植物中钼的吸收转运及钼辅因子与钼酶的研究进展

钼是植物生长发育所必需的微量元素,只有和蛋白质或者蝶呤结合形成钼辅因子才能产生生物活性。自然界存在2种钼辅因子:以铁硫簇为基础的铁钼辅因子(Fe Moco)和以钼蝶呤为基础的钼辅因子(MPT/Moco)。植物对钼的吸收有2种转运蛋白系统,一种是专一性转运蛋白,如MOTl和MOT2;另一种是共转运蛋白,如磷酸盐转运蛋白(PHT)和硫酸盐转运蛋白(SULTR)。最近研究发现一种钼酶——线粒体氨肟还原蛋白(m ARC)。本文综述了近年来植物体内钼的吸收与转运机制、钼辅因子的合成过程以及钼酶的研究进展,并提出了今后的重点研究方向。     

钼铁蛋白铁钼辅因子的有机组分对其功能的影响

棕色固氮菌(Azotobacter vinelandii)固氮酶的钼铁蛋白经邻菲啰啉在厌氧或有氧环境中处理后,变为 P-cluster 单一缺失或 P-cluster 和 FeMoco 同时缺失的失活钼铁蛋白。含柠檬酸盐或高柠檬酸盐的重组液都使这两种失活蛋白能恢复固氮酶重组的 H~ 和 C_2H_2还原活性,活性恢复程度随反映钼铁蛋白中金属原子簇含量变化的圆二色和磁圆二色谱及金属含量的恢复程度的提高而提高,但它们固 N_2能力的恢复程度则不相同:P-cluster 单一缺失的蛋白用两种重组液重组后均可恢复其固 N_2能力,而 P-cluster 和 FeMoco 同时缺失的蛋白,只有用含高柠檬酸盐的重组液重组才恢复其固 N_2能力,表明含不同有机组分的重组液所组装的 P-cluster 均与天然状态相同,只有含高柠檬酸盐的重组液所组装的 FeMoco 才与天然状态相同,从而证明高柠檬酸盐是 FeMoco 的必需的有机组分。     

固氮酶铁钼辅因子FeMo-co在水与非水体系内重组活力与催化活力的比较研究

 本文提出一种测定FeMo-co催化活力的反应体系,用此反应体系,在测定FeMo-co催化活力的过程中,FeMo-co与变种UW45抽提液的重组活性始终保持不变。讨论了水含量、还原剂对FeMo-co催化活力和重组活力的影响。     

维生素B型氨基酸衍生维生素的生物合成及功能研究

氨基酸衍生维生素是以氨基酸为前体合成的维生素,主要为维生素B和E家族的维生素,其生物合成方式主要为氨基酸整合和转氨作用。氨基酸衍生维生素在大多数真核生物中主要是作为辅被用物和辅因子,缺乏某些维生素会使动植物患病。本文主要对氨基酸衍生维生素中的维生素B家族的生物合成及功能进行综述,概述了氨基酸在B族维生素生物合成中的作用、不同物种中B族维生素的含量水平以及B族维生素的作用。     

潜在的防治肥胖及糖尿病靶标——DGAT

二酰基甘油酰基转移酶(DGAT)是甘油三酯(TG)合成的关键酶,催化TG合成的最后一步。DGAT有两种亚型:DGAT1和DGAT2。DGAT1缺陷的小鼠对胰岛素和瘦素的敏感性增加且可以抵抗饮食诱导的肥胖;DGAT2功能下调可明显降低肥胖小鼠肝脏TG含量,改善脂肪肝的形成。DGAT抑制剂可改善动物模型的高脂血症和脂肪肝。因此,DGAT有可能成为防治肥胖、糖尿病等代谢性疾病的新的药物靶标。该文详细阐述了DGAT的生理功能研究及其抑制剂的研究进展。     

