Mechanism and applications of phosphite dehydrogenase

Phosphite dehydrogenase catalyzes the NAD+-dependent oxidation of hydrogen phosphonate (common name phosphite) to phosphate in what amounts to a formal phosphoryl transfer reaction from hydride to hydroxide. This review places the enzyme in the context of phosphorus redox metabolism in nature and discusses the results of mechanistic investigations into its reaction mechanism. The potential of the enzyme as a NAD(P)H cofactor regeneration system is discussed as well as efforts to engineer the cofactor specificity of the protein.     

Production and characterization of recombinant perdeuterated cholesterol oxidase

Cholesterol oxidase (CO) is a FAD (flavin adenine dinucleotide) containing enzyme that catalyzes the oxidization and isomerization of cholesterol. Studies directed toward elucidating the catalytic mechanism of CO will provide an important general understanding of Flavin-assisted redox catalysis. Hydrogen atoms play an important role in enzyme catalysis; however, they are not readily visualized in protein X-ray diffraction structures. Neutron crystallography is an ideal method for directly visualizing hydrogen positions at moderate resolutions because hydrogen and deuterium have comparable neutron scattering lengths to other heavy atoms present in proteins. The negative coherent and large incoherent scattering lengths of hydrogen atoms in neutron diffraction experiments can be circumvented by replacing hydrogen atoms with its isotope, deuterium. The perdeuterated form of CO was successfully expressed from minimal medium, purified, and crystallized. X-ray crystallographic structures of the enzyme in the perdeuterated and hydrogenated states confirm that there are no apparent structural differences between the two enzyme forms. Kinetic assays demonstrate that perdeuterated and hydrogenated enzymes are functionally identical. Together, structural and functional studies indicate that the perdeuterated protein is suitable for structural studies by neutron crystallography directed at understanding the role of hydrogen atoms in enzyme catalysis.     

Membrane cholesterol uptake by recombinant lipoproteins

Recombinant lipoproteins, prepared with apo A-I isolated from human high density lipoprotein (HDL) and various phospholipids (PLs), were compared with respect to their ability to remove cholesterol (Chol) from labelled erythrocyte ghost membranes. It was found that uptake of Chol was essentially complete following an 8 h incubation at 37 degrees C. Quantitation of the amount of cholesterol taken up showed that recombinants prepared from bovine brain sphingomyelin (BBSM) or dipalmitoyl phosphatidylcholine (DPPC) acquired the highest proportion of Chol (80-140 mol/mol protein), whereas shorter chain phospholipids like dimyristoyl phosphatidylcholine (DMPC) acquired little or no membrane Chol. Chemical analysis of the incubation products indicated that this latter result was due to loss of PL, presumably to the membrane, with consequent disruption of the recombinant particle. Results with DPPC:A-I recombinants of differing PL/protein ratios and sizes showed that Chol uptake was fairly constant at 0.70 mol Chol/mol PL. It is concluded that discoidal, phospholipid-rich recombinant lipoproteins can effectively take up substantial amounts of Chol from physiological membranes, provided that the PLs utilized form micellar complexes which are capable of retaining their structural integrity during the incubation with the membranes.     

Characteristics and biotechnological applications of microbial cholesterol oxidases

Microbial cholesterol oxidase is an enzyme of great commercial value, widely employed by laboratories routinely devoted to the determination of cholesterol concentrations in serum, other clinical samples, and food. In addition, the enzyme has potential applications as a biocatalyst which can be used as an insecticide and for the bioconversion of a number of sterols and non-steroidal alcohols. The enzyme has several biological roles, which are implicated in the cholesterol metabolism, the bacterial pathogenesis, and the biosynthesis of macrolide antifungal antibiotics. Cholesterol oxidase has been reported from a variety of microorganisms, mostly from actinomycetes. We recently reported cholesterol oxidases from gram-negative bacteria such as Burkholderia and Chromobacterium. These enzymes possess thermal, detergent, and organic solvent tolerance. There are two forms of cholesterol oxidase, one containing a flavin adenine dinucleotide cofactor non-covalently bound to the enzyme (class I) and the other containing the cofactor covalently linked to the enzyme (class II). These two enzymes have no significant sequence homology. The phylogenetic tree analyses show that both class I and class II enzymes can be further divided into at least two groups.     

Catabolism and biotechnological applications of cholesterol degrading bacteria

Cholesterol is a steroid commonly found in nature with a great relevance in biology, medicine and chemistry, playing an essential role as a structural component of animal cell membranes. The ubiquity of cholesterol in the environment has made it a reference biomarker for environmental pollution analysis and a common carbon source for different microorganisms, some of them being important pathogens such as Mycobacterium tuberculosis. This work revises the accumulated biochemical and genetic knowledge on the bacterial pathways that degrade or transform this molecule, given that the characterization of cholesterol metabolism would contribute not only to understand its role in tuberculosis but also to develop new biotechnological processes that use this and other related molecules as starting or target materials.     

