Burkholderia xenovorans LB400联苯双加氧酶及突变体对DDTs的降解

DDTs（dichlorodiphenyltrichloroethane,1,1,1-三氯-2,2-双氯苯基乙烷）是一种典型的持久性有机污染物,曾在疟疾防治和农业除虫方面被广泛应用。虽然包括我国在内的很多国家已经禁止使用DDTs,但目前对环境中DDTs的检测发现它仍然广泛存在且具有新的输入源。DDTs的持续存在对近海生态系统和人类健康具有一定危害,因此它所造成的环境污染问题仍然值得关注。由于Rieske型芳香羟化双加氧酶能够起始多种持久性污染物的降解,过去的几十年里一直是芳香化合物降解领域的焦点。[目的] 为探讨联苯双加氧酶对DDTs的降解特性及机制,本研究选取了食异生素伯克霍尔德氏菌LB400（Burkholderia xenovorans）联苯双加氧酶及突变体对p,p''-DDT和o,p''-DDT的降解过程进行研究。[方法] 以BphAE_LB400为亲本,通过两步定点突变将283位的丝氨酸突变为蛋氨酸,获得突变体BphAE_S283M。通过比较亲本酶与突变体对DDTs的催化性能,模拟突变蛋白结构和分子对接等方法,探究其降解特性及机制。[结果] BphAE_LB400和突变体BphAE_S283M都无法降解对位的p,p''-DDT,但突变体BphAE_S283M可以代谢o,p''-DDT并产生2个立体异构体。对接p,p''-DDT的BphAE_LB400和BphAE_S283M的结构分析表明,BphAE_LB400和BphAE_S283M中p,p''-DDT的反应环均不与原晶体结构中的联苯反应环重合。而对接o,p''-DDT的BphAE_S283M的结构分析表明o,p''-DDT的反应环与晶体结构中的联苯反应环距离很近,且2、3位的碳原子与单核铁原子催化中心的距离在0.5 nm以内,此外,BphAE_S283M的催化腔表面积和体积比BphAE_LB400更大,这很可能有助于BphAE_S283M与o,p''-DDT的结合。[结论] 283位氨基酸是影响BphAE_LB400对DDTs的催化代谢能力的关键氨基酸残基,它可以通过调节反应碳原子与催化中心的距离以及催化腔的大小来影响底物特异性。本次研究进一步阐明了283位氨基酸残基的影响机理,为更有效修复DDTs污染提供理论依据和技术支持。     

Subtractive phage display technology identifies zebrafish marcksb that is required for gastrulation

In the present study, we used a phage display technique to screen differentially expressed proteins from zebrafish post-gastrula embryos. With a subtractive screening approach, 6 types of single-chain Fv fragments (scFvs) were screened out from an scFv antibody phage display library by biopanning against zebrafish embryonic homogenate. Four scFv fragments (scFv1, scFv3, scFv4 and scFv6) showed significantly stronger binding to the tailbud embryos than to the 30%-epiboly embryos. A T7 phage display cDNA library was constructed from zebrafish tailbud embryos and used to identify the antigens potentially recognized by scFv1, which showed the highest frequency and strongest binding against the tailbud embryos. We acquired 4 candidate epitopes using scFv1 and the corresponding genes showed significantly higher expression levels at tailbud stage than at 30%-epiboly. The most potent epitope of scFv1 was the clone scFv1-2, which showed strong homology to zebrafish myristoylated alanine-rich C-kinase substrate b (Marcksb). Western blot analysis confirmed the high expression of marcksb in the post-gastrula embryos, and the endogenous expression of Marcksb was interfered by injection of scFv1. Zebrafish marcksb showed dynamic expression patterns during embryonic development. Knockdown of marcksb strongly affected gastrulation movements. Moreover, we revealed that zebrafish marcksb is required for cell membrane protrusion and F-actin alignment. Thus, our study uncovered 4 types of scFvs binding to zebrafish post-gastrula embryos, and the epitope of scFv1 was found to be required for normal gastrulation of zebrafish. To our knowledge, this was the first attempt to combine phage display technique with the embryonic and developmental study of vertebrates, and we were able to identify zebrafish marcksb that was required for gastrulation.     

π–π Stacking mediated drug–drug interactions in human CYP2E1

Because of having many low molecular mass substrates, CYP2E1 is of particular interests to the pharmaceutical industry. Many evidences showed that this enzyme can adopt multiple substrates to significantly reduce the oxidation rate of the substrates. The detailed mechanism for this observation is still unclear. In the current study, we employed GPU‐accelerated molecular dynamics simulations to study the multiple‐binding mode of human CYP2E1, with an aim of offering a mechanistic explanation for the unexplained multiple‐substrate binding. Our results showed that Thr303 and Phe478 were key factors for the substrate recognition and multiple‐substrate binding. The former can form a significant hydrogen bond to recognize and position the substrate in the productive binding orientation in the active site. The latter acted as a mediator for the substrate communications via π–π stacking interactions. In the multiple‐binding mode, the aforementioned π–π stacking interactions formed by the aromatic rings of both substrates and Phe478 drove the first substrate far away from the catalytic center, orienting in an additional binding position and going against the substrate metabolism. All these findings could give atomic insights into the detailed mechanism for the multiple‐substrate binding in human CYP2E1, providing useful information for the drug metabolism mechanism and personalized use of clinical drugs. Proteins 2013; © 2012 Wiley Periodicals, Inc.     

