Use of haloacetate dehalogenase genes as selection markers forEscherichia coli andPseudomonas vectors

The haloacetate dehalogenase gene,dehH2, cloned fromMoraxella sp. strain B could be used as a selection marker gene for vectors inEscherichia coli andPseudomonas putida. Haloacetates, especially iodoacetate, inhibit the growth of some microorganisms. ThedehH2 gene introduced into the cells conferred iodoacetate resistance on them. Therefore,E. coli andP. putida transformed with vectors marked withdehH2 could be easily selected on plates containing iodoacetate.     

α-氯丙酸脱卤酶发酵动力学模型

对α-氯丙酸脱卤酶发酵动力学进行了研究。基于Logistic方程和Luedeking-Piret方程,得到了描述Pseudomonas W20菌发酵过程菌体生长、α-氯丙酸脱卤酶生成及基质消耗的动力学数学模型和模型参数,对试验数据与模型进行了验证比较,模型计算值与试验结果拟合良好,平均相对误差大部分小于10%;对脱卤酶反应动力学进行了研究,结果表明脱卤酶的脱卤反应基本符合米氏方程,并求得最大反应速率V_(max)=1.11×10~(-5)mol/(g·min),表观米氏常数K_m=3.72×10~(-3)mol/L。     

Purification, characterization, and gene cloning of a novel fluoroacetate dehalogenase from Burkholderia sp. FA1

Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of the highly stable carbon–fluorine bond in an aliphatic compound. The bacterial isolate FA1, which was identified as Burkholderia, grew on fluoroacetate as the sole carbon source to produce fluoroacetate dehalogenase (FAc-DEX FA1). The enzyme was purified to homogeneity and characterized. The molecular weights were estimated to be 79,000 and 34,000 by gel filtration and SDS-polyacrylamide gel electrophoresis (PAGE), respectively, suggesting that the enzyme is a dimer. The purified enzyme was specific to haloacetates, and fluoroacetate was the best substrate. The activities toward chloroacetate and bromoacetate were less than 5% of the activity toward fluoroacetate. The K_m and V_max values for the hydrolysis of fluoroacetate were 5.1 mM and 11 μmol per minute milligram, respectively. The gene coding for the enzyme was isolated, and the nucleotide sequence was determined. The open reading frame consisted of 912 nucleotides, corresponding to 304 amino acid residues. Although FAc-DEX FA1 showed high sequence similarity to fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX H1) (61% identity), the substrate specificity of FAc-DEX FA1 was significantly different from that of FAc-DEX H1: FAc-DEX FA1 was more specific to fluoroacetate than FAc-DEX H1.     

Immobilisation of the D-2-haloacid dehalogenase fromPseudomonas putida strain AJ1/23

A variety of procedures were used to immobilise D-2-haloacid dehalogenase. Natural polymer supports were insufficiently robust to withstand degradation by high concentrations of 2-chloropropionate. The best results were obtained with enzyme covalently attached to controlled-pore glass via a diazo linkage. The immobilisation procedure was optimised with respect to enzyme loading, pH, temperature and the presence of substrate during attachment. Immobilisation significantly modified the kinetics of the enzyme, in particular improving its temperature stability and ability to withstand mildly alkaline conditions where it is most active. The performance of the immobilised preparation in batch and plug-flow bioreactors was assessed. Biocatalyst half-life in plug-flow reactors was better than in batch bioreactors whereas effectiveness factors, although concentration dependent in the batch reactor, were similar at least with 200 mM D,L-2-CPA as substrate.     

