生物分子水溶液的蒙特卡罗模拟——Ⅱ.缬氨酸溶液

本文对由115个水分子包围一个中性缬氨酸分子所组成的分子集团做了蒙特卡罗模拟.模拟的温度是298K.对缬氨酸羧基区、氨基区和异丙基区分别提取了平均水—水和水—缬氨酸分子的相互作用能的径向分布函数,水分子的氧原子和氢原子的径向分布函数以及水分子偶极矩的取向关联函数.此外,我们还求出了所模拟溶液的构型比热和缬氨酸分子分区及全体的第一个水化层的平均水分子数.     

深海放线菌Actinomadura sp.01119的代谢产物研究

采用硅胶柱色谱、制备薄层色谱和凝胶色谱等分离方法,对分离自深海沉积物中的海洋放线菌Actinomadura sp.01119的代谢产物进行研究,从中分离得到9个化合物,经1H NMR、13C NMR和MS等波谱分析并结合相关文献鉴定为环(L-苯丙-L-脯)二肽(1),环(L-脯-L-脯)二肽(2),环(L-酪-L-脯)二肽(3),环(D-酪-L-脯)二肽(4),环(L-异亮-L-脯)二肽(5),环(L-缬-L-脯)二肽(6),棕榈酸(7),对羟基苯甲酸(8),尿嘧啶(9)。其中化合物1~6为环二肽类化合物。     

发酵法生产 L—酪氨酸

<正> 1、发明的名称:发酵法制备L—酪氨酸2、专利申请范围:采用谷氨酸棒状杆菌属的L—苯丙氨酸缺陷型,以及对L—苯丙氨酸类似物及磺酰胺有抗性的突变株制造酪氨酸的方法,该突变株在培养基中可以生成和积累L—酪氨酸。3、本发明的详细说明:本发明是关于发酵法制备L—酪氨酸。用发酵法生产L—酪氨酸已知的有谷氨酸微球菌的L—苯丙氨酸缺陷型的制造方法、短杆菌属等的对一氟苯丙氨酸抗性株或棒状杆菌属的L—酪氮酸及L—苯丙氨酸的类似物的抗性菌株的制造方法等。     

发酵法生产 L—酪氨酸

<正> 1、发明的名称:发酵法制备L—酪氨酸2、专利申请范围:采用谷氨酸棒状杆菌属的L—苯丙氨酸缺陷型,以及对L—苯丙氨酸类似物及磺酰胺有抗性的突变株制造酪氨酸的方法,该突变株在培养基中可以生成和积累L—酪氨酸。3、本发明的详细说明:本发明是关于发酵法制备L—酪氨酸。用发酵法生产L—酪氨酸已知的有谷氨酸微球菌的L—苯丙氨酸缺陷型的制造方法、短杆菌属等的对一氟苯丙氨酸抗性株或棒状杆菌属的L—酪氮酸及L—苯丙氨酸的类似物的抗性菌株的制造方法等。     

L—谷氨酸的综合利用 Ⅱ报:从L—谷氨酸合成N—乙酰—L—谷氨酰胺~#

<正> N—乙酰—L—谷氨酰胺(N—Acetyl—L—Glutamine)为L—谷氨酰胺的乙酰化衍生物。具有抗痉挛活性。它比L—谷氨酰胺更易透过“血脑屏障”。改善脑的功能以维持其良好应激机能。适用于肝昏迷及脑外伤、脑肿瘤、神经外科手术等引起的昏迷等。并可调整变态代谢,在体内不会很快消     

海洋真菌与细菌发酵物中的环二肽

从红树林真菌1356攻株发酵液中分离到4个环二肽,它们是环Pro-Tyr,环Pro-Ile,环Gly-Phe和环Ala-Phe。另从海洋细菌110菌株发酵液中分离到5个环二肽,它们是环Leu-Ile,环Val-Leu,环Ala-Val,环Ala-Ile和环Ala-Leu。这些环二肽多数是新发现的微生物产物。本比较了分离的环二肽与已经报道的环二肽在来源、结构、功能等方面的区别。     

