Thiamin pyrophosphokinase is required for thiamin cofactor activation in Arabidopsis

Thiamin pyrophosphate (TPP) is an essential enzyme cofactor required for the viability of all organisms. Whether derived from exogenous sources or through de novo synthesis, thiamin must be pyrophosphorylated for cofactor activation. The enzyme thiamin pyrophosphokinase (TPK) catalyzes the conversion of free thiamin to TPP in plants and other eukaryotic organisms and is central to thiamin cofactor activation. While TPK activity has been observed in a number of plant species, the corresponding gene/protein has until now not been identified or characterized for its role in thiamin metabolism. Here we report the functional identification of two Arabidopsis TPK genes, AtTPK1 and AtTPK2 and the enzymatic characterization of the corresponding proteins. AtTPK1 and AtTPK2 are biochemically redundant cytosolic proteins that are similarly expressed throughout different plant tissues. The essential nature of TPKs in plant metabolism is reflected in the observation that while single gene knockouts of either AtTPK1 or AtTPK2 were viable, the double mutant possessed a seedling lethal phenotype. HPLC analysis revealed the double mutant is nearly devoid of TPP and instead accumulates the precursor of the TPK reaction, free thiamin. These results suggest that TPK activity provides the sole mechanism by which exogenous and de novo derived thiamin is converted to the enzyme cofactor TPP.     

激酶组学在原核生物中的研究进展

激酶组学是近年研究较多的一类组学。激酶的磷酸化在调控细胞代谢和生理进程等方面起到重要作用,但原核生物激酶组学研究存在研究时间比真核生物晚、组氨酸激酶磷酸化无法检测等问题。通常通过对真核生物中激酶的研究,可以类比到原核生物中的激酶研究。对丝氨酸/苏氨酸激酶、酪氨酸激酶、组氨酸激酶在原核生物中的研究进展进行分析,并对激酶磷酸化的研究方法进行总结,阐明了激酶在原核生物中的重要调控作用。     

Thiamin dynamics during the adult life cycle of Atlantic salmon (Salmo salar)

Thiamin is an essential water-soluble B vitamin known for its wide range of metabolic functions and antioxidant properties. Over the past decades, reproductive failures induced by thiamin deficiency have been observed in several salmonid species worldwide, but it is unclear why this micronutrient deficiency arises. Few studies have compared thiamin concentrations in systems of salmonid populations with or without documented thiamin deficiency. Moreover, it is not well known whether and how thiamin concentration changes during the marine feeding phase and the spawning migration. Therefore, samples of Atlantic salmon (Salmo salar) were collected when actively feeding in the open Baltic Sea, after the sea migration to natal rivers, after river migration, and during the spawning period. To compare populations of Baltic salmon with systems without documented thiamin deficiency, a population of landlocked salmon located in Lake Vänern (Sweden) was sampled as well as salmon from Norwegian rivers draining into the North Atlantic Ocean. Results showed the highest mean thiamin concentrations in Lake Vänern salmon, followed by North Atlantic, and the lowest in Baltic populations. Therefore, salmon in the Baltic Sea seem to be consistently more constrained by thiamin than those in other systems. Condition factor and body length had little to no effect on thiamin concentrations in all systems, suggesting that there is no relation between the body condition of salmon and thiamin deficiency. In our large spatiotemporal comparison of salmon populations, thiamin concentrations declined toward spawning in all studied systems, suggesting that the reduction in thiamin concentration arises as a natural consequence of starvation rather than to be related to thiamin deficiency in the system. These results suggest that factors affecting accumulation during the marine feeding phase are key for understanding the thiamin deficiency in salmonids.     

Mutagenesis studies on TenA: a thiamin salvage enzyme from Bacillus subtilis

TenA catalyzes the hydrolysis of 4-amino-5-aminomethyl-2-methylpyrimidine and participates in the salvage of base-degraded thiamin. Here, we describe mutagenesis of the active site of TenA guided by structures of the enzyme complexed to a substrate analog and to the product. Catalytic roles for each of the active site residues are identified and a mechanism for the reaction is described.     