Molecular Mechanism of Photoperiodic Time Measurement in the Brain of Japanese Quail

In most organisms living in temperate zones, reproduction is under photoperiodic control. Although photoperiodic time measurement has been studied in organisms ranging from plants to vertebrates, the underlying molecular mechanism is not well understood. The Japanese quail (Coturnix japonica) represents an excellent model to study this problem because of the rapid and dramatic photoperiodic response of its hypothalamic‐pituitary‐gonadal axis. Recent investigations of Japanese quail show that long‐day‐induced type 2 deiodinase (Dio2) expression in the mediobasal hypothalamus (MBH) plays an important role in the photoperiodic gonadal regulation by catalyzing the conversion of the prohormone thyroxine (T₄) to bioactive 3,5,3′‐triiodothyronine (T₃). The T₃ content in the MBH is approximately 10‐fold higher under long than short days and conditions, and the intracerebroventricular infusion of T₃ under short days and conditions mimics the photoperiodic gonadal response. While Dio2 generates active T₃ from T₄ by outer ring deiodination, type 3 deiodinase (Dio3) catalyzes the conversion of both T₃ and T₄ into inactive forms by inner ring deiodination. In contrast to Dio2 expression, Dio3 expression in the MBH is suppressed under the long‐day condition. Photoperiodic changes in the expression of both genes during the photoinduction process occur before the changes in the level of luteinizing hormone (LH) secretion, suggesting that the reciprocal changes in Dio2 and Dio3 expression act as gene switches of the photoperiodic molecular cascade to trigger induction of LH secretion.     

Molecular Characterization of Monochloroacetate-Degrading Arthrobacter sp. Strain D2 Isolated from Universiti Teknologi Malaysia Agricultural Area

Arthrobacter sp. strains D2 and D3 and Labrys sp. strain D1 capable of degrading 20 mM monochloroacetic acid (MCA) were isolated from soil contaminated with herbicides and pesticides. All three isolates were able to grow on MCA as the sole source of carbon and energy with concomitant chloride ion release in the growth medium (19 mM). Strains D2 and D3 (cells doubling time 7 ± 0.3 h) grew four times faster than D1 (26 ± 0.1 h). Strain D2 was then further investigated and could also grow in 10 mM of monobromoacetic acid (MBA), 2,2-dichloropropionic acid (2,2DCP), d,l-2-chloropropionic acid (D,L2CP), l-2-chloropropionic acid (L-2CP), d-2-chloropropionic acid (D-2CP), and glycolate as the sole sources of carbon and energy. Dehalogenase gene amplification using group I primers revealed a 410-bp polymerase chain reaction (PCR) product, but there was none using group II primers. The partial amino acid sequence analysis of group I DehD2 dehalogenase showed at least 32% identity to the corresponding regions of DehE, DhlIV, DehI, and D,L-DEX, with key amino acid residues Ser188, Ala187, and Asp189. These amino acid residues were involved in substrate binding and catalysis and were conserved in the partial amino acid sequence.     

还原性脱卤酶的生物信息学分析

【背景】有机卤呼吸细菌介导的还原性脱卤过程对于卤素循环与有机卤污染场地的修复至关重要。还原性脱卤酶(reductive dehalogenases, RDases)作为有机卤呼吸的末端电子受体还原酶,在脱卤过程中起着关键作用。【目的】系统地了解还原性脱卤酶的序列与结构特征,为阐明还原性脱卤酶及有机卤呼吸细菌作用机制及生物学功能奠定基础。【方法】应用多种生物信息学分析工具对44个还原性脱卤酶蛋白的基本理化性质、跨膜结构、信号肽、磷酸化位点、系统发育关系、多序列比对的同源性矩阵、保守基序、保守结构域、二级结构、三级结构及无序区域进行预测分析。【结果】不同还原性脱卤酶蛋白的理化性质存在差异,但呈现出一定的保守性。不同还原性脱卤酶蛋白的磷酸化位点、保守结构域分布、保守基序分布,以及二级结构的保守性较高。底物类别相似或相同的还原性脱卤酶在蛋白序列和三级结构上更为相似。大多数还原性脱卤酶蛋白的亲缘关系较近。还原性脱卤酶蛋白主要为分泌型蛋白、非膜定位蛋白和固有无序蛋白。【结论】不同还原性脱卤酶蛋白具有一定的保守性,因此能够发挥相似的生物学功能。研究结果对进一步研究还原性脱卤酶具有一定的参考价值,尤其是为基于还原性脱卤酶的有机卤呼吸细菌应用于实际场地修复提供科学依据与理论参考。     

Molecular Modelling of Human DT-Diaphorase For Enzyme-Directed Bioreductive Drug Design

Abstract

The enzyme DT-Diaphorase (NAD(P)H:quinone acceptor oxidoreductase, EC 1.6.99.2.; DTD) has been recognised as a good target for enzyme-directed bioreductive drug development. This is due to elevated levels of enzyme activity in several human tumour types and its role in the bioreductive activation of several quinone-based anti-cancer drugs.

