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1.
Designed retroaldolases have utilized a nucleophilic lysine to promote carbon–carbon bond cleavage of β-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest kcat (> 10− 3 s− 1) and kcat/KM (11–25 M− 1 s− 1) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the > 105-fold rate accelerations that were achieved are within 1–3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (kcat/kuncat = 106 to 108) and an extensively evolved computational design (kcat/kuncat > 107). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches.  相似文献   
2.
Single-stranded DNA binding proteins (SSBs) selectively bind single-stranded DNA (ssDNA) and facilitate recruitment of additional proteins and enzymes to their sites of action on DNA. SSB can also locally diffuse on ssDNA, which allows it to quickly reposition itself while remaining bound to ssDNA. In this work, we used a hybrid instrument that combines single-molecule fluorescence and force spectroscopy to directly visualize the movement of Escherichia coli SSB on long polymeric ssDNA. Long ssDNA was synthesized without secondary structure that can hinder quantitative analysis of SSB movement. The apparent diffusion coefficient of E. coli SSB thus determined ranged from 70,000 to 170,000 nt2/s, which is at least 600 times higher than that determined from SSB diffusion on short ssDNA oligomers, and is within the range of values reported for protein diffusion on double-stranded DNA. Our work suggests that SSB can also migrate via a long-range intersegment transfer on long ssDNA. The force dependence of SSB movement on ssDNA further supports this interpretation.  相似文献   
3.
Kaposi sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies. KSHV latency-associated nuclear antigen (LANA) mediates persistence of viral episomes in latently infected cells. LANA mediates KSHV DNA replication and segregates episomes to progeny nuclei. The structure of the LANA DNA binding domain was recently solved, revealing a positive electrostatic patch opposite the DNA binding surface, which is the site of BET protein binding. Here we investigate the functional role of the positive patch in LANA-mediated episome persistence. As expected, LANA mutants with alanine or glutamate substitutions in the central, peripheral, or lateral portions of the positive patch maintained the ability to bind DNA by EMSA. However, all of the substitution mutants were deficient for LANA DNA replication and episome maintenance. Mutation of the peripheral region generated the largest deficiencies. Despite these deficiencies, all positive patch mutants concentrated to dots along mitotic chromosomes in cells containing episomes, similar to LANA. The central and peripheral mutants, but not the lateral mutants, were reduced for BET protein interaction as assessed by co-immunoprecipitation. However, defects in BET protein binding were independent of episome maintenance function. Overall, the reductions in episome maintenance closely correlated with DNA replication deficiencies, suggesting that the replication defects account for the reduced episome persistence. Therefore, the electrostatic patch exerts a key role in LANA-mediated DNA replication and episome persistence and may act through a host cell partner(s) other than a BET protein or by inducing specific structures or complexes.  相似文献   
4.
光皮木瓜果实发育期间主要成分含量的变化   总被引:1,自引:0,他引:1  
对光皮木瓜果实发育期间主要成分含量的变化进行了分析.结果显示:(1)光皮木瓜果实发育期间,可溶性固形物含量总体呈上升趋势,花后151 d达到最大值14.8%;干物质、粗纤维和多酚含量的变化均呈单峰型,分别于花后95、67和95 d达到峰值(26.49%、14.96%、81.843 1 mg/gDW),并分别于花后165、179和179 d降至最低值(18.3%、6.47%、42.014 6 mg/gDW);黄酮、多糖、齐墩果酸含量均呈逐渐增加趋势,在果实发育后期略有下降,其中多糖和齐墩果酸含量均于花后137 d达到最大值,分别为110.431 2 mg/gDW和10.312 1 mg/gDW,黄酮含量在花后151 d达到最大值3.201 1 mg/gDW.(2)多酚的单果产量在果实发育过程中的变化趋势与多酚含量一致,于花后95 d多酚单果产量达到最高值(5.448 6 g);齐墩果酸、多糖及黄酮的单果产量在果实发育期间逐渐增加,花后165 d多糖的单果产量达到最高值(8.508 1 g),花后179 d黄酮和齐墩果酸的单果产量均达到最高值(0.261 2 g和0.747 4 g).研究表明,花后137 d(8月25日)至151 d(9月8日)木瓜果实青绿,齐墩果酸、多糖、黄酮含量最高,为加工木瓜饮片等的最佳采收期;花后165 d(9月22日)至179 d(10月6日)时,木瓜果实基本转黄,其中齐墩果酸、多糖、黄酮的单果产量最高,且多酚和粗纤维含量最低,是药用有效成分提取和加工果酒、果脯、果醋等木瓜食品的最佳采收期.  相似文献   
5.