硫胺素、核黄素和烟酰胺对大鼠体重增长及脂代谢影响的实验研究

目的：B族维生素以辅酶的形式参与糖、脂肪和蛋白质代谢,本文观察硫胺素、核黄素和烟酰胺补充对高脂饲料诱导大鼠肥胖的影响。方法：采用预防肥胖模型法,2×2×2析因设计分为8组：高脂对照组（F0组）,高脂＋硫胺素（F1组）,高脂＋核黄素（F2组）,高脂＋烟酰胺（F3组）,高脂＋硫胺素＋核黄素（F4组）,高脂＋硫胺素＋烟酰胺（F5组）,高脂＋核黄素＋烟酰胺（F6组）,高脂＋硫胺素＋核黄素＋烟酰胺（F7组）,每组12只大鼠,给予高脂饲料喂养,同时硫胺素（100mg／kgbw／d）、核黄素（100mg／kgbw／d）、烟酰胺（250mg／kgbw／d）灌胃,另设正常对照组（C组）12只,普通饲料喂养,自来水灌胃,15周后,分析其体重、摄食量、体脂重量、血脂等实验前后的变化情况及各组动物之间的差别。结果：经过15周喂养后,高脂喂养大鼠体重比正常对照组平均增加了15．7％;而补充烟酰胺（F3）及联合硫胺素（F5）或核黄素（F6）以及三者联合补充（F7）组高脂喂养大鼠与高脂对照组（F0）相比,体重分别降低了35．0％,30．0％,30．1％和30．6％（P值均小于0．05）;甚至比正常对照组大鼠平均体重分别下降了22．8％,17．0％,17．0％和17．7％（P值均小于0．05）;而补充核黄素或和硫胺素组大鼠体重没有明显增加或降低（P值均大于0．05）。血脂分析结果显示高脂喂养并联合补充核黄素或／和烟酰胺和／或硫胺素组大鼠血清CHOL和LDL水平明显低于高脂对照组;而高脂喂养并联合补充烟酰胺（F3）及联合硫胺素（F5）或核黄素（F6）以及三者联合补充（F7）组大鼠LDL／HDL比值分别0．29、0．26、0．25和0．26,明显低于F0组的0．37（P值均小于0．05）。结论：大剂量烟酰胺可有效地调节血脂水平和控制肥胖大鼠的体重增长,而核黄素及硫胺素对控制肥胖     

The ABO, Lewis and related blood group antigens; a review of structure and biosynthesis

Abstract Numerous studies have shown that the antigenic determinants of the ABO blood group system are closely related in biochemical terms to the antigenic determinants of the Hh, P, Lewis and Ii blood group systems. The blood group antigens of each of these systems are formed by the addition of specific sugars to an oligosaccharide precursor chain which may be bound through sphingosine to fatty acids (glycolipid) or through serine or threonine to a peptide chain (glycoproteins). The direct gene products of each of these blood group systems are the glycosyltransferase enzymes which catalyse the addition of the specific sugar thus conferring the specified blood group activity to the glycolipid or glycoprotein molecule. The antigenic determinants of the ABO and Lewis systems in addition to red cells also exist in the body secretions in soluble form when the relevant genes are expressed in the phenotype. The antigens expressed on both the red cells and in the secretions are determined by the interaction of Hh, Sese, ABO and Lele genes.     

硫胺素、核黄素和烟酰胺对大鼠体重增长及脂代谢影响的实验研究(英文)

