Structural analysis of an "open" form of PBP1B from Streptococcus pneumoniae

The class A PBP1b from Streptococcus pneumoniae is responsible for glycosyltransferase and transpeptidase (TP) reactions, forming the peptidoglycan of the bacterial cell wall. The enzyme has been produced in a stable, soluble form and undergoes time-dependent proteolysis to leave an intact TP domain. Crystals of this TP domain were obtained, diffracting to 2.2 A resolution, and the structure was solved by using molecular replacement. Analysis of the structure revealed an "open" active site, with important conformational differences to the previously determined "closed" apoenzyme. The active-site nucleophile, Ser460, is in an orientation that allows for acylation by beta-lactams. Consistent with the productive conformation of the conserved active-site catalytic residues, adjacent loops show only minor deviation from those of known acyl-enzyme structures. These findings are discussed in the context of enzyme functionality and the possible conformational sampling of PBP1b between active and inactive states.     

耐盐氨基甲酸乙酯水解酶的分离纯化及酶学性质

氨基甲酸乙酯是发酵食品中存在的一种致癌物质,酶法去除发酵食品中的氨基甲酸乙酯是消除氨基甲酸乙酯危害的一种重要方法。从小鼠的胃部获得了一株产氨基甲酸乙酯水解酶的肺炎克雷伯氏菌,为了解该氨基甲酸乙酯水解酶的酶学性质,从肺炎克雷伯氏菌中提取获得氨基甲酸乙酯水解酶粗酶液,经硫酸铵沉淀、离子交换层析和凝胶过滤层析分离得到氨基甲酸乙酯水解酶纯酶。通过十二烷基硫酸钠聚丙烯酰胺电泳(SDS-PAGE)分析,估计该酶的分子量约为55 kDa。其水解氨基甲酸乙酯的Km值为74 mmol/L。酶反应的最适温度为55℃,最适pH为7.0。乙二胺四乙酸(EDTA)和二硫苏糖醇(DTT)对该酶有较强的激活作用,而Cu2+和Zn2+则有较强的抑制作用。该酶可耐受高浓度NaCl,对低浓度乙醇也有一定的耐受性,对于酱油中氨基甲酸乙酯的消除有一定的参考意义。     

Expression, Purification, and Characterization of Recombinant Drosophila Choline Acetyltransferase

Abstract: A cDNA for Drosophila choline acetyltransferase (EC 2.3.1.6; ChAT) was fused with a polyhistidine sequence and expressed in Escherichia coli. The recombinant enzyme was purified to a specific activity of 500 μmol/min/mg of protein using metal affinity chromatography and ion exchange chromatography. Kinetic properties of the recombinant enzyme did not differ significantly from those previously determined. Circular dichroism (CD) spectra revealed that the secondary structure of the enzyme is largely μ-helical. Intrinsic fluorescence spectra of the enzyme indicate that its tryptophan residues are buried. Neither CD nor fluorescence spectra changed significantly in the presence of substrates. The cysteine content of the recombinant Drosophila ChAT was determined to be 16 in the absence and 22 in the presence of 6 M guanidine hydrochloride. Finally, crystallization of recombinant Drosophila ChAT was achieved.     

Evolving the naturally compromised chorismate mutase from Mycobacterium tuberculosis to top performance