钼靶、多模态MRI对乳腺癌及肿块型浆细胞性乳腺炎的鉴别诊断价值研究

摘要 目的：探讨钼靶、多模态MRI对乳腺癌及肿块型浆细胞性乳腺炎（PCM）的鉴别诊断价值。方法：回顾性分析2017年4月-2021年2月我院经病理证实的98例乳腺癌患者及31例肿块型PCM患者的钼靶和MRI资料,比较乳腺癌和肿块型PCM的钼靶及MRI形态学表现、ADC值、时间-信号强度曲线（TIC）的差异。采用受试者工作特征曲线（ROC）分析钼靶、多模态MRI鉴别诊断乳腺癌及肿块型PCM的效能。结果：钼靶形态学显示：乳腺癌、肿块型PCM在病灶形态及边缘表现上差异有统计学意义（P＜0.05）;乳腺癌、肿块型PCM在密度、伴随征象以及是否有斑点、泥沙样钙化表现上差异无统计学意义（P＞0.05）。多模态MRI形态学显示：乳腺癌、肿块型PCM在形态、边缘、导管扩张、强化方式、TIC曲线类型及ADC信号表现上差异有统计学意义（P＜0.05）;乳腺癌、肿块型PCM在T2WI信号及伴随征象表现上差异无统计学意义（P＞0.05）。ROC曲线分析结果显示：多模态MRI成像检查对乳腺癌及肿块型PCM的鉴别诊断价值明显优于钼靶检查,其曲线下面积（AUC）、准确度、敏感度、特异度及Youden指数分别为0.921、90.63%、100%、89.37%、0.89。结论：钼靶主要通过形态学表现鉴别诊断乳腺癌和肿块型PCM,多模态MRI则可通过病灶形态学表现、ADC值、动态增强表现及TIC客观性判断病灶性质,因此其鉴别诊断价值优于钼靶检查。     

棕色固氮菌((?)zotobacter vinelandii)产生的钼配位化合物——Ⅰ.分离、纯化及其特性

棕色固氮菌在正常培养条件下能产生一种含肽物质,经浓缩及CM-和DEAE-纤维素柱分离纯化后,在蔗糖密度梯度离心中处于5％蔗糖溶液中(ρ=1.010～1.017gcm~(-3)),在聚丙烯酰胺凝胶电泳中呈现一条带。含肽物质是由脂类、多肽和糖所组成。脂:肽:糖比例为1.76:1.00:0.27。脂类以磷脂为主,主要成分为磷脂酰乙醇胺。多肽含有十三种氨基酸,分子量约为4000道尔顿,圆二色散谱表明只有α螺旋结构。糖为乳糖。电子显微镜观察含肽物质为大小形状不一的颗粒状(直径为20～25 nm)和盘绕的聚合体。这种含肽物质可能是来自该细菌胞壁质中的脂多糖络合物。     

细胞药物存在的问题、解决对策及发展前景——细胞药物连载之六

细胞药物具有自身特点和优势,对肿瘤、自身免疫性疾病等疑难病症具有较好疗效,起到了其他药物难以发挥的作用。但细胞药物在研发过程中,也存在伦理、安全等问题。针对这些问题,提出了一些解决策略,并介绍了细胞药物最新临床研发情况,对其广阔的发展前景进行了展望。     

肠道病毒A71型3C蛋白结构、功能及抗3C蛋白药物的研究进展

肠道病毒A71型(Enterovirus A71,EV-A71)可引起手足口病(Hand,foot,and mouth disease,HFMD),严重者伴有神经系统并发症,如无菌性脑膜炎、神经源性肺水肿等。EV-A71引起的HFMD自2007年以来在全世界,尤其是亚太地区多次暴发,已成为亚太地区公共健康的主要威胁之一。目前尚无有效的抗病毒药物或疫苗。EVA71的致病机制尚未完全研究清楚,而非结构蛋白3C在病毒的复制和抑制天然免疫方面发挥了不可替代的作用。EV-A71 3C蛋白的研究在进一步了解EV-A71的致病机制以及研制抗病毒药物方面发挥着重要的作用。本文将对EV-A71 3C蛋白的结构、功能以及抗3C蛋白病毒药物的研究进展做出综述。     