目的:B族维生素以辅酶的形式参与糖、脂肪和蛋白质代谢,本文观察硫胺素、核黄素和烟酰胺补充对高脂饲料诱导大鼠肥胖的影响。方法:采用预防肥胖模型法,2×2×2析因设计分为8组:高脂对照组(F0组),高脂+硫胺素(F1组),高脂+核黄素(F2组),高脂+烟酰胺(F3组),高脂+硫胺素+核黄素(F4组),高脂+硫胺素+烟酰胺(F5组),高脂+核黄素+烟酰胺(F6组),高脂+硫胺素+核黄素+烟酰胺(F7组),每组12只大鼠,给予高脂饲料喂养,同时硫胺素(100 mg/kg bw/d)、核黄素(100mg/kg bw/d)、烟酰胺(250 mg/kg bw/d)灌胃,另设正常对照组(C组)12只,普通饲料喂养,自来水灌胃,15周后,分析其体重、摄食量、体脂重量、血脂等实验前后的变化情况及各组动物之间的差别。结果:经过15周喂养后,高脂喂养大鼠体重比正常对照组平均增加了15.7%;而补充烟酰胺(F3)及联合硫胺素(F5)或核黄素(F6)以及三者联合补充(F7)组高脂喂养大鼠与高脂对照组(F0)相比,体重分别降低了35.0%,30.0%,30.1%和30.6%(P值均小于0.05);甚至比正常对照组大鼠平均体重分别下降了22.8%,17.0%,17.0%和17.7%(P值均小于0.05);而补充核黄素或和硫胺素组大鼠体重没有明显增加或降低(P值均大于0.05)。血脂分析结果显示高脂喂养并联合补充核黄素或/和烟酰胺和/或硫胺素组大鼠血清CHOL和LDL水平明显低于高脂对照组;而高脂喂养并联合补充烟酰胺(F3)及联合硫胺素(F5)或核黄素(F6)以及三者联合补充(F7)组大鼠LDL/HDL比值分别0.29、0.26、0.25和0.26,明显低于F0组的0.37(P值均小于0.05)。结论:大剂量烟酰胺可有效地调节血脂水平和控制肥胖大鼠的体重增长,而核黄素及硫胺素对控制肥胖大鼠体重增长的作用不明显,尚待进一步研究;核黄素只能够降低血脂水平,提示大剂量烟酰胺可通过增加机体的能量代谢来控制体重的增长。     

Genes specifying cytokinin biosynthesis in prokaryotes

Cytokinins are plant hormones which have long been associated with cell division and plastid differentiation. Recently, they have been found to play a central role also in the growth of plant tumors. Certain phytopathogenic bacteria, notably Agrobacterium tumefaciens and Pseudomonas syringae pv. savastanoi, can incite tumors on dicotyledonous plants and such tumors exhibit growth which is characteristic of the presence of excess auxin and cytokinin. Genes specifying cytokinin biosynthesis have now been isolated from both sets of bacteria. The genes encode prenyl transferase responsible for cytokinin biosynthesis which, upon expression in E. coli,cause the production of the active cytokinin, zeatin. Expression of these genes in association with the plant is responsible for at least part of the tumor phenotype, although the molecular mechanisms of infection by these bacteria are apparently quite dissimilar. There is extensive homology between the cytokinin biosynthetic genes from the two sets of bacteria.     

植物芥子油苷代谢及其转移

芥子油苷是一类含氮、含硫的植物次生代谢产物,主要分布于十字花科植物。芥子油苷及其降解产物具有多种生化活性,在植物防御方面也有重要作用。简要介绍芥子油苷的分布、合成、降解和在植物体内的转移。     

Genomic GC level, optimal growth temperature, and genome size in prokaryotes

Two years ago, we showed that positive correlations between optimal growth temperature (T(opt)) and genome GC are observed in 15 out of the 20 families of prokaryotes we analyzed, thus indicating that "T(opt) is one of the factors that influence genomic GC in prokaryotes". Our results were disputed, but these criticisms were demonstrated to be mistaken and based on misconceptions. In a recent report, Wang et al. [H.C. Wang, E. Susko, A.J. Roger, On the correlation between genomic G+C content and optimal growth temperature in prokaryotes: data quality and confounding factors, Biochem. Biophys. Res. Commun. 342 (2006) 681-684] criticize our results by stating that "all previous simple correlation analyses of GC versus temperature have ignored the fact that genomic GC content is influenced by multiple factors including both intrinsic mutational bias and extrinsic environmental factors". This statement, besides being erroneous, is surprising because it applies in fact not to ours but to the authors' article. Here, we rebut the points raised by Wang et al. and review some issues that have been a matter of debate, regarding the influence of environmental factors upon GC content in prokaryotes. Furthermore, we demonstrate that the relationship that exists between genome size and GC level is valid for aerobic, facultative, and microaerophilic species, but not for anaerobic prokaryotes.     