Chorismate mutase (CM), an essential enzyme at the branch-point of the shikimate pathway, is required for the biosynthesis of phenylalanine and tyrosine in bacteria, archaea, plants, and fungi. MtCM, the CM from Mycobacterium tuberculosis, has less than 1% of the catalytic efficiency of a typical natural CM and requires complex formation with 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase for high activity. To explore the full potential of MtCM for catalyzing its native reaction, we applied diverse iterative cycles of mutagenesis and selection, thereby raising k_cat/K_m 270-fold to 5 × 10⁵ m⁻¹s⁻¹, which is even higher than for the complex. Moreover, the evolutionarily optimized autonomous MtCM, which had 11 of its 90 amino acids exchanged, was stabilized compared with its progenitor, as indicated by a 9 °C increase in melting temperature. The 1.5 Å crystal structure of the top-evolved MtCM variant reveals the molecular underpinnings of this activity boost. Some acquired residues (e.g. Pro⁵² and Asp⁵⁵) are conserved in naturally efficient CMs, but most of them lie beyond the active site. Our evolutionary trajectories reached a plateau at the level of the best natural enzymes, suggesting that we have exhausted the potential of MtCM. Taken together, these findings show that the scaffold of MtCM, which naturally evolved for mediocrity to enable inter-enzyme allosteric regulation of the shikimate pathway, is inherently capable of high activity.     

Large Conformational Changes of Insertion 3 in Human Glycyl-tRNA Synthetase (hGlyRS) during Catalysis

Glycyl-tRNA synthetase (GlyRS) is the enzyme that covalently links glycine to cognate tRNA for translation. It is of great research interest because of its nonconserved quaternary structures, unique species-specific aminoacylation properties, and noncanonical functions in neurological diseases, but none of these is fully understood. We report two crystal structures of human GlyRS variants, in the free form and in complex with tRNA^Gly respectively, and reveal new aspects of the glycylation mechanism. We discover that insertion 3 differs considerably in conformation in catalysis and that it acts like a “switch” and fully opens to allow tRNA to bind in a cross-subunit fashion. The flexibility of the protein is supported by molecular dynamics simulation, as well as enzymatic activity assays. The biophysical and biochemical studies suggest that human GlyRS may utilize its flexibility for both the traditional function (regulate tRNA binding) and alternative functions (roles in diseases).     

Purification and Characterization of a Cold-adapted Isocitrate Lyase and a Malate Synthase from Colwellia maris,a Psychrophilic Bacterium

Isocitrate lyase (ICL) and malate synthase (MS) of a psychrophilic marine bacterium, Colwellia maris, were purified to electrophoretically homogeneous state. The molecular mass of the ICL was found to be 240 kDa, composed of four identical subunits of 64.7 kDa. MS was a dimeric enzyme composed of 76.3 kDa subunits. N-Terminal amino acid sequences of the ICL and MS were analyzed. Purified ICL had its maximum activity at 20°C and was rapidly inactivated at the temperatures above 30°C, but the optimum temperature for the activity of MS was 45°C. NaCl was found to protect ICL from heat inactivation above 30°C, but the salt did not stabilize MS. Effects of temperatures on the kinetic parameters of both the enzymes were examined. The K_m for the substrate (isocitrate) of ICL was decreased with decreasing temperature. On the other hand, the K_m for the substrate (glyoxylate) of MS was increased with decreasing temperature. The calculated value of free energy of activation of ICL was on the same level as that of MS.     

Cryoelectron-Microscopic Structure of the pKpQIL Conjugative Pili from Carbapenem-Resistant Klebsiella pneumoniae

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Differential Effects of Antibiotic Therapy on the Structure and Function of Human Gut Microbiota

The human intestinal microbiota performs many essential functions for the host. Antimicrobial agents, such as antibiotics (AB), are also known to disturb microbial community equilibrium, thereby having an impact on human physiology. While an increasing number of studies investigate the effects of AB usage on changes in human gut microbiota biodiversity, its functional effects are still poorly understood. We performed a follow-up study to explore the effect of ABs with different modes of action on human gut microbiota composition and function. Four individuals were treated with different antibiotics and samples were taken before, during and after the AB course for all of them. Changes in the total and in the active (growing) microbiota as well as the functional changes were addressed by 16S rRNA gene and metagenomic 454-based pyrosequencing approaches. We have found that the class of antibiotic, particularly its antimicrobial effect and mode of action, played an important role in modulating the gut microbiota composition and function. Furthermore, analysis of the resistome suggested that oscillatory dynamics are not only due to antibiotic-target resistance, but also to fluctuations in the surviving bacterial community. Our results indicated that the effect of AB on the human gut microbiota relates to the interaction of several factors, principally the properties of the antimicrobial agent, and the structure, functions and resistance genes of the microbial community.     