植物二酰甘油酰基转移酶基因(DGAT)研究进展   总被引:2,自引:0,他引:2  
三酰甘油(TAG)是油料作物最主要的储藏脂类,二酰甘油酰基转移酶(DGAT,EC2.3.1.20)是TAG合成途径的限速酶,其主要作用是催化二酰甘油加上酰基脂肪酸形成三酰甘油.在植物中已发现了3种不同类型的DGAT基因,分别为DGAT1、DGAT2和DGAT3.该文对近年来国内外有关植物DGAT相关基因及其蛋白分类、定位、结构及其在脂肪酸合成、种子发育与萌发、幼苗发育、叶片新陈代谢等过程中的作用等研究进展进行综述.为提高油料作物种子油含量以及特定脂肪酸积累提供理论参考.  相似文献   
6.
长期以来,螨类主要依靠其形态特征进行系统学研究。DNA标记是指能反映生物个体或物种间基因组中某种差异特征的DNA片段。近年来,DNA标记技术在螨类系统学研究中得到越来越广泛的应用。本文综述了随机扩增多态性RAPD、限制性内切酶片段长度多态性RFLP、微卫星SSR、核酸序列扩增、扩增片段长度多态性AFLP和直接扩增片段长度多态性DALP等6种DNA标记技术在螨类系统学研究中的应用现状及前景。  相似文献   
7.
8.
We have analyzed the cell wall of the radio-resistant bacterium Deinococcus radiodurans. Unexpectedly, the bacterial envelope appears to be organized in different complexes of high molecular weight. Each complex is composed of several proteins, most of which are coded by genes of unknown function and the majority are constituents of the inner/outer membrane system. One of the most abundant complexes is constituted by the gene DR_0774. This protein is a type of secretin which is a known subunit of the homo-oligomeric channel that represents the main bulk of the type IV piliation family. Finally, a minor component of the pink envelope consists of several inner-membrane proteins. The implications of these findings are discussed.  相似文献   
9.
Novel synthetic mimics of antimicrobial peptides have been developed to exhibit structural properties and antimicrobial activity similar to those of natural antimicrobial peptides (AMPs) of the innate immune system. These molecules have a number of potential advantages over conventional antibiotics, including reduced bacterial resistance, cost-effective preparation, and customizable designs. In this study, we investigate a family of nylon-3 polymer-based antimicrobials. By combining vesicle dye leakage, bacterial permeation, and bactericidal assays with small-angle X-ray scattering (SAXS), we find that these polymers are capable of two interdependent mechanisms of action: permeation of bacterial membranes and binding to intracellular targets such as DNA, with the latter necessarily dependent on the former. We systemically examine polymer-induced membrane deformation modes across a range of lipid compositions that mimic both bacteria and mammalian cell membranes. The results show that the polymers' ability to generate negative Gaussian curvature (NGC), a topological requirement for membrane permeation and cellular entry, in model Escherichia coli membranes correlates with their ability to permeate membranes without complete membrane disruption and kill E. coli cells. Our findings suggest that these polymers operate with a concentration-dependent mechanism of action: at low concentrations permeation and DNA binding occur without membrane disruption, while at high concentrations complete disruption of the membrane occurs. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.  相似文献   
10.
Here we describe a modified version of the digestion–ligation approach for efficient molecular cloning. In comparison with the original method, the modified method has the additional steps of gel purification and a second ligation after the first ligation of the linearized vector and DNA insert. During this process, the efficiency and reproducibility could be significantly improved for both stick-end cloning and blunt-end cloning. As an improvement of the very important molecular cloning technique, this method may find a wide range of applications in bioscience and biotechnology.  相似文献   
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