The reductive reaction mechanism of tobacco nitrite reductase derived from a combination of crystal structures and ultraviolet-visible microspectroscopy

Assimilatory nitrite reductase (aNiR) reduces nitrite to an ammonium ion and has siroheme and a [Fe(4)S(4)] cluster as prosthetic groups. A reaction mechanism for Nii3, an aNiR from tobacco, is proposed based on high resolution X-ray structures and UV-Vis (ultraviolet-visible) microspectroscopy of Nii3-ligand complexes. Analysis of UV-Vis spectral changes in Nii3 crystals with increasing X-ray exposure showed prosthetic group reductions. In Nii3-NO2(-) structures, X-ray irradiation enhanced the progress of the reduction reaction, and cleavage of the N-O bond was observed when X-ray doses were increased. Crystal structures of Nii3 with other bound ligands, such as Nii3-NO and Nii3-NH(2)OH, were also determined. Further, by combining information from these Nii3 ligand-bound structures, including that of Nii3-NO2(-), with UV-Vis microspectral data obtained using different X-ray doses, a reaction mechanism for aNiR was suggested. Cleavage of the two N-O bonds of nitrite was envisaged as a two-step process: first, the N-O bond close to Lys224 was cleaved, followed by cleavage of the N-O bond close to Arg109. X-ray structures also indicated that aNiR-catalyzed nitrite reduction proceeded without the need for conformation changes in active site residues. Geometrical changes in the ligand molecules and the placement of neighboring water molecules appeared to be important to the stability of the active site residue interactions (Arg109, Arg179, and Lys224) and the ligand molecule. These interactions may contribute to the efficiency of aNiR reduction reactions.     

Crystal structure of a trimeric archaeal adenylate kinase from the mesophile Methanococcus maripaludis with an unusually broad functional range and thermal stability

The structure of the trimeric adenylate kinase from the Archaebacteria Methanococcus mariplaludis (AK_MAR) has been solved to 2.5‐Å resolution and the temperature dependent stability and kinetics of the enzyme measured. The K_M and V_max of AK_MAR exhibit only modest temperature dependence from 30°–60°C. Although M. mariplaludis is a mesophile with a maximum growth temperature of 43°C, AK_MAR has a very broad functional range and stability (T_m = 74.0°C) that are more consistent with a thermophilic enzyme with high thermostability and exceptional activity over a wide range of temperatures, suggesting that this microbe may have only recently invaded a mesophilic niche and has yet to fully adapt. A comparison of the Local Structural Entropy (LSE) for AK_MAR to the related adenylate kinases from the mesophile Methanococcus voltae and thermophile Methanococcus thermolithotrophicus show that changes in LSE are able to fully account for the intermediate stability of AK_MAR and highlights a general mechanism for protein adaptation in this class of enzymes. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Proposed amino acid sequence and the 1.63 A X-ray crystal structure of a plant cysteine protease,ervatamin B: some insights into the structural basis of its stability and substrate specificity

The crystal structure of a cysteine protease ervatamin B, isolated from the medicinal plant Ervatamia coronaria, has been determined at 1.63 A. The unknown primary structure of the enzyme could also be traced from the high-quality electron density map. The final refined model, consisting of 215 amino acid residues, 208 water molecules, and a thiosulfate ligand molecule, has a crystallographic R-factor of 15.9% and a free R-factor of 18.2% for F > 2sigma(F). The protein belongs to the papain superfamily of cysteine proteases and has some unique properties compared to other members of the family. Though the overall fold of the structure, comprising two domains, is similar to the others, a few natural substitutions of conserved amino acid residues at the interdomain cleft of ervatamin B are expected to increase the stability of the protein. The substitution of a lysine residue by an arginine (residue 177) in this region of the protein may be important, because Lys --> Arg substitution is reported to increase the stability of proteins. Another substitution in this cleft region that helps to hold the domains together through hydrogen bonds is Ser36, replacing a conserved glycine residue in the others. There are also some substitutions in and around the active site cleft. Residues Tyr67, Pro68, Val157, and Ser205 in papain are replaced by Trp67, Met68, Gln156, and Leu208, respectively, in ervatamin B, which reduces the volume of the S2 subsite to almost one-fourth that of papain, and this in turn alters the substrate specificity of the enzyme.     