Immunoaffinity Purification of Human Choline Acetyltransferase: Comparison of the Brain and Placental Enzymes

A rapid and efficient immunoaffinity purification procedure has been developed for human placental choline acetyltransferase (ChAT). Using this procedure, human placental ChAT was purified to homogeneity with high recovery of enzyme activity (50-60%). Purified ChAT was used to raise a monospecific anti-human ChAT polyclonal antibody in rabbits. A comparison of the physical properties of ChAT was made between the enzymes purified from human brain and human placenta. Only one form of the enzyme exists in either tissue, having identical molecular weights of 68,000 and a single apparent pI of 8.1. A more detailed comparison of the two enzymes using peptide mapping and epitope mapping indicates identity between the brain and placental enzymes.     

Two-step Ligand Binding in a (βα)8 Barrel Enzyme: SUBSTRATE-BOUND STRUCTURES SHED NEW LIGHT ON THE CATALYTIC CYCLE OF HisA*

HisA is a (βα)₈ barrel enzyme that catalyzes the Amadori rearrangement of N′-[(5′-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) to N′-((5′-phosphoribulosyl) formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in the histidine biosynthesis pathway, and it is a paradigm for the study of enzyme evolution. Still, its exact catalytic mechanism has remained unclear. Here, we present crystal structures of wild type Salmonella enterica HisA (SeHisA) in its apo-state and of mutants D7N and D7N/D176A in complex with two different conformations of the labile substrate ProFAR, which was structurally visualized for the first time. Site-directed mutagenesis and kinetics demonstrated that Asp-7 acts as the catalytic base, and Asp-176 acts as the catalytic acid. The SeHisA structures with ProFAR display two different states of the long loops on the catalytic face of the structure and demonstrate that initial binding of ProFAR to the active site is independent of loop interactions. When the long loops enclose the substrate, ProFAR adopts an extended conformation where its non-reacting half is in a product-like conformation. This change is associated with shifts in a hydrogen bond network including His-47, Asp-129, Thr-171, and Ser-202, all shown to be functionally important. The closed conformation structure is highly similar to the bifunctional HisA homologue PriA in complex with PRFAR, thus proving that structure and mechanism are conserved between HisA and PriA. This study clarifies the mechanistic cycle of HisA and provides a striking example of how an enzyme and its substrate can undergo coordinated conformational changes before catalysis.     

The comparative specificity of the inner and outer substrate transfer sites in the choline carrier of human erythrocytes

Summary The substrate specificities on the inner and outer surfaces of the cell membrane have been compared by determining the relative affinities, inside and outside, of a series of choline analogs. The results of two different methods were in agreement: (1) the carrier distribution was determined in the presence of a saturating concentration of an equilibrated analog, using N-ethylmaleimide as a probe for the inward-facing carrier; (2) the degree of competition was measured between an equilibrated analog and choline in the external solution. The carrier sites are found to have markedly different specificities: the outer site is more closely complementary to the structure of choline than is the inner, and even a slight enlargement of either the trimethylammonium or hydroxyethyl group gives rise to preferential binding inside. It is also found that a nonpolar binding region, which is adjacent to the outer site, is absent from the inner site. As the transport mechanism involves the exposure of only one site at a time, first on one surface and then the other, it follows that an extensive reorganization of the structure of the substrate site may occur during the carrier-reorientation step, or alternatively that two distinct sites may be present, only one of which is exposed at a time.     