Bioreductive drugs are designed to exploit one of the features of solid tumours, namely tumour hypoxia. However, selectivity of bioreductive drugs is not only governed by oxygen levels, but also by the levels of the enzymes catalysing bioreductive activation, leading to the concept of “enzyme-directed bioreductive drug development” introduced by Workman and Walton in 1990. This concept requires the identification of tumours within a patient that have elevated levels of enzyme activity (enzyme profiling) and treating the patient with drugs activated by such enzymes. DTD has been singled out as a particularly good candidate for such targeting. In order to rationalise the design of drugs to target DTD, molecular modelling techniques have been employed.

The human DTD three-dimensional structure has been modelled with homology to the known rat DTD structure (about 85% identity) and the model refined using energy minimisation. Drug-binding orientations have been determined and molecular dynamics simulations performed. Using data from a series of quinone based compounds with a broad range of substrate specificity we examine drug-enzyme interactions and suggest how DTD substrate specificity might be further optimised.     

Effect of chronic ethanol administration on the rat pineal N-acetyltransferase and thyroxine type II 5′-deiodinase activities

Chronic ethanol intake resulted in a significant decrease in the rate of rat ponderal growth and an impaired nyctohemeral profile of pineal N-acetyltransferase (NAT) activity. In ethanol-treated animals, the onset of the nocturnal NAT increase is delayed by 2 hours when compared to control animals. Moreover, pineal NAT nocturnal peak was reached at 4 h (2 hours later than controls), while pineal type II thyroxine 5-deiodinase (5-D) nyctohemeral profile was not modified by ethanol administration. The effect of ethanol administration (12 weeks) on 5-D activity in different tissues was also studied. Ethanol induced a 5-D activity increase in hypothesis and brain frontal cortex, when compared to control animals. No change in 5-D activity is observed in either pineal gland, Harderian gland, or brown adipose tissue. Since basal values of 5-D activity in hypophysis or brain frontal cortex are particularly dependent on serum thyroxine (T₄) concentration, the effect of chronic ethanol administration on thyroid hormone levels was studied. Serum T₄ levels in ethanol-treated animals were significantly decreased when compared to controls at any time point studied. However, no change in serum 3,3,5-triiodothyronine (T₃) levels were found.     

The Desulfitobacterium genus

Desulfitobacterium spp. are strictly anaerobic bacteria that were first isolated from environments contaminated by halogenated organic compounds. They are very versatile microorganisms that can use a wide variety of electron acceptors, such as nitrate, sulfite, metals, humic acids, and man-made or naturally occurring halogenated organic compounds. Most of the Desulfitobacterium strains can dehalogenate halogenated organic compounds by mechanisms of reductive dehalogenation, although the substrate spectrum of halogenated organic compounds varies substantially from one strain to another, even with strains belonging to the same species. A number of reductive dehalogenases and their corresponding gene loci have been isolated from these strains. Some of these loci are flanked by transposition sequences, suggesting that they can be transmitted by horizontal transfer via a catabolic transposon. Desulfitobacterium spp. can use H2 as electron donor below the threshold concentration that would allow sulfate reduction and methanogenesis. Furthermore, there is some evidence that syntrophic relationships occur between Desulfitobacterium spp. and sulfate-reducing bacteria, from which the Desulfitobacterium cells acquire their electrons by interspecies hydrogen transfer, and it is believed that this relationship also occurs in a methanogenic consortium. Because of their versatility, desulfitobacteria can be excellent candidates for the development of anaerobic bioremediation processes. The release of the complete genome of Desulfitobacterium hafniense strain Y51 and information from the partial genome sequence of D. hafniense strain DCB-2 will certainly help in predicting how desulfitobacteria interact with their environments and other microorganisms, and the mechanisms of actions related to reductive dehalogenation.