L—谷氨酰胺的化学合成

本文叙述了以L—谷氨酸为原料,经过酯化、氨解、酸解等工艺路线制备L—谷氨酰胺的方法。本工艺综合了德国和日本专利所报导的合成方法,结合我们的工作实践,在酰化反应后不将所生成的L—谷氨酸—r—甲酯分离出来,而采用低溫酯化以控制L—谷氨酸二甲酯的生成,并在产品精制过程中采用树脂方法,分离少量未反应完全而混入产品中的L—谷氨酸。此法优点是:中间产物不用分离,减少了工艺步骤;操作简便,易实施工业大生产;减少了设备投资。采用本法生产的L—谷氨酰胺质量符合日本味之素公司的质量标准,收率达到理论值的52.8%。本工艺于一九八三年十二月在武汉通过技术鉴定。     

L—谷氨酰胺的化学合成

本文叙述了以L—谷氨酸为原料,经过酯化、氨解、酸解等工艺路线制备L—谷氨酰胺的方法。本工艺综合了德国和日本专利所报导的合成方法,结合我们的工作实践,在酰化反应后不将所生成的L—谷氨酸—r—甲酯分离出来,而采用低溫酯化以控制L—谷氨酸二甲酯的生成,并在产品精制过程中采用树脂方法,分离少量未反应完全而混入产品中的L—谷氨酸。此法优点是:中间产物不用分离,减少了工艺步骤;操作简便,易实施工业大生产;减少了设备投资。采用本法生产的L—谷氨酰胺质量符合日本味之素公司的质量标准,收率达到理论值的52.8%。本工艺于一九八三年十二月在武汉通过技术鉴定。     

Streptomyces aidingensis CGMCC 4.5739中环二肽氧化酶DmtD3_E3的体外生化功能

【背景】环二肽合酶(cyclodipeptide synthase, CDPS)途径中新颖后修饰酶的挖掘对获得结构新颖活性良好的二酮哌嗪类化合物具有重要意义。前期研究中发现来源于Streptomyces aidingensis CGMCC 4.5739的环二肽合酶基因簇dmt3中dmtA3B3C3可编码二酮哌嗪—萜类化合物drimentines (DMTs),推测其下游环二肽氧化酶基因dmtD3_E3也参与了DMTs的生物合成,但其功能一直未鉴定。【目的】对S.aidingensisCGMCC 4.5739中环二肽合酶基因簇dmt3内的环二肽氧化酶DmtD3_E3的功能进行表征,为增加二酮哌嗪类化合物结构多样性提供功能元件。【方法】从S.aidingensisCGMCC 4.5739的基因组中克隆环二肽氧化酶基因dmtD3_E3,构建重组表达质粒pWLI209,并在大肠杆菌BL21(DE3)中可溶性表达。通过建立体外酶促反应,运用液质联用(high performance liquid chromatography-mass spectrometry,HPLC-MS)和核磁共振(nuclear magnetic resonance,NMR)等方法确定催化产物结构。【结果】环二肽氧化酶DmtD3_E3可催化环二肽cyclo-(L-Trp-L-Leu) (cWL)的C14-C17位氧化脱氢形成cyclo-(L-Trp-L-ΔLeu) (cWΔL)。此外DmtD3_E3还可以催化环二肽cyclo-(L-Trp-L-Ala) (cWA)的C10-C11位脱氢生成cyclo-(L-Trp-L-ΔAla) (cΔWA),具有底物宽泛性。【结论】本研究通过对环二肽合酶生物合成途径中新颖环二肽氧化酶的挖掘和表征,为后续通过组合生物合成及合成生物学手段生成“非天然”二酮哌嗪类化合物衍生物奠定了基础。     

L—异亮氨酸发酵对氧需求的研究

L—异亮氨酸主要用于医药和食品行业,是人体八种必需氨基酸之一.合理地供氧L—异亮氨酸产酸多,杂氨基酸量低.以钝齿棒杆菌(Corynebacterium Crenatum)为菌株,在71自动控制发酵罐中分批培养,在1.0—1.5vvm通气量范围内,测得了L—异亮氨酸发酵的多项参数,并对它们之间的关系及控制手段进行综合分析,提出了工业发酵过程中继续提高发酵水平的依据.     