Elucidating the reaction mechanism of the benzoate oxidation pathway encoded aldehyde dehydrogenase from Burkholderia xenovorans LB400

Oxidation of cis-3,4-dehydroadipyl-CoA semialdehyde to cis-3,4-dehydroadipyl-CoA by the aldehyde dehydrogenase, ALDH(C) (EC.1.2.1.77), is an essential step in the metabolism of benzoate in Burkholderia xenovorans LB400. In a previous study, we established a structural blueprint for this novel group of ALDH enzymes. Here, we build significantly on this initial work and propose a detailed reaction mechanism for ALDH(C) based on comprehensive structural and functional investigations of active site residues. Kinetic analyses reveal essential roles for C296 as the nucleophile and E257 as the associated general base. Structural analyses of E257Q and C296A variants suggest a dynamic charge repulsion relationship between E257 and C296 that contributes to the inherent flexibility of E257 in the native enzyme, which is further regulated by E496 and E167. A proton relay network anchored by E496 and supported by E167 and K168 serves to reset E257 for the second catalytic step. We also propose that E167, which is unique to ALDH(C) and its homologs, serves a critical role in presenting the catalytic water to the newly reset E257 such that the enzyme can proceed with deacylation and product release. Collectively, the reaction mechanism proposed for ALDH(C) promotes a greater understanding of these novel ALDH enzymes, the ALDH super-family in general, and benzoate degradation in B. xenovorans LB400.     

Identification of the bona fide DHDPS from a common plant pathogen

Agrobacterium tumefaciens is a Gram‐negative soil‐borne bacterium that causes Crown Gall disease in many economically important crops. The absence of a suitable chemical treatment means there is a need to discover new anti‐Crown Gall agents and also characterize bona fide drug targets. One such target is dihydrodipicolinate synthase (DHDPS), a homo‐tetrameric enzyme that catalyzes the committed step in the metabolic pathway yielding meso‐diaminopimelate and lysine. Interestingly, there are 10 putative DHDPS genes annotated in the A. tumefaciens genome, including three whose structures have recently been determined (PDB IDs: 3B4U, 2HMC, and 2R8W). However, we show using quantitative enzyme kinetic assays that nine of the 10 dapA gene products, including 3B4U, 2HMC, and 2R8W, lack DHDPS function in vitro. A sequence alignment showed that the product of the dapA7 gene contains all of the conserved residues known to be important for DHDPS catalysis and allostery. This gene was cloned and the recombinant product expressed and purified. Our studies show that the purified enzyme (i) possesses DHDPS enzyme activity, (ii) is allosterically inhibited by lysine, and (iii) adopts the canonical homo‐tetrameric structure in both solution and the crystal state. This study describes for the first time the structure, function and allostery of the bona fide DHDPS from A. tumefaciens, which offers insight into the rational design of pesticide agents for combating Crown Gall disease. Proteins 2014; 82:1869–1883. © 2014 Wiley Periodicals, Inc.     

根域限制下水氮供应对膜下滴灌棉花根系及叶片衰老特性的影响

在新疆的气候生态条件下, 选用北疆2个棉花(Gossypium hirsutum)主栽品种‘新陆早13号’和‘新陆早33号’为供试材料, 设置限根(RR)与对照(CK)处理, 每个处理设置4个水氮水平: 水氮亏缺(W₀N₀)、水分亏缺(W₀N₁)、氮素亏缺(W₁N₀)与水氮适量(W₁N₁), 组成再裂区试验方案。采用管栽方法, 通过人工改变根系垂直生长深度和水氮供应, 在棉花产量形成期测定根系及叶片抗氧化保护酶系活性、生物量累积及分配等, 探讨根域限制及水氮供应对棉花根系生长及叶片衰老的影响机理。结果表明: 根域限制条件下, 棉花根系生物量、根系与叶片超氧化物歧化酶(SOD)、过氧化物酶(POD)与过氧化氢酶(CAT)活性、棉株总生物量、根冠比均低于对照, 而地上部生物量与籽棉产量显著高于对照。水氮供应能有效地调节根系及叶片的生长, 不同水氮处理间棉花根系与叶片抗氧化保护酶系活性、叶绿素含量、地上部生物量及籽棉产量均表现为W₁N₁ > W₀N₁ > W₁N₀ > W₀N₀, 根冠比与根系生物量的表现与之相反。根域限制与水氮供应表现出互作优势, 根域限制下适量水氮供应处理的地上部生物量与籽棉产量均明显高于其他处理, 根冠比较低。因此, 在棉花根系生长受限的条件下, 优化生育期间水氮供应, 可以增强根系及叶片的抗氧化保护酶系活性、增加光合产物向地上部的分配比例、增加产量, 是进一步挖掘膜下滴灌棉花增产潜力的有效途径。     