Purification and crystallization of recombinant Escherichia coli malate dehydrogenase

Malate dehydrogenase from Escherichia coli has been crystallized with polyethylene glycol and citrate buffer at pH 5.7. The enzyme was obtained from an E. coli strain in which the chromosomal malate dehydrogenase gene was contained on a pBR322 vector. Two types of crystals have been observed; a monoclinic C2 form and an orthorhombic C222(1) form, which is found infrequently. Monoclinic crystals were used as seeds in several rounds of crystallization until large crystals suitable for diffraction analysis were available. A complete X-ray data set to 2.0 A has been collected.     

细菌纤维素性质及应用的研究进展

细菌纤维素是由微生物合成的多孔性网状纳米级生物高分子材料,由于它具备高持水性、高透气性、良好生物相容性、高机械强度、三维网络结构等独特性质,因此在纺织、医用敷料、组织工程、食品、导电材料等行业具有广阔的应用前景。本文主要从性质和应用两方面对其近年来的研究进展做了综述,并对未来的发展做了展望。     

Expression of 11 beta-hydroxysteroid dehydrogenase using recombinant vaccinia virus

Ligand specificity of the type I steroid receptor is apparently conferred by the activity of 11 beta-hydroxysteroid dehydrogenase. To determine the kinetic properties of this enzyme, rat liver cDNA was expressed in cultured cells using recombinant vaccinia virus. Although this enzyme catalyzes only dehydrogenation when purified from rat liver, the recombinant enzyme obtained from cell lysates catalyzed both 11 beta-dehydrogenation of corticosterone to 11-dehydrocorticosterone and the reverse 11-oxoreduction reaction. At pH 8.5, the first order rate constant Kcat/Km for dehydrogenase activity exceeded that for reductase (63 vs. 38 min-1 x 10(-4], whereas the rate constants for the two reactions were nearly equal (48 vs. 47 min-1 x 10(-4] at pH 7.0. These results are consistent with the previously determined pH optima for these activities in liver microsomes. Removal (with glucose-6-phosphate dehydrogenase) of NADP+ produced by the reductase reaction significantly increased reductase activity. Glycyrrhetinic acid, a known inhibitor of the dehydrogenase reaction, also inhibited the reductase reaction at slightly higher concentrations (50% inhibitory concentration, less than 5 nM for dehydrogenase, 10-20 nM for reductase). Partial inhibition of glycosylation with A1-tunicamycin decreased dehydrogenase activity 50% without affecting reductase activity. The data demonstrate that a single polypeptide catalyzes both dehydrogenation and reduction, although the presence of additional enzyme forms catalyzing one or the other activity has not been ruled out.     

Homologous expression of recombinant cellobiose dehydrogenase in Phanerochaete chrysosporium

Cellobiose dehydrogenase (CDH) is a novel extracellular hemoflavoenzyme from Phanerochaete chrysosporium and is produced only in cultures supplemented with cellulose. In this report, CDH from P. chrysosporium has been homologously expressed in cultures supplemented with glucose as the sole carbon source when no endogenous CDH is expressed. This was achieved by placing the cdh-1 gene under the control of the D-glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter (1.1 kb) fused upstream of the ATG start codon of cdh-1. The gpd promoter-chd-1 construct was inserted into the multiple cloning site of the expression vector pOGI18, which contained the Schizophyllum commune ade5 as a selectable marker. The P. chrysosporium ade1 auxotrophic strain OGC107-1 was transformed with the pAGC1 construct, and the prototrophic transformants were assayed for CDH activity. Approximately 50% of the Ade(+) transformants exhibited CDH activity in the extracellular medium of stationary cultures. At least one of the transformants produced high levels (500-600 U/liter) of recombinant CDH (rCDH). Purification by ammonium sulfate precipitation, Sephacryl S-200 chromatography, and FPLC using a Mono-Q 5/5 column yielded homogeneous rCDH. Physical, spectral, and kinetic characteristics of purified homologously expressed rCDH were similar to those of wild-type CDH. This expression system will enable site-directed mutagenesis studies to be carried out on CDH.     

Characterization of the oxidative 3alpha-hydroxysteroid dehydrogenase activity of human recombinant 11-cis-retinol dehydrogenase.