Hydration of phospholipid bilayers in the presence and absence of cholesterol

The number of water molecules bound (unfreezable) by a molecule of dipalmitoyl phosphatidylserine (DPPS) or by a molecule of dipalmitoyl phosphatidylcholine (DPPC) alone or in mixtures with cholesterol was determined by differential scanning calorimetry (DSC). When the phospholipids are in the gel state and in the absence of cholesterol, molecule of DPPS binds about 3.5 molecules of water and molecule of DPPC binds about 6 molecules of water. Number of water molecules bound increases when cholesterol crystallites are formed in the bilayer. For DPPS-cholesterol mixture at X(chol) -0.5, as well as for DPPC-cholesterol mixture at X(chol) -0.5 about 7 water molecules are bound.     

Active-site gorge and buried water molecules in crystal structures of acetylcholinesterase from Torpedo californica

Buried water molecules and the water molecules in the active-site gorge are analyzed for five crystal structures of acetylcholinesterase from Torpedo californica in the resolution range 2.2-2.5 A (native enzyme, and four inhibitor complexes). A total of 45 buried hydration sites are identified, which are populated with between 36 and 41 water molecules. About half of the buried water is located in a distinct region neighboring the active-site gorge. Most of the buried water molecules are very well conserved among the five structures, and have low displacement parameters, B, of magnitudes similar to those of the main-chain atoms of the central beta-sheet structure. The active-site gorge of the native enzyme is filled with over 20 water molecules, which have poor hydrogen-bond coordination with an average of 2.9 polar contacts per water molecule. Upon ligand binding, distinct groups of these water molecules are displaced, whereas the others remain in positions similar to those that they occupy in the native enzyme. Possible roles of the buried water molecules are discussed, including their possible action as a lubricant to allow large-amplitude fluctuations of the loop structures forming the gorge wall. Such fluctuations are required to facilitate traffic of substrate, products and water molecules to and from the active-site. Because of their poor coordination, the gorge water molecules can be considered as "activated" as compared to bulk water. This should allow their easy displacement by incoming substrate. The relatively loose packing of the gorge water molecules leaves numerous small voids, and more efficient space-filling by substrates and inhibitors may be a major driving force of ligand binding.     

WaterScore: a novel method for distinguishing between bound and displaceable water molecules in the crystal structure of the binding site of protein-ligand complexes

We have performed a multivariate logistic regression analysis to establish a statistical correlation between the structural properties of water molecules in the binding site of a free protein crystal structure, with the probability of observing the water molecules in the same location in the crystal structure of the ligand-complexed form. The temperature B-factor, the solvent-contact surface area, the total hydrogen bond energy and the number of protein-water contacts were found to discriminate between bound and displaceable water molecules in the best regression functions obtained. These functions may be used to identify those bound water molecules that should be included in structure-based drug design and ligand docking algorithms.FIGURE The binding site ( thin sticks) of penicillopepsin (3app) with its crystallographically determined water molecules ( spheres) and superimposed ligand (in thick sticks, from complexed structure 1ppk). Water molecules sterically displaced by the ligand upon complexation are shown in cyan. Bound water molecules are shown in blue. Displaced water molecules are shown in yellow. Water molecules removed from the analysis due to a lack of hydrogen bonds to the protein are shown in white. WaterScore correctly predicted waters in blue as Probability=1 to remain bound and waters in yellow as Probability<1x10(-20) to remain bound.     

