One site fits both: A model for the ternary complex of folate + NADPH in R67 dihydrofolate reductase, a D2 symmetric enzyme

R67 dihydrofolate reductase (DHFR) is a novel enzyme that confers resistance to the antibiotic trimethoprim. The crystal structure of R67 DHFR displays a toroidal structure with a central active-site pore. This homotetrameric protein exhibits 222 symmetry, with only a few residues from each chain contributing to the active site, so related sites must be used to bind both substrate (dihydrofolate) and cofactor (NADPH) in the productive R67 DHFR?NADPH?dihydrofolate complex. Whereas the site of folate binding has been partially resolved crystallographically, an interesting question remains: how can the highly symmetrical active site also bind and orient NADPH for catalysis? To model this ternary complex, we employed DOCK and SLIDE, two methods for docking flexible ligands into proteins using quite different algorithms. The bound pteridine ring of folate (Fol I) from the crystal structure of R67 DHFR was used as the basis for docking the nicotinamide-ribose-P_i (NMN) moiety of NADPH. NMN was positioned by both DOCK and SLIDE on the opposite side of the pore from Fol I, where it interacts with Fol I at the pore's center. Numerous residues serve dual roles in binding. For example, Gln 67 from both the B and D subunits has several contacts with the pteridine ring, while the same residue from the A and C subunits has several contacts with the nicotinamide ring. The residues involved in dual roles are generally amphipathic, allowing them to make both hydrophobic and hydrophilic contacts with the ligands. The result is a `hot spot' binding surface allowing the same residues to co-optimize the binding of two ligands, and orient them for catalysis.     

基于细菌脱氢酶活性法的水质综合毒性快速测定仪

在无氧条件下,生物染料亚甲基蓝可以作为生物新陈代谢过程中体内脱氢酶催化反应的电子受体,亚甲基蓝与细菌悬液共存时,其退色速度标志着细菌脱氢酶活性的大小。脱氢酶的活性大小在一定程度上能够反映出微生物新代谢的能力,微生物所处的水环境中毒物的存在会引起微生物脱氢酶活性变化,毒物的毒性与脱氢酶的活性相关。本文以动力学比色法测定亚甲基蓝的退色速度为基础设计并制造了一台水质综合毒性相关。本文以动力学比色法测定亚     

Norcholanic acids as substrates for recombinant 3β-hydroxysteroid dehydrogenase and progesterone 5β-reductase, enzymes of the 5β-cardenolide biosynthesis

The conversion of 23-nor-5,20(22)E-choladienic acid-3β-ol and other intermediates of the putative norcholanic acid pathway of cardenolide biosynthesis by recombinant 3β-hydroxysteroid dehydrogenase from Digitalis lanata in dehydrogenation and reduction reactions was investigated. 23-nor-4,20(22)E-choladienic acid-3-one was found to be a substrate of recombinant progesterone 5β-reductases from D. lanata and Arabidopsis thaliana. The role of various substrates in cardenolide biosynthesis is discussed.     

Assay of Serum Glutamic-Oxaloacetic Transaminase Using Malate Dehydrogenase Preparation Obtained from Streptomyces aureofaciens

Abstract

A modified spectrophotometric method for serum glutamic-oxaloacetic transaminase (SGOT) assay was developed. A crude cell-free extract from Streptomyces aureofaciens which showed a high level of malate dehydrogenase (MDH) activity (E.C. 1.1.1.37) was used as the enzymatic indicator. The lyophilized microbial preparation was used without previous purification and was quite stable under refrigeration for one year. Serum sample assays using both the method utilizing the crude cell extract and an enzymatic commercial kit showed good correlation.     

Effects of Micronutrient Niacin on Treatment Efficiency of Textile Wastewater

0 IntroductionMioclroogincuatlri pernotsce pslsaeys afo vrer wyais tmepwoarttaernt t rroelaetmine nbti- .A widerange of micronutrientsis required byactiva-ted sludge[1-5]. Adequate trace nutrient concentra-tions ,including metal ions and vitamins , are re-quiredtosupport all the genera presentinthe activa-ted sludgetreatment systemfor a diverse communityto survive. The lack of specific nutrients may resultin an unbalanced mixed culture,leading to poor-quality effluent , reduced treatment effic…     

Effects of He－Ne laser irradiation on enzyme activity in milking Goats＇ seminal plasma