Molecular basis for redox-Bohr and cooperative effects in cytochrome c3 from Desulfovibrio desulfuricans ATCC 27774: crystallographic and modeling studies of oxidized and reduced high-resolution structures at pH 7.6

The tetraheme cytochrome c3 is a small metalloprotein with ca. 13,000 Da found in sulfate-reducing bacteria, which is believed to act as a partner of hydrogenase. The three-dimensional structure of the oxidized and reduced forms of cytochrome c3 from Desulfovibrio desulfuricans ATCC 27774 at pH 7.6 were determined using high-resolution X-ray crystallography and were compared with the previously determined oxidized form at pH 4.0. Theoretical calculations were performed with both structures, using continuum electrostatic calculations and Monte Carlo sampling of protonation and redox states, in order to understand the molecular basis of the redox-Bohr and cooperativity effects related to the coupled transfer of electrons and protons. We were able to identify groups that showed redox-linked conformational changes. In particular, Glu61, His76, and propionate D of heme II showed important contributions to the redox-cooperativity, whereas His76, propionate A of heme I, and propionate D of heme IV were the key residues for the redox-Bohr effect. Upon reduction, an important movement of the backbone region surrounding hemes I and II was also identified, that, together with a few redox-linked conformational changes in side-chain residues, results in a significant decrease in the solvent accessibility of hemes I and II.     

Structural and biochemical characterization of the nucleoside hydrolase from C. elegans reveals the role of two active site cysteine residues in catalysis

Nucleoside hydrolases (NHs) catalyze the hydrolysis of the N‐glycoside bond in ribonucleosides and are found in all three domains of life. Although in parasitic protozoa a role in purine salvage has been well established, their precise function in bacteria and higher eukaryotes is still largely unknown. NHs have been classified into three homology groups based on the conservation of active site residues. While many structures are available of representatives of group I and II, structural information for group III NHs is lacking. Here, we report the first crystal structure of a purine‐specific nucleoside hydrolase belonging to homology group III from the nematode Caenorhabditis elegans (CeNH) to 1.65Å resolution. In contrast to dimeric purine‐specific NHs from group II, CeNH is a homotetramer. A cysteine residue that characterizes group III NHs (Cys253) structurally aligns with the catalytic histidine and tryptophan residues of group I and group II enzymes, respectively. Moreover, a second cysteine (Cys42) points into the active site of CeNH. Substrate docking shows that both cysteine residues are appropriately positioned to interact with the purine ring. Site‐directed mutagenesis and kinetic analysis proposes a catalytic role for both cysteines residues, with Cys253 playing the most prominent role in leaving group activation.     

Structure and oligomerization of the PilC type IV pilus biogenesis protein from Thermus thermophilus

Type IV pili are expressed from a wide variety of Gram‐negative bacteria and play a major role in host cell adhesion and bacterial motility. PilC is one of at least a dozen different proteins that are implicated in Type IV pilus assembly in Thermus thermophilus and a member of a conserved family of integral inner membrane proteins which are components of the Type II secretion system (GspF) and the archeal flagellum. PilC/GspF family members contain repeats of a conserved helix‐rich domain of around 100 residues in length. Here, we describe the crystal structure of one of these domains, derived from the N‐terminal domain of Thermus thermophilus PilC. The N‐domain forms a dimer, adopting a six helix bundle structure with an up‐down‐up‐down‐up‐down topology. The monomers are related by a rotation of 170°, followed by a translation along the axis of the final α‐helix of approximately one helical turn. This means that the regions of contact on helices 5 and 6 in each monomer are overlapping, but different. Contact between the two monomers is mediated by a network of hydrophobic residues which are highly conserved in PilC homologs from other Gram‐negative bacteria. Site‐directed mutagenesis of residues at the dimer interface resulted in a change in oligomeric state of PilC from tetramers to dimers, providing evidence that this interface is also found in the intact membrane protein and suggesting that it is important to its function. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

