution. ABA-bound PYL3 greatly promotes the generation of monomeric PYL3, which can excessively increase the efficiency of inhibiting PP2Cs. Structure-guided biochemical experiments show that Ser195 accounts for the key intermediate. Interestingly, pyrabactin binds to PYL3 in a distinct nonproductive mode with gate closure, which sheds light on the design of agonists and antagonists for abscisic acid receptors. According to different conformations of ligand-bound PYLs, the PYLs family can be divided into three subclasses, among which the trans-dimeric subclass, represented by PYL3, reveals a distinct regulatory mechanism.     

An anticomplementary agent, K-76 monocarboxylic acid: its site and mechanism of inhibition of the complement activation cascade.

A monocarboxylic acid derivative (K-76 COOH) of K-76, purified from the culture filtrate of Stachybotrys complement I nov. sp. K-76, inhibits complement (C) activity. Its inhibitory action is mainly on C5 step. It strongly inhibits the generation of EAC1,4b,2a,3b,5b from C5 and EAC1,4b,2a,3b, and accelerates the decay of EAC1,4b,2a,3b,5b. It also causes some inhibition of the reactions of the reactions of C2,C3,C6,C7 and C9 with their respective preceding intermediate cells. It has no effect on the generation of EAC1,4b from C4 and EAC1, or of EAC-8 from C8 and EAC-7, and apparently increases the generation of EAC1,4b from C1 and EAC4b probably by inhibiting transfer or turnover of C1. It does not affect the rate of decay of EAC1,4b,2a or the T max of generation of EAC1,4b,2a, and it inhibits immune adherence only at high concentration. K-76 COOH also strongly inhibits hemolysis through the alternative pathway of C activation by cobra venom factor, but it does not seem to inhibit the early steps of the alternative pathway, because it has little affect on the consumption of C3 or the conversion of beta 1C to beta 1A on treatment of C serum with zymosan. K-76 COOH probably combines with C5 molecules, forming the inactive complexes, or it causes the structural alteration of C5.     

Adenosine triphosphatase of bean plastids: its properties and site of formation

Extracts of bean (Phaseolus vulgaris L.) etioplasts and chloroplasts contain a dithiothreitol-activated Ca²⁺-dependent adenosine triphosphatase which is inhibited by Dio-9. The chloroplast and etioplast enzymes have identical R_F values upon disc gel electrophoresis. Optimum extraction of the enzyme from either plastid preparation is accomplished with 1 mm ethylenediamine tetraacetic acid. Photophosphorylation capacity can be partially restored to depleted chloroplast preparations by addition of either the chloroplast or etioplast extract. These results suggest that the adenosine triphosphatase from etioplasts and chloroplasts represents a modified coupling factor for photophosphorylation.