ＥｆｆｅｃｔｓｏｆＨｅ－ＮｅｌａｓｅｒｉｒｒａｄｉａｔｉｏｎｏｎｅｎｚｙｍｅａｃｔｉｖｉｔｙｉｎｍｉｌｋｉｎｇＧｏａｔｓ＇ｓｅｍｉｎａｌｐｌａｓｍａＹＵＥＷｅｎｂｉｎ；ＺＨＥＮＧＸｉａｏｚｈｏｎｇ；ＣＵＩＢａｏｗｅｉ；ＬＩＫｅｌｕ（ＳｈａｎｘｉＡｇｒ...     

谷氨酸氧化酶粗品的制备,性质及应用

谷氨酸氧化酶发酵液经（ＨＮ４）２ＳＯ４沉淀后用透析法获得酶粗品。利用ＧＯ测定谷氨酸发酵液中谷氨酸的含量与传统的瓦氏法结果一致，相关系数为０．９８６，相关方程为：ｙ－０．９２８ｘ＋０．１２７（其中ｙ，ｘ分别表示氧化酶法与瓦氏法测定的值）。且这种方法比瓦氏法方便、简单、分析成本低、耗时少。利用ＧＯ法测定血清中谷草转氨酶和谷丙转氨酶的含量，与传统的试剂盒法比较，相关系数分别为０．９７８、０．９８４，相关     

Polymorphisms in genes involved in folate metabolism as maternal risk factors for Down syndrome in China

Objective： To explore the relationship between genetic polymorphisms in methylenetetrahydrofolate reductase （MTHFR）, methionine synthase reductase （MTRR）, the central enzymes in folate metabolism that affects DNA methylation and synthesis, and the risk of Down syndrome in China. Methods： Genomic DNA was isolated from the peripheral lymphocytes of 64 mothers of children with Down syndrome and 70 age matched control subjects. Polymerase chain reaction and restriction fragment length polymorphism were used to examine the polymorphisms of MTHFR 677C→T, MTRR 66A→G and the relationship between these genotypes and the risk of Down syndrome was analyzed. Results： The results show that the MTHFR 677C→T polymorphism is more prevalent among mothers of children with Down syndrome than among control mothers, with an odds ratio of 3.78 （95% confidence interval （CI）, 1.78-8.47）. In addition, the homozygous MTRR 66A→G polymorphism was independently associated with a 5.2-fold increase in estimated risk （95% CI, 1.90-14.22）. The combined presence of both polymorphisms was associated with a greater risk of Down syndrome than the presence of either alone, with an odds ratio of 6.0 （95% CI, 2.058-17.496）. The two polymorphisms appear to act without a multiplicative interaction. Conclusion： MTHFR and MTRR gene mutation alleles are related to Down syndrome, and CT, TT and GG gene mutation types increase the risk of Down syndrome.     

Amperometric l-glutamate biosensor based on bacterial cell-surface displayed glutamate dehydrogenase

A novel l-glutamate biosensor was fabricated using bacteria surface-displayed glutamate dehydrogenase (Gldh-bacteria). Here the cofactor NADP⁺-specific dependent Gldh was expressed on the surface of Escherichia coli using N-terminal region of ice nucleation protein (INP) as the anchoring motif. The cell fractionation assay and SDS-PAGE analysis indicated that the majority of INP-Gldh fusion proteins were located on the surface of cells. The biosensor was fabricated by successively casting polyethyleneimine (PEI)-dispersed multi-walled carbon nanotubes (MWNTs), Gldh-bacteria and Nafion onto the glassy carbon electrode (Nafion/Gldh-bacteria/PEI-MWNTs/GCE). The MWNTs could not only significantly lower the oxidation overpotential towards NAPDH, which was the product of NADP⁺ involving in the oxidation of glutamate by Gldh, but also enhanced the current response. Under the optimized experimental conditions, the current–time curve of the Nafion/Gldh-bacteria/PEI-MWNTs/GCE was performed at +0.52 V (vs. SCE) by amperometry varying glutamate concentration. The current response was linear with glutamate concentration in two ranges (10 μM–1 mM and 2–10 mM). The low limit of detection was estimated to be 2 μM glutamate (S/N = 3). Moreover, the proposed biosensor is stable, specific, reproducible and simple, which can be applied to real samples detection.