一种新的抗菌药物靶标蛋白质(3，4-二羟基-2-丁酮-4-磷酸合成酶)的克隆、表达、纯化及酶活性鉴定

[目的]核黄素( vitamin B12,riboflavin)是辅因子黄素腺嘌呤二核苷酸(flavin adenine dinucleotide,FAD)和黄素单核苷酸(flavin mononucleotide,FMN)的前体物,对生物体的生物合成至关重要.如果细菌不能够从外界摄取足够的黄素( flavin)就需要自身合成核黄素以维持菌体的生存与增殖.3,4-二羟基-2-丁酮-4-磷酸合成酶(3,4-Dihydroxy-2-butanone-4-phosphate synthase,DHBPs)为核黄素生物合成途径中关键酶之一.在镁离子存在的情况下,DHBPs将5-磷酸核酮糖(ribulose-5 -phosphate,Ru5P)转换成3,4-二羟基-2-丁酮4-磷酸(3,4-dihydroxy-2-Bu-tanone-4-Pho-sphate,DHBP)和甲酸盐(formate),生成的DHBP为核黄素合成的必需原料之一.人类没有合成核黄素的相关途径,因此细菌参与合成核黄素的DHBPs等相关酶就有望成为抗菌药物作用的靶位点.本课题通过对肺炎链球菌的DHBPs进行克隆表达纯化与酶学性质鉴定,为开展其三维结构的解析和抗菌药物设计提供重要的工作基础.[方法]利用PCR技术扩增DHBPs基因,构建重组表达载体pW28-DHBPs.将其转入大肠杆菌(Escherichia coli)BL21( DE3)中表达,用Ni离子亲和层析及离子交换(DEAE)纯化获得有活性的DHBPs后,进行酶学性质鉴定.[结果]酶切和测序证实成功构建了质粒pW28-DHBPs,在E.coli BL21中表达了可溶性DHBPs,纯化后获得了纯度为95％的靶蛋白质,经分子筛分析DHBPs在溶液中以二聚体形式存在.对DHBPs进行酶学性质分析表明,在25℃、pH为7.5和Mg2+存在的情况下,DHBPs具有将5-磷酸核酮糖转换成DHBP和甲酸盐的活性.[结论]第一次成功克隆并在E.coli BL21中表达了一种肺炎链球菌合成核黄素的相关酶—DHBPs,纯化后的重组DHBPs具有较好的5-磷酸核酮糖分解活性,这为解析其三维结构和基于结构进行的新一代抗菌药物设计提供重要的工作基础.     

Molybdenum Metabolism in Plants

Abstract: Among the micronutrients essential for plant growth and for microsymbionts, Mo is required in minute amounts. However, since Mo is often sequestered by Fe- or Al-oxihydrox-ides, especially in acidic soils, the concentration of the water-soluble molybdate anion available for uptake by plants may be limiting for the plant, even when the total Mo content of the soil is sufficient. In contrast to bacteria, no specific molybdenum uptake system is known for plants, but since molybdate and sulfate behave similarly and have similar structure, uptake of molybdate could be mediated unspecifically by one of the sulfate transporters. Transport into the different plant organs proceeds via xylem and phloem. A pterin-bound molybdenum is the cofactor of important plant enzymes involved in redox processes: nitrate reductase, xanthine dehydrogenase, aIdehyde oxidase, and probably sulfite oxidase. Biosynthesis of the molybdenum cofactor (Moco) starts with a guanosine-X-phos-phate. Subsequently, a sulfur-free pterin is synthesized, sulfur is added, and finally molybdenum is incorporated. In addition to the molybdopterin enzymes, small molybdopterin binding proteins without catalytic function are known and are probably involved in the storage of Moco. In symbiotic systems the nitrogen supply of the host plant is strongly influenced by the availability of Mo in soil, since both bacterial nitrogenase and NADPH-dependent nitrate reductase of mycorrhizal fungi are Mo enzymes.     

Isolation,Purification, and Characterization of Nitrate Reductase from a Salt-Tolerant <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis> Yeast Strain Grown in the Presence of Tungsten

The salt-tolerant Rhodotorula glutinis yeast strain grew in medium containing nitrate, 1 mM tungsten, and trace amounts of molybdenum (as impurities from the reagents used). Isolation of electrophoretically homogenous preparation of nitrate reductase from the Rh. glutinis cells grown under these growth conditions is described. The isolated nitrate reductase is a molybdenum-containing homodimer with molecular mass of 130 kD, containing 0.177 mol of Mo per mol of the enzyme. The activity of the enzyme is maximal at pH 7.0 and 35-45 degrees C and is inhibited by low concentrations of azide and cyanide. The enzyme is almost insensitive to 1 mM tungsten.     

Nitrate reductases: Structure,functions, and effect of stress factors

Structural and functional peculiarities of four types of nitrate reductases are considered: assimilatory nitrate reductase of eukaryotes, as well as cytoplasmic assimilatory, membrane-bound respiratory, and periplasmic dissimilatory bacterial nitrate reductases. Arguments are presented showing that eukaryotic organisms are capable of nitrate dissimilation. Data concerning new classes of extremophil nitrate reductases, whose active center does not contain molybdocofactor, are summarized.     