Exploring the role of large conformational changes in the fidelity of DNA polymerase beta

The relationships between the conformational landscape, nucleotide insertion catalysis and fidelity of DNA polymerase beta are explored by means of computational simulations. The simulations indicate that the transition states for incorporation of right (R) and wrong (W) nucleotides reside in substantially different protein conformations. The protein conformational changes that reproduce the experimentally observed fidelity are significantly larger than the small rearrangements that usually accompany motions from the reactant state to the transition state in common enzymatic reactions. Once substrate binding has occurred, different constraints imposed on the transition states for insertion of R and W nucleotides render it highly unlikely that both transition states can occur in the same closed structure, because the predicted fidelity would then be many orders of magnitude too large. Since the conformational changes reduce the transition state energy of W incorporation drastically they decrease fidelity rather than increase it. Overall, a better agreement with experimental data is attained when the R is incorporated through a transition state in a closed conformation and W is incorporated through a transition state in one or perhaps several partially open conformations. The generation of free energy surfaces for R and W also allow us to analyze proposals about the relationship between induced fit and fidelity.     

Structure and Mechanism of Metallocarboxypeptidases

Metallocarboxpeptidases cleave C-terminal residues from peptide substrates and participate in a wide range of physiological processes, but they also contribute to human pathology. On the basis of structural information, we can distinguish between two groups of such metallopeptidases: cowrins and funnelins. Cowrins comprise protozoan, prokaryotic, and mammalian enzymes related to both neurolysin and angiotensin-converting enzyme and their catalytic domains contain 500–700 residues. They are ellipsoidal and traversed horizontally by a long, deep, narrow active-site cleft, in which the C-terminal residues are cut from oligopeptides and unstructured protein tails. The consensus cowrin structure contains a common core of 17 helices and a three-stranded β-sheet, which participates in substrate binding. This protease family is characterized by a set of spatially conserved amino acids involved in catalysis, HEXXH+EXXS/G+H+Y/R+Y. Funnelins comprise structural relatives of the archetypal bovine carboxypeptidase A1 and feature mammalian, insect and bacterial proteins with strict carboxypeptidase activity. Their ～ 300-residue catalytic domains evince a consensus central eight-stranded β-sheet flanked on either side by a total of eight helices. They also contain a characteristic set of conserved residues, HXXE+R+NR+H+Y+E, and their active-site clefts are rather shallow and lie at the bottom of a funnel-like cavity. Therefore, these enzymes act on a large variety of well-folded proteins. In both cowrins and funnelins, substrate hydrolysis follows a common general base/acid mechanism. A metal-bound solvent molecule ultimately performs the attack on the scissile peptide bond with the assistance of a strictly conserved glutamate residue.     

Structure predictions and surface charge of nitrogenase flavodoxins from Klebsiella pneumoniae and Azotobacter vinelandii

A first approximation to the tertiary structure of the nitrogenase flavodoxins of Klebsiella pneumoniae and Azotobacter vinelandii can be obtained by superimposing their amino acid sequences upon the crystallographically determined structure of the long-chain flavodoxin from Anacystis nidulans. This procedure is validated by secondary structure predictions based on the sequence alone and by the distribution of polar and hydrophobic residues. It reveals, among other things, a distinctive distribution of surface charge peculiar to the nitrogenase flavodoxins, which is probably important in determining the kinetics of electron transfer with their physiological redox partners. The most likely positions of the phosphodiester bridge which has been described in the A. vinelandii molecule can also be assessed.     

Structure and Function of 2,3-Dimethylmalate Lyase, a PEP Mutase/Isocitrate Lyase Superfamily Member