中国亚热带不同菌根树种的根叶形态学性状特征与生长差异: 以江西新岗山为例

探究植物功能性状的种内和种间变异不仅有助于揭示植物对环境的适应, 也能够反映植物的生态策略, 但不同菌根类型树木生长过程中根叶形态学功能性状的适应策略仍有待探究。本研究依托中国亚热带森林生物多样性与生态系统功能实验研究平台(BEF-China)选取7种丛枝菌根(AM)树木和7种外生菌根(EM)树木的纯林, 测定各个树种的比叶面积、叶干物质含量、比根长、根系直径、树高生长速率、地径生长速率及细根生物量等根叶形态学功能性状和生长指标, 探讨了两种菌根类型树种间的根叶形态学特征的差异。结果表明: 与AM树种相比, EM树种具有较小的比叶面积、吸收根平均直径和生长速率, 但具有更大的叶干物质含量; 两种菌根树种之间的比根长和细根生物量无显著差异。比叶面积、叶干物质含量、树高生长速率、地径生长速率和细根生物量等功能性状及生长指标在不同菌根类型、树种及二者的交互作用中均存在显著差异; 且树种、根功能型、菌根类型及三者之间的交互作用均对根功能性状有显著影响。EM树种地上指标的种内变异均大于种间变异, 而AM树种地上指标的种内和种间变异程度类似; 但两种菌根树种细根生物量的种间变异均大于种内变异。尽管两种菌根树种地上部分生长速率较快通常表现为较低的叶干物质含量, 但AM树种通常拥有较高的吸收根比根长, 而EM树种拥有较粗的运输根平均直径。吸收根比根长越低, 两类菌根树种的细根生物量就越多。由此可见, 根叶功能性状对植物地上部分的生长具有一定的协同效应, 其中运输根主要在EM树种地上生长过程中发挥重要作用, 吸收根主要与AM树种的地上部分生长有关; 但两类菌根树种的地下细根生物量均与吸收根有关。     

Structure and vasorelaxant activity of floranol, a flavonoid isolated from the roots of Dioclea grandiflora

The structure of the prenylated flavanonol, floranol (1=(2R,3R)-3,5,7-trihydroxy-2-(2-hydroxyphenyl)-6-methoxy-8-(3-methylbut-2-enyl)-4H-1-benzopyran-4-one), isolated from the roots of Dioclea grandiflora (Fabaceae), was unambiguously determined by X-ray analysis. The compound was tested for vasorelaxant activity. In endothelium-containing aortic rings, floranol (1) induced a concentration-dependent vasodilator effect in vessels precontracted with 0.1 microM phenylephrine with an IC(50) value of 19.9+/-2.4 microM. The removal of endothelium or pretreatment of vessels with the NO-synthase inhibitor L-NAME did not change the IC(50) and E(max) values for floranol-induced vasorelaxation. We conclude that floranol (1) should be acting directly in the rat-aorta smooth muscle cells to produce its vasorelaxant effect. The structure-activity relationship was discussed in terms of the 3-D floranol structure determined by X-ray crystallography.     

Influence of (hydroxymethyl)pyridine and pyridine-carboxylic acids, in trans-position to the isopropylamine and amine ligands, on the cytotoxicity of platinum complexes

trans-Pt(II) Complexes with aliphatic amines and planar amines such as (hydroxymethyl)pyridines, and pyridine-3- and pyridine-4-carboxylic acids were synthesized and screened for their potential cytotoxic activity in different cancer cell lines used at the NCI for in vitro screens, i.e., MCF7, NCIH460, and SF268. The complexes studied were designed to differ in geometrical parameters such as the position of the phenyl-group substituents and the nature of the substituents themselves for gathering information about the structure-activity relationships in the trans-complexes. The variation of the substituents turns to be crucial for their biological activity, as both pyridine-3- and pyridine-4-carboxylic acids in trans-position to both amine and isopropylamine ligands provided complexes which displayed no specificity toward any type of cell tested, while (hydroxymethyl)pyridine in trans-position to isopropylamine ligands led to complexes that were clearly more effective against the cell lines tested.     