Molybdenum cofactor amounts in Chlamydomonas reinhardtii depend on the Nit5 gene function related to molybdate transport

Strain 21gr from Chlamydomonas reinhardtii is a cryptic mutant defective in the Nit5 gene related to the biosynthesis of molybdenum cofactor (MoCo). In spite of this mutation, this strain has active MoCo and can grow on nitrate media. In genetic crosses, the Nit5 mutation cosegregated with a phenotype of resistance to high concentrations of molybdate and tungstate. Molybdate/tungstate toxicity was much higher in nitrate than in ammonium media. Strain 21gr showed lower amounts of MoCo activity than the wild type both when grown in nitrate and after growth in ammonium and nitrate induction. However, nitrate reductase (NR) specific activity was similar in wild type and 21gr cells. Tungstate, either at nanomolar concentrations in nitrate media or at micromolar concentrations during growth in ammonium and nitrate induction, strongly decreased MoCo and NR amounts in wild‐type cells but had a slight effect in 21gr cells. Molybdate uptake activity of ammonium‐grown cells from both the wild‐type and 21gr strains was small and blocked by sulphate 0·3 mM . However, cells from nitrate medium showed a molybdate uptake activity insensitive to sulphate. This uptake activity was much higher and more sensitive to inhibition by tungstate in the wild type than in strain 21gr. These results suggest that strain 21gr has a high affinity and low capacity molybdate transport system able to discriminate efficiently tungstate, and lacks a high capacity molybdate/tungstate transport system, which operates in wild‐type cells upon nitrate induction. This high capacity molybdate transport system would account for both the stimulating effect of molybdate on MoCo amounts and the toxic effects of tungstate and molybdate when present at high concentrations.     

Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase

We test the hypothesis that pyranopterin (PPT) coordination plays a critical role in defining molybdenum active site redox chemistry and reactivity in the mononuclear molybdoenzymes. The molybdenum atom of Escherichia coli nitrate reductase A (NarGHI) is coordinated by two PPT-dithiolene chelates that are defined as proximal and distal based on their proximity to a [4Fe-4S] cluster known as FS0. We examined variants of two sets of residues involved in PPT coordination: (i) those interacting directly or indirectly with the pyran oxygen of the bicyclic distal PPT (NarG-Ser⁷¹⁹, NarG-His¹¹⁶³, and NarG-His¹¹⁸⁴); and (ii) those involved in bridging the two PPTs and stabilizing the oxidation state of the proximal PPT (NarG-His¹⁰⁹² and NarG-His¹⁰⁹⁸). A S719A variant has essentially no effect on the overall Mo(VI/IV) reduction potential, whereas the H1163A and H1184A variants elicit large effects (ΔE_m values of −88 and −36 mV, respectively). Ala variants of His¹⁰⁹² and His¹⁰⁹⁸ also elicit large ΔE_m values of −143 and −101 mV, respectively. An Arg variant of His¹⁰⁹² elicits a small ΔE_m of +18 mV on the Mo(VI/IV) reduction potential. There is a linear correlation between the molybdenum E_m value and both enzyme activity and the ability to support anaerobic respiratory growth on nitrate. These data support a non-innocent role for the PPT moieties in controlling active site metal redox chemistry and catalysis.     

Lectins of members of the Amaryllidaceae are encoded by multigene families which show extensive homology

Nitrate uptake and reduction are highly regulated processes. In many plant species, nitrate uptake is induced by nitrate, Little, however, is known about the genetic and molecular aspects of nitrate transport. Reduction of nitrate to ammonia is carried out by nitrate and nitrite reductases. Nitrate and light enhance expression of the nitrate and nitrite reductase genes in most species. Mutants have been selected and characterized to identify genes controlling nitrate reductase in several higher plant species. Six loci are known to control the synthesis or assembly of the molybdenum cofactor of nitrate reductase, xanthine dehydrogenase and aldehyde oxidase. The nitrate reductase apoenzyme is encoded by a single gene, except in allopolyploid species and in those species possessing both NADH-specific and NAD(P)H-bispecific nitrate reductases. Comparison of NADH-specific nitrate reductase amino acid sequences deduced from cloned genes reveals considerable sequence conservation in regions believed to encode the functional domains of nitrate reductase, but less conservation in the N-terminal and hinge regions of the enzyme. For both nitrate and nitrite reductases, sequence identity is greater among species of the same subclass than between Monocotyledoneae and Dicotyledoneae subclass species.     