Dynamics of hydration in hen egg white lysozyme

We investigate the hydration dynamics of a small globular protein, hen egg-white lysozyme. Extensive simulations (two trajectories of 9 ns each) were carried out to identify the time-scales and mechanism of water attachment to this protein. The location of the surface and integral water molecules in lysozyme was also investigated. Three peculiar temporal scales of the hydration dynamics can be discerned: two among these, with sub-nanosecond mean residence time, tau(w), are characteristic of surface hydration water; the slower time-scale (tau(w) approximately 2/3 ns) is associated with buried water molecules in hydrophilic pores and in superficial clefts. The computed tau(w) values in the two independent runs fall in a similar range and are consistent with each other, thus adding extra weight to our result. The tau(w) of surface water obtained from the two independent trajectories is 20 and 24 ps. In both simulations only three water molecules are bound to lysozyme for the entire length of the trajectories, in agreement with nuclear magnetic relaxation dispersion estimates. Locations other than those identified in the protein crystal are found to be possible for these long-residing water molecules. The dynamics of the hydration water molecules observed in our simulations implies that each water molecule visits a multitude of residues during the lifetime of its bound with the protein. The number of residues seen by a single water molecule increases with the time-scale of its residence time and, on average, is equal to one only for the water molecules with shorter residence time. Thus, tau(w) values obtained from inelastic neutron scattering and based on jump-diffusion models are likely not to account for the contribution of water molecules with longer residence time.     

Computational study on the function of water within a beta-helix antifreeze protein dimer and in the process of ice-protein binding

Antifreeze proteins (AFPs) help many organisms protect themselves from freezing in subzero temperatures. The most active AFPs found to date are those from insects, which possess exceptionally regular beta-helical structures. On the ice-binding surface of these proteins, regularly arrayed water molecules are observed within the repeating Thr-Xxx-Thr motif, but the exact role of these water molecules remains unknown. In this work, we have employed a number of computational methods to examine the role of these water molecules in an AFP from Tenebrio molitor (TmAFP). Our investigation involved a combination of molecular and quantum mechanical approaches. Properties such as stability, interaction energy, orbital overlap, and conformational analysis of various systems, including TmAFP-water, TmAFP-water-ice, and TmAFP-ice, were systematically evaluated and compared. The regularly arrayed water molecules were found to remain associated with TmAFP before ice binding, demonstrating that they are an intrinsic part of the protein. These water molecules may assist TmAFP in the process of ice recognition and binding. However, after facilitating the initial stages of ice recognition and binding, these water molecules are excluded in the final formation of the AFP-ice complex. The departure of these water molecules enables a better two-dimensional match between TmAFP and ice. These results agree with experimental observations showing that although these water molecules are aligned with the ice-binding hydroxyl groups of Thr residues in one dimension, they are in fact positioned slightly off in the second dimension, making a good two-dimensional match impossible.     

Protein-bound water molecules in primate red- and green-sensitive visual pigments

Protein-bound water molecules play crucial roles in the structure and function of proteins. The functional role of water molecules has been discussed for rhodopsin, the light sensor for twilight vision, on the basis of X-ray crystallography, Fourier transform infrared (FTIR) spectroscopy, and a radiolytic labeling method, but nothing is known about the protein-bound waters in our color visual pigments. Here we apply low-temperature FTIR spectroscopy to monkey red (MR)- and green (MG)-sensitive color pigments at 77 K and successfully identify water vibrations using D(2)O and D(2)(18)O in the whole midinfrared region. The observed water vibrations are 6-8 for MR and MG, indicating that several water molecules are present near the retinal chromophore and change their hydrogen bonds upon retinal photoisomerization. In this sense, color visual pigments possess protein-bound water molecules essentially similar to those of rhodopsin. The absence of strongly hydrogen-bonded water molecules (O-D stretch at <2400 cm(-1)) is common between rhodopsin and color pigments, which greatly contrasts with the case of proton-pumping microbial rhodopsins. On the other hand, two important differences are observed in water signal between rhodopsin and color pigments. First, the water vibrations are identical between the 11-cis and 9-cis forms of rhodopsin, but different vibrational bands are observed at >2550 cm(-1) for both MR and MG. Second, strongly hydrogen-bonded water molecules (2303 cm(-1) for MR and 2308 cm(-1) for MG) are observed for the all-trans form after retinal photoisomerization, which is not the case for rhodopsin. These specific features of MR and MG can be explained by the presence of water molecules in the Cl(-)-biding site, which are located near positions C11 and C9 of the retinal chromophore. The averaged frequencies of the observed water O-D stretching vibrations for MR and MG are lower as the λ(max) is red-shifted, suggesting that water molecules are involved in the color tuning of our vision.     