Human MICAL1: Activation by the small GTPase Rab8 and small‐angle X‐ray scattering studies on the oligomerization state of MICAL1 and its complex with Rab8

Human MICAL1 is a member of a recently discovered family of multidomain proteins that couple a FAD‐containing monooxygenase‐like domain to typical protein interaction domains. Growing evidence implicates the NADPH oxidase reaction catalyzed by the flavoprotein domain in generation of hydrogen peroxide as a second messenger in an increasing number of cell types and as a specific modulator of actin filaments stability. Several proteins of the Rab families of small GTPases are emerging as regulators of MICAL activity by binding to its C‐terminal helical domain presumably shifting the equilibrium from the free – auto‐inhibited – conformation to the active one. We here extend the characterization of the MICAL1–Rab8 interaction and show that indeed Rab8, in the active GTP‐bound state, stabilizes the active MICAL1 conformation causing a specific four‐fold increase of k_cat of the NADPH oxidase reaction. Kinetic data and small‐angle X‐ray scattering (SAXS) measurements support the formation of a 1:1 complex between full‐length MICAL1 and Rab8 with an apparent dissociation constant of approximately 8 μM. This finding supports the hypothesis that Rab8 is a physiological regulator of MICAL1 activity and shows how the protein region preceding the C‐terminal Rab‐binding domain may mask one of the Rab‐binding sites detected with the isolated C‐terminal fragment. SAXS‐based modeling allowed us to propose the first model of the free full‐length MICAL1, which is consistent with an auto‐inhibited conformation in which the C‐terminal region prevents catalysis by interfering with the conformational changes that are predicted to occur during the catalytic cycle.     

Breaking up and making up: The secret life of the vacuolar H+‐ATPase

The vacuolar ATPase (V‐ATPase; V₁V_o‐ATPase) is a large multisubunit proton pump found in the endomembrane system of all eukaryotic cells where it acidifies the lumen of subcellular organelles including lysosomes, endosomes, the Golgi apparatus, and clathrin‐coated vesicles. V‐ATPase function is essential for pH and ion homeostasis, protein trafficking, endocytosis, mechanistic target of rapamycin (mTOR), and Notch signaling, as well as hormone secretion and neurotransmitter release. V‐ATPase can also be found in the plasma membrane of polarized animal cells where its proton pumping function is involved in bone remodeling, urine acidification, and sperm maturation. Aberrant (hypo or hyper) activity has been associated with numerous human diseases and the V‐ATPase has therefore been recognized as a potential drug target. Recent progress with moderate to high‐resolution structure determination by cryo electron microscopy and X‐ray crystallography together with sophisticated single‐molecule and biochemical experiments have provided a detailed picture of the structure and unique mode of regulation of the V‐ATPase. This review summarizes the recent advances, focusing on the structural and biophysical aspects of the field.     

低氧胁迫下外源硝态氮对樱桃根系功能及氮代谢相关酶活性的影响

以樱桃组培苗‘吉塞拉5号’（Prunun cerasus × P. canescens）为试材,采用营养液水培控制溶氧浓度的方法,研究了短期低氧胁迫下外源硝态氮对其根系功能及氮代谢相关酶活性的影响.结果表明：与对照（7.5 mmol NO₃^-·L^-1）相比,低氧加氮处理（15和22.5 mmol NO₃^-·L^-1）使樱桃体内代谢原料充足,保证了各类酶蛋白的合成,使植株根系活力升高,根系呼吸未受到明显抑制,与氮代谢相关的硝酸还原酶（NR）、谷氨酰胺合成酶（GS）及谷氨酸脱氢酶（NADH-GDH）活性升高,从而为低氧逆境下樱桃根系的吸收作用提供了足够的能量,保证了糖酵解和电子传递的顺利进行,并及时同化了NO₃^-还原生成NH₄⁺,避免了铵毒害,缓解了樱桃的低氧伤害,且22.5 mmol NO₃^-·L^-1处理的缓解效果优于15 mmol·L^-1处理;低氧缺氮处理（0 mmol NO₃^-·L^-1）的樱桃植株根系活力下降,根系呼吸受到抑制,NR、GS及NADH-GDH活性降低.这说明低氧胁迫下,适当提高生长介质中的NO₃^-浓度可调控樱桃的根系功能及氮代谢,缓解低氧胁迫对樱桃根系的伤害.     