原核微生物之杆菌和小杆菌

原核微生物的发现已有约340年,成千上万的原核微生物被发现和命名。近年来,随着分子生物学技术的发展,众多新的种属已经得到鉴定和命名,对这些微生物系统的总结并对中文命名系统适当调整已迫在眉睫。本文试图对人类发现最早、总量最多的350多属1 500多种杆菌和120多属1 200多种小杆菌进行总结和归纳,从生境、形态学(动静态、形状和尺寸)、生物化学、细菌和古菌属性等特征层次规范这类菌属的命名,期盼有助于推动国内外微生物分类学发展,有利于推动国际交流与合作。     

Thiamin transport in Escherichia coli: the mechanism of inhibition by the sulfhydryl-specific modifier N-ethylmaleimide

Active transport of thiamin (vitamin B₁) into Escherichia coli occurs through a member of the superfamily of transporters known as ATP-binding cassette (ABC) transporters. Although it was demonstrated that the sulfhydryl-specific modifier N-ethylmaleimide (NEM) inhibited thiamin transport, the exact mechanism of this inhibition is unknown. Therefore, we have carried out a kinetic analysis of thiamin transport to determine the mechanism of inhibition by NEM. Thiamin transport in vivo exhibits Michaelis-Menten kinetics with K_M=15 nM and V_max=46 U mg⁻¹. Treatment of intact E. coli KG33 with saturating NEM exhibited apparent noncompetitive inhibition, decreasing V_max by approximately 50% without effecting K_M or the apparent first-order rate constant (k_obsd). Apparent noncompetitive inhibition is consistent with an irreversible covalent modification of a cysteine(s) that is critical for the transport process. A primary amino acid analysis of the subunits of the thiamin permease combined with our kinetic analysis suggests that inhibition of thiamin transport by NEM is different from other ABC transporters and occurs at the level of protein-protein interactions between the membrane-bound carrier protein and the ATPase subunit.     

Class IIa bacteriocins: biosynthesis, structure and activity

In the last decade, a variety of ribosomally synthesized antimicrobial peptides or bacteriocins produced by lactic acid bacteria have been identified and characterized. As a result of these studies, insight has been gained into fundamental aspects of biology and biochemistry such as producer self protection, membrane-protein interactions, and protein modification and secretion. Moreover, it has become evident that these peptides may be developed into useful antimicrobial additives. Class IIa bacteriocins can be considered as the major subgroup of bacteriocins from lactic acid bacteria, not only because of their large number, but also because of their activities and potential applications. They have first attracted particular attention as listericidal compounds and are now believed to be the next in line if more bacteriocins are to be approved in the future. The present review attempts to provide an insight into general knowledge available for class IIa bacteriocins and discusses common features and recent findings concerning these substances.     

Radical S-Adenosylmethionine (SAM) Enzymes in Cofactor Biosynthesis: A Treasure Trove of Complex Organic Radical Rearrangement Reactions

In this minireview, we describe the radical S-adenosylmethionine enzymes involved in the biosynthesis of thiamin, menaquinone, molybdopterin, coenzyme F₄₂₀, and heme. Our focus is on the remarkably complex organic rearrangements involved, many of which have no precedent in organic or biological chemistry.     

A Structural and Biochemical Model of Processive Chitin Synthesis

Chitin synthases (CHS) produce chitin, an essential component of the fungal cell wall. The molecular mechanism of processive chitin synthesis is not understood, limiting the discovery of new inhibitors of this enzyme class. We identified the bacterial glycosyltransferase NodC as an appropriate model system to study the general structure and reaction mechanism of CHS. A high throughput screening-compatible novel assay demonstrates that a known inhibitor of fungal CHS also inhibit NodC. A structural model of NodC, on the basis of the recently published BcsA cellulose synthase structure, enabled probing of the catalytic mechanism by mutagenesis, demonstrating the essential roles of the DD and QXXRW catalytic motifs. The NodC membrane topology was mapped, validating the structural model. Together, these approaches give insight into the CHS structure and mechanism and provide a platform for the discovery of inhibitors for this antifungal target.     