Fourth polymorph of [Phe4 Val6] antamanide (pentahydrate)

The cyclic decapeptide antamanide and the synthetic, biologically active analog [Phe4 Val6]antamanide (cyclic[ValProProPhePhe]2) crystallize in various crystal forms as a function of the solvent. The present crystalline polymorph obtained from acetone/water (also from ethanol/water and DMSO/water) crystallizes in space group P2(1)2(1)2(1) with a = 20.194 (30) A, b = 21.118 (31) A, c = 16,132 (25) A and four molecules of peptide in the unit cell. There are five cocrystallized water molecules per peptide molecule, of which four water molecules are intrinsic to the peptide molecule. Although the molecular packing is entirely different in each of the polymorphs, the conformation of the peptide molecule, including the intrinsic water molecules, is very similar in all the polymorphs.     

Intercalation of water molecules between nucleic acid bases in the crystal structures of 6-azathymine hemihydrate and 5-amino-2-thiocytosine dihydrochloride dihydrate

The crystal structure of 6-azathymine hemihydrate (6AzTH) exhibits a novel intercalation of water molecules interposed half-way between the modified bases 6.3 to 6.7 A apart. The crystal contains four molecules of 6-azathymine (6AzT) and two water molecules as the independent repeating unit. These two water molecules together with the four bases form two separate water sandwiches. In the crystal structure these sandwiches form two sets of local clusters. The anhydrous crystalline form of 6AzT, on the other hand, is stabilized by base stacking interactions. Both the water molecules in 6AzTH that are involved in sandwich formation have trigonal coordination around them. A reexamination of the crystal structure of 5-amino-2-thiocytosine (5A2TC) revealed that one of the water molecules in this structure also forms a water sandwich and has trigonal coordination whereas the other water molecule with tetrahedral coordination does not form a sandwich. The environment and the characteristics of the intercalated water molecule in these structures suggest a possible role for such water intercalations in the dynamics of DNA. Crystals of 6AzTH are monoclinic, space group P21/n, with unit cell parameters a = 8.861 (1), b = 13.177 (3), c = 20.662 (2) A, beta = 93.35 (1) degrees, and Z = 16. From diffractometer data (2503 reflections, greater than or equal to 3 sigma), the crystal structure was solved and refined to an R of 0.056.     

Structural and dynamic roles of permanent water molecules in ligand molecular recognition by chicken liver bile acid binding protein

Chicken liver bile acid binding protein (cL-BABP) crystallizes with water molecules in its binding site. To obtain insights on the role of internal water, we performed two 100 ns molecular dynamics (MD) simulations in explicit solvent for cL-BABP, as apo form and as a complex with two molecules of cholic acid, and analyzed in detail the dynamics properties of all water molecules. The diffusion coefficients of the more persistent internal water molecules are significantly different from the bulk, but similar between the two protein forms. A different number of molecules and a different organization are observed for apo- and holo-cL-BABP. Most water molecules identified in the binding site of the apo-crystal diffuse to the bulk during the simulation. In contrast, almost all the internal waters of the holo-crystal maintain the same interactions with internal sidechains and ligands, which suggests they have a relevant role in protein-ligand molecular recognition. Only in the presence of these water molecules we were able to reproduce, by a classical molecular docking approach, the structure of the complex cL-BABP::cholic acid with a low ligand root mean square deviation (RMSD) with respect to its reference positioning. Literature data reported a conserved pattern of hydrogen bonds between a single water molecule and three amino acid residues of the binding site in a series of crystallized FABP. In cL-BABP, the interactions between this conserved water molecule and the three residues are present in the crystal of both apo- and holo-cL-BABP but are lost immediately after the start of molecular dynamics. Copyright (c) 2008 John Wiley & Sons, Ltd.