The Aspergillus niger genome contains four genes that encode proteins exhibiting greater than 30% amino acid sequence identity to the confirmed oxaloacetate acetyl hydrolase (OAH), an enzyme that belongs to the phosphoenolpyruvate mutase/isocitrate lyase superfamily. Previous studies have shown that a mutant A. niger strain lacking the OAH gene does not produce oxalate. To identify the function of the protein sharing the highest amino acid sequence identity with the OAH (An07g08390, Swiss-Prot entry Q2L887, 57% identity), we produced the protein in Escherichia coli and purified it for structural and functional studies. A focused substrate screen was used to determine the catalytic function of An07g08390 as (2R,3S)-dimethylmalate lyase (DMML): k_cat = 19.2 s^− 1 and K_m = 220 μM. DMML also possesses significant OAH activity (k_cat = 0.5 s^− 1 and K_m = 220 μM). DNA array analysis showed that unlike the A. niger oah gene, the DMML encoding gene is subject to catabolite repression. DMML is a key enzyme in bacterial nicotinate catabolism, catalyzing the last of nine enzymatic steps. This pathway does not have a known fungal counterpart. BLAST analysis of the A. niger genome for the presence of a similar pathway revealed the presence of homologs to only some of the pathway enzymes. This and the finding that A. niger does not thrive on nicotinamide as a sole carbon source suggest that the fungal DMML functions in a presently unknown metabolic pathway. The crystal structure of A. niger DMML (in complex with Mg²⁺ and in complex with Mg²⁺ and a substrate analog: the gem-diol of 3,3-difluoro-oxaloacetate) was determined for the purpose of identifying structural determinants of substrate recognition and catalysis. Structure-guided site-directed mutants were prepared and evaluated to test the contributions made by key active-site residues. In this article, we report the results in the broader context of the lyase branch of the phosphoenolpyruvate mutase/isocitrate lyase superfamily to provide insight into the evolution of functional diversity.     

重组人巨噬细胞集落刺激因子的结构与功能

重组人粒细胞──巨噬细胞集落刺激因子(rhGM-CSF) 已经在原核、真核细胞表达并得到纯品,为它的结构与功能研究提供了有利条件．目前国内外对于rhGM-CSF的结构与功能研究主要集中在晶体结构、化学修饰、溶液中构象和稳定性以及突变和分子设计方面．分子结构与功能的关系以及与GM-CSF受体作用机理研究也取得了突破性进展．     

血管紧张素转换酶的结构功能及相关抑制剂

血管紧张素转化酶(angiotensin converting enzyme, ACE, EC 3.4.15.1)是一种位于细胞膜上, 依赖锌离子的羧二肽酶, 催化水解十肽血管紧张素I羧基末端两个氨基酸, 生成具有血管收缩作用的八肽血管紧张素II。ACE在血压调节系统renin - angiotensin system (RAS系统)中具有重要作用, 从ACE的结构功能、基因多态性及其抑制剂等方面进行了详细综述。发现体细胞ACE两个活性中心催化血管紧张素I和缓激肽的机制不同, 因此以体细胞ACE单个活性中心为靶点的研究, 将会为研制开发副作用更少, 安全性更高的ACE抑制剂提供新的途径。     

NMR solution structure of KP-TerB, a tellurite-resistance protein from Klebsiella pneumoniae

Klebsiella pneumoniae (KP), a Gram-negative bacterium, is a common cause of hospital-acquired bacterial infections worldwide. Tellurium (Te) compounds, although relatively rare in the environment, have a long history as antimicrobial and therapeutic agents. In bacteria, tellurite (TeO(3) (-2)) resistance is conferred by the ter (Te(r)) operon (terZABCDEF). Here, on the basis of 2593 restraints derived from NMR analysis, we report the NMR structure of TerB protein (151 amino acids) of KP (KP-TerB), which is mainly composed of seven alpha-helices and a 3(10) helix, with helices II to V apparently forming a four-helix bundle. The ensemble of 20 NMR structures was well-defined, with a RMSD of 0.32 +/- 0.06 A for backbone atoms and 1.11 +/- 0.07 A for heavy atoms, respectively. A unique property of the KP-TerB structure is that the positively and negatively charged clusters are formed by the N-terminal positively and C-terminal negatively charged residues, respectively. To the best of our knowledge, the protein sequence and structures of KP-TerB are unique.