Terminal alkene formation by the thioesterase of curacin A biosynthesis: structure of a decarboxylating thioesterase

Curacin A is a polyketide synthase (PKS)-non-ribosomal peptide synthetase-derived natural product with potent anticancer properties generated by the marine cyanobacterium Lyngbya majuscula. Type I modular PKS assembly lines typically employ a thioesterase (TE) domain to off-load carboxylic acid or macrolactone products from an adjacent acyl carrier protein (ACP) domain. In a striking departure from this scheme the curacin A PKS employs tandem sulfotransferase and TE domains to form a terminal alkene moiety. Sulfotransferase sulfonation of β-hydroxy-acyl-ACP is followed by TE hydrolysis, decarboxylation, and sulfate elimination (Gu, L., Wang, B., Kulkarni, A., Gehret, J. J., Lloyd, K. R., Gerwick, L., Gerwick, W. H., Wipf, P., Håkansson, K., Smith, J. L., and Sherman, D. H. (2009) J. Am. Chem. Soc. 131, 16033–16035). With low sequence identity to other PKS TEs (<15%), the curacin TE represents a new thioesterase subfamily. The 1.7-Å curacin TE crystal structure reveals how the familiar α/β-hydrolase architecture is adapted to specificity for β-sulfated substrates. A Ser-His-Glu catalytic triad is centered in an open active site cleft between the core domain and a lid subdomain. Unlike TEs from other PKSs, the lid is fixed in an open conformation on one side by dimer contacts of a protruding helix and on the other side by an arginine anchor from the lid into the core. Adjacent to the catalytic triad, another arginine residue is positioned to recognize the substrate β-sulfate group. The essential features of the curacin TE are conserved in sequences of five other putative bacterial ACP-ST-TE tridomains. Formation of a sulfate leaving group as a biosynthetic strategy to facilitate acyl chain decarboxylation is of potential value as a route to hydrocarbon biofuels.     

Cytokinin biosynthesis and interconversion

To maintain hormone homeostasis, the rate of cytokinin biosynthesis, interconversion, and degradation is regulated by enzymes in plant cells. Cytokinins can be synthesized via direct (de novo) or indirect (tRNA) pathways. In the de novo pathway, a cytokinin nucleotide is synthesized from 5'-AMP and isopentenyl pyrophosphate; a key enzyme which catalyzes this synthesis has been isolated from plant tissues, slime mold, and some microorganisms. Studies on the in vitro synthesis of the isopentenyl side chain of cytokinin in tRNA demonstrated that the isopentenyl group was derived from mevalonate, and turnover of the cytokinin-containing tRNA may serve as a minor source of free cytokinins in plant cells. The interconversion of cytokinin bases, nucleosides and nucleotides is a major feature of cytokinin metabolism; and enzymes that regulate the interconversion have been identified. The N⁶-side chain and purine moiety of cytokinins are often modified and some of the enzymes involved in the modifications have been isolated. Most of the cytokinin metabolites have been characterized but very few enzymes regulating their metabolism have been purified to homogeneity. It remains a significant challenge to isolate plant genes involved in the regulation of cytokinin biosynthesis, interconversion and degradation.     

Structure and electron transfer mechanism of pyruvate:ferredoxin oxidoreductase

The first crystal structure of pyruvate:ferredoxin oxidoreductase to be determined has provided significant new information on its structural organization and redox chemistry. Spectroscopic analyses of a radical reaction intermediate have shed more light on its thiamin-based mechanism of catalysis. Different approaches have been used to study the interaction between the enzyme and ferredoxin, its redox partner.     

New enzymes for peptide biosynthesis in microorganisms

Peptides, biologically occurring oligomers of amino acids linked by amide bonds, are essential for living organisms. Many peptides isolated as natural products have biological functions such as antimicrobial, antivirus and insecticidal activities. Peptides often possess structural features or modifications not found in proteins, including the presence of nonproteinogenic amino acids, macrocyclic ring formation, heterocyclization, N-methylation and decoration by sugars or acyl groups. Nature employs various strategies to increase the structural diversity of peptides. Enzymes that modify peptides to yield mature natural products are of great interest for discovering new enzyme chemistry and are important for medicinal chemistry applications. We have discovered novel peptide modifying enzymes and have identified: (i) a new class of amide bond forming-enzymes; (ii) a pathway to biosynthesize a carbonylmethylene-containing pseudodipeptide structure; and (iii) two distinct peptide epimerases. In this review, an overview of our findings on peptide modifying enzymes is presented.