New GATEWAY vectors for High Throughput Analyses of Protein-Protein Interactions by Bimolecular Fluorescence Complementation

Complex protein interaction networks constitute plant metabolic and signaling systems. Bimolecular fluorescence complementation （BiFC） is a suitable technique to investigate the formation of protein complexes and the localization of protein-protein interactions in planta. However, the generation of large plasmid collections to facilitate the exploration of complex interaction networks is often limited by the need for conventional cloning techniques. Here, we report the implementation of a GATEWAY vector system enabling large-scale combination and investigation of candidate proteins in BiFC studies. We describe a set of 12 GATEWAY-compatible BiFC vectors that efficiently permit the combination of candidate protein pairs with every possible N-or C-terminal sub-fragment of S（CFP）3A or Venus, respectively, and enable the performance of multicolor BiFC （mcBiFC）. We used proteins of the plant molybdenum metabolism, in that more than 20 potentially interacting proteins are assumed to form the cellular molybdenum network, as a case study to establish the functionality of the new vectors. Using these vectors, we report the formation of the molybdopterin synthase complex by interaction of Arabidopsis proteins Cnx6 and Cnx7 detected by BiFC as well as the simultaneous formation of Cnx6/Cnx6 and Cnx6/Cnx7 complexes revealed by mcBiFC. Consequently, these GATEWAY-based BiFC vector systems should significantly facilitate the large-scale investigation of complex regulatory networks in plant cells.     

Biosynthesis of the Polyoxins,Nucleoside Peptide Antibiotics: Formation of 5-Carboxyuracil Nucleosides by Streptomyces cacaoi

Biosynthetic origin of the 5-carboxyuracil base of the polyoxins and the octosyl acids is described. These unusual nucleosides are the metabolites of Streptomyces cacaoi var. asoensis. In vivo experiments show that the 5-carboxyuracil base of the polyoxins and the octosyl acids is biosynthesized from uracil and carbon-3 of serine. [2-¹⁴C]Thymine is incorporated into DNA-thymine but not into the 5-carboxyuracil base of these nucleosides. Exogenously supplied [2-¹⁴C]5-carboxyuracil is not taken up by cells. The biosynthesis of this base in the polyoxins is also shown to occur predominantly in the latest stage of the fermentation. Possible formation of this base through oxidation of thymine or 5-hydroxymethyluracil at the nucleoside level is discussed.     

Molybdoenzymes and Molybdenum Cofactor in Plants

The transition element molybdenum is essential for (nearly) all organisms and occurs in more than 30 enzymes catalyzing diverse redox reactions; however, only three Mo-enzymes have been found in plants so far. (1) Nitrate reductase catalyzes the key step in inorganic nitrogen assimilation, (2) aldehyde oxidase(s) recently have been shown to catalyze the last step in the biosynthesis of the phytohormones indole acetic acid and abscisic acid, respectively, and (3) xanthine dehydrogenase is involved in purine catabolism. These enzymes are homodimeric proteins harboring an electron transport chain that involves different prosthetic groups (FAD, heme, or Fe-S, Mo). Among different Mo-enzymes, the alignment of amino acid sequences helps to define regions that are well conserved (domains) and other regions that are highly variable in sequence (interdomain hinge regions). The existence of additional plant Mo-enzymes (like sulfite oxidase) also has to be considered. In this review we focus on structure-function relationships and stress the functional importance of the enzymes for the plant. With the exception of bacterial nitrogenase, Mo-enzymes share a similar pterin compound at their catalytic sites, the molybdenum cofactor. Molybdenum itself seems to be biologically inactive unless it is complexed by the cofactor. This molybdenum cofactor combines with diverse apoproteins where it is responsible for the correct anchoring and positioning of the Mo-center within the holo-enzyme so that the Mo-center can interact with other components of the enzyme's electron transport chain. The organic moiety of the molybdenum cofactor is a unique pterin named molybdopterin. The core structure of molybdopterin is conserved in all organisms. Accordingly, its biosynthetic pathway seems to be conserved because a similar set of cofactor genes has been found in bacteria and higher plants. We describe a model for the biosynthesis of the plant molybdenum cofactor involving the complex interaction of seven proteins.     

Structural and mechanistic insights on nitrate reductases

Nitrate reductases (NR) belong to the DMSO reductase family of Mo‐containing enzymes and perform key roles in the metabolism of the nitrogen cycle, reducing nitrate to nitrite. Due to variable cell location, structure and function, they have been divided into periplasmic (Nap), cytoplasmic, and membrane‐bound (Nar) nitrate reductases. The first crystal structure obtained for a NR was that of the monomeric NapA from Desulfovibrio desulfuricans in 1999. Since then several new crystal structures were solved providing novel insights that led to the revision of the commonly accepted reaction mechanism for periplasmic nitrate reductases. The two crystal structures available for the NarGHI protein are from the same organism (Escherichia coli) and the combination with electrochemical and spectroscopic studies also lead to the proposal of a reaction mechanism for this group of enzymes. Here we present an overview on the current advances in structural and functional aspects of bacterial nitrate reductases, focusing on the mechanistic implications drawn from the crystallographic data.