LabCaS: Labeling calpain substrate cleavage sites from amino acid sequence using conditional random fields

The calpain family of Ca²⁺‐dependent cysteine proteases plays a vital role in many important biological processes which is closely related with a variety of pathological states. Activated calpains selectively cleave relevant substrates at specific cleavage sites, yielding multiple fragments that can have different functions from the intact substrate protein. Until now, our knowledge about the calpain functions and their substrate cleavage mechanisms are limited because the experimental determination and validation on calpain binding are usually laborious and expensive. In this work, we aim to develop a new computational approach (LabCaS) for accurate prediction of the calpain substrate cleavage sites from amino acid sequences. To overcome the imbalance of negative and positive samples in the machine‐learning training which have been suffered by most of the former approaches when splitting sequences into short peptides, we designed a conditional random field algorithm that can label the potential cleavage sites directly from the entire sequences. By integrating the multiple amino acid features and those derived from sequences, LabCaS achieves an accurate recognition of the cleave sites for most calpain proteins. In a jackknife test on a set of 129 benchmark proteins, LabCaS generates an AUC score 0.862. The LabCaS program is freely available at: http://www.csbio.sjtu.edu.cn/bioinf/LabCaS . Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Characterisation of a thermo-alkali-stable lipase from oil-contaminated soil using a metagenomic approach

Lipases are widely used for a variety of biotechnological applications. Screening these industrial enzymes directly from environmental microorganisms is a more efficient and practical approach than conventional cultivation-dependent methods. Combined with activity-based functional screening, six clones with lipase activity were detected and a gene (termed lipZ01) isolated from a target clone with the highest lipase activity was cloned from an oil-contaminated soil-derived metagenomic library and then sequenced. Gene lipZ01 was expressed in Pichia pastoris GS115 and the molecular weight of the recombinant lipase LipZ01 was estimated by electrophoresis analysis to be approximately 50 kDa. The maximum activity of the purified lipase was 42 U/mL, and the optimum reaction temperature and pH value were 45 °C and 8.0, respectively. The enzyme was highly stable in the temperature range 35–60 °C and under alkaline conditions (pH 7–10). The presence of Ca²⁺ and Mn²⁺ ions could significantly enhance the activity of the lipase. The purified lipase preferentially hydrolysed triacylglycerols with acyl chain lengths ≥8 carbon atoms, and the conversion degree of biodiesel production was nearly 92% in a transesterification reaction using olive oil and methanol. Some attractive properties suggested that the recombinant lipase may be valuable in industrial applications.     

3D-QSAR with CoMFA Model of Prolylendopeptidase Substrates

Abstract

The prolylendopeptidase (PEP) is the proteolytic enzyme, which plays an essential role in the regulation of some processes in central nervous system, such as memory, learning and behavior. It was shown that PEP activity changes at different diseases, like Parkinsons or Alzheimer's diseases, and some PEP inhibitors are used in therapy. At present time the discovery of new types of PEP inhibitors are the actual task.

In this study the structure of PEP active site was analyzed by 3D-QSAR with CoMFA methods using of 12 PEP substrates. The designed pharmacophore model assumes that substrates interact with PEP active site by pyrrolidol ring of proline residue and by hydrogen bonding.

The 3-D-QSAR + CoMFA model of PEP substrates propose that the hydrophobic bonds play the essential role in substrate interaction with enzyme. This model reveals the important steric and electrostatic areas around the molecules and the presence of substituents controls the PEP activity for substrates. Analysis of obtained data allows to assume, that substrate binding in PEP active site causes essential perturbations of substrate structure. This effect mainly depends on chemical nature of the amino acid side chain, located near to proline.     

A sensitive fluorigenic substrate for selective in vitro and in vivo assay of leukotriene A4 hydrolase activity

Leukotriene A4 hydrolase (LTA4H) is a bifunctional zinc-dependent metalloprotease bearing both an epoxide hydrolase, producing the pro-inflammatory LTB4 leukotriene, and an aminopeptidase activity, whose physiological relevance has long been ignored. Distinct substrates are commonly used for each activity, although none is completely satisfactory; LTA4, substrate for the hydrolase activity, is unstable and inactivates the enzyme, whereas aminoacids β-naphthylamide and para-nitroanilide, used as aminopeptidase substrates, are poor and nonselective. Based on the three-dimensional structure of LTA4H, we describe a new, specific, and high-affinity fluorigenic substrate, PL553 [l-(4-benzoyl)phenylalanyl-β-naphthylamide], with both in vitro and in vivo applications. PL553 possesses a catalytic efficiency (k_cat/K_m) of 3.8 ± 0.5 × 10⁴ M⁻¹ s⁻¹ using human recombinant LTA4H and a limit of detection and quantification of less than 1 to 2 ng. The PL553 assay was validated by measuring the inhibitory potency of known LTA4H inhibitors and used to characterize new specific amino-phosphinic inhibitors. The LTA4H inhibition measured with PL553 in mouse tissues, after intravenous administration of inhibitors, was also correlated with a reduction in LTB4 levels. This authenticates the assay as the first allowing the easy measurement of endogenous LTA4H activity and in vitro specific screening of new LTA4H inhibitors.