Divergent patterns of foliar δ13C and δ15N in Quercus aquifolioides with an altitudinal transect on the Tibetan Plateau: an integrated study based on multiple key leaf functional traits

Aims With a close association with plant water availability, foliar δ 13 C had been investigated extensively in alpine regions; however, foliar δ 15 N has rarely been concurrently used as an indicator of plant nitrogen availability. Due to the positive correlations between leaf nitrogen content and foliar δ 13 C and δ 15 N found in previous studies, we expected that they should show consistent patterns along an altitudinal gradient.Methods To test our hypothesis, we measured foliar δ 13 C and δ 15 N in conjunction with multiple key leaf functional traits of Quercus aquifolioides, a dominant species of alpine forest on the eastern slopes of the Sygera Mountains, southeastern Tibetan Plateau from 2500 to 3800 m.Important findings (i) Contrary to our hypothesis, foliar δ 13 C exhibited a significant positive linear relationship with altitude; in contrast, foliar δ 15 N initially increased and subsequently decreased with altitude, the change in trend occurring around 3300 m. (ii) Our analyses indicated that leaf internal resistance and stomatal conductance, rather than photosynthetic capacity indicated by leaf N concentration, apparently explained the altitudinal variation in foliar δ 13 C, while differences in foliar δ 15 N were likely the result of soil N availability. (iii) Principal component analysis revealed a clear association between δ 13 C and a tradeoff between water loss and carbon gain, indicated by traits related to gas exchange such as leaf thickness, density, stomatal properties. In contrast, the second axis was associated with δ 15 N and nitrogen acquisition strategy in Q. aquifolioides across its altitudinal distribution, represented by traits related to nitrogen concentration and stomata per gram of leaf nitrogen.     

Combined effects of solvation and aggregation propensity on the final supramolecular structures adopted by hydrophobic,glycine‐rich,elastin‐like polypeptides

Previous work on elastin‐like polypeptides (ELPs) made of hydrophobic amino acids of the type XxxGlyGlyZzzGly (Xxx, Zzz = Val, Leu) has consistently shown that differing dominant supramolecular structures were formed when the suspending media were varied: helical, amyloid‐like fibers when suspended in water and globules evolving into “string of bead” structures, poly(ValGlyGlyValGly), or cigar‐like bundles, poly(ValGlyGlyLeuGly), when suspended in methyl alcohol. Comparative experiments with poly(LeuGlyGlyValGly) have further indicated that the interface energy plays a significant role and that solvation effects act in concomitance with the intrinsic aggregation propensity of the repeat sequence. Continuing our investigation on ELPs using surface (X‐ray photoelectron spectroscopy, atomic force microscopy) and bulk (circular dichroism, Fourier transform infrared spectroscopy) techniques for their characterization, here we have compared the effect of suspending solvents (H₂O, dimethylsulfoxide, ethylene glycol, and MeOH) on poly(ValGlyGlyValGly), the polypeptide most inclined to form long and well‐refined helical fibers in water, searching for the signature of intermolecular interactions occurring between the polypeptide chains in the given suspension. The influence of sequence specificities has been studied by comparing poly(ValGlyGlyValGly) and poly(LeuGlyGlyValGly) with a similar degree of polymerization. Deposits on substrates of the polypeptides were characterized taking into account the differing evaporation rate of solvents, and tests on their stability in ultra high vacuum were performed. Finally, combining experimental and computational studies, we have revaluated the three‐dimensional modeling previously proposed for the supramolecular assembly in water of poly(ValGlyGlyValGly). The results were discussed and rationalized also in the light of published data. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 292–292, 2013.