11-cis-Retinol dehydrogenase catalyzes the oxidation of cis-retinols, a rate-limiting step in the biosynthesis of 9-cis-retinoic acid. It is also active toward 3alpha-hydroxysteroids, and thus might be involved in steroid metabolism. To better understand the role of this enzyme, we produced stable transfectants expressing 11-cis-retinol dehydrogenase in human embryonic kidney 293 cells. In vitro enzymatic assays have demonstrated that, with an appropriate exogenous cofactor, the enzyme catalyzes the interconversion of 5alpha-androstane-3alpha,17beta-diol and dihydrotestosterone and that of androsterone and androstanedione. However, using intact transfected cells, we found that the enzyme catalyzes reactions only in the oxidative direction. Thus, it is possible that 5alpha-androstane-3alpha,17beta-diol (an inactive androgen) can be converted into dihydrotestosterone, the most potent androgen, by the action of 11-cis-retinol dehydrogenase. This reaction could constitute a non-classical pathway of production of active androgens in the peripheral tissues. We also showed that all-trans-, 9-cis- and 13-cis-retinol inhibit the oxidative 3alpha-hydroxysteroid steroid activity of 11-cis-retinol dehydrogenase with similar K(i) values. Since all-trans-retinol is a precursor of cis-retinols, its inhibitory effect on the activity suggests that it could play an important role in modulating the formation of 9-cis-retinoic acid. In addition, we examined the effect of several known enzyme modulators, namely carbenoxolone, phenylarsine oxide and phosphatidylcholine, on 11-cis-retinol dehydrogenase activity. Taken together, our results suggest that, in humans, this enzyme might play a role in the biosynthesis of both 9-cis-retinoic acid and dihydrotestosterone.     

铜绿假单胞菌二氢硫辛酸酰胺脱氢酶的重组表达及其与脂蛋白(a)的相互作用

【目的】二氢硫辛酸酰胺脱氢酶(Dihydrolipoamide dehydrogenase,Lpd)是铜绿假单胞菌(Pseudomonas aeruginosa)表面的一种纤溶酶原(Plasminogen,Plg)受体,旨在研究Lpd与脂蛋白(a)[Lipoprotein(a),Lp(a)]以及Plg之间的相互作用。【方法】用大肠杆菌表达rLpd及其突变分子(rLpd K476A、rLpd K477A、rLpdΔKKR),用酶联免疫吸附实验(ELISA)、亲和色谱层析及Western blot等技术检测rLpd及其突变分子与Lp(a)、Plg的相互作用。【结果】ELISA及亲和色谱层析实验结果表明,rLpd可以与Lp(a)结合但不与LDL结合,Lp(a)与rLpdΔKKR的结合能力显著低于其与rLpd的结合能力。1 mmol/L的赖氨酸类似物6-氨基己酸(EACA)对rLpd与Lp(a)的结合有显著的抑制作用。1 000μg/L的Lp(a)对rLpd与Plg的结合起到显著的抑制作用。【结论】Lpd能够与Lp(a)特异性结合,其476和477两个相邻的赖氨酸残基是与Lp(a)结合的主要位点,Lp(a)可以竞争性地抑制rLpd与Plg的结合。     

Selective binding of cholesterol by recombinant fatty acid binding proteins

The sterol binding specificity of rat recombinant liver fatty acid binding protein (L-FABP) and intestinal fatty acid binding protein (I-FABP) was characterized with [3H]cholesterol and a fluorescent sterol analog dehydroergosterol. Ligand binding analysis, fluorescence spectroscopy, and activation of microsomal acyl-CoA:cholesterol acyltransferase activity showed that L-FABP-bound sterols. 1) Lipidex-1000 assay showed a dissociation constant Kd = 0.78 +/- 0.18 microM and stoichiometry of 0.47 +/- 0.16 mol/mol for [3H]cholesterol binding to L-PABP. 2) With [3H]cholesterol/phosphatidylcholine liposomes, the cholesterol binding parameters for L-FABP were Kd = 1.53 +/- 0.28 microM and stoichiometry 0.83 +/- 0.07 mol/mol. 3) L-FABP interaction with dehydroergosterol altered the fluorescence intensity and polarization of dehydroergosterol. Dehydroergosterol bound to L-FABP with Kd = 0.37 microM and a stoichiometry of 0.83 mol/mol. 4) Cholesterol and dehydroergosterol decreased L-FABP tyrosine lifetime. Dehydroergosterol binding produced sensitized emission of bound dehydroergosterol with longer lifetime.5) L-FABP bound two cis-parinaric acid molecules/molecule of protein. Cholesterol displaced one of these bound cis-parinaric acids. 6) L-FABP enhanced acyl-CoA:cholesterol acyltransferase in a concentration-dependent manner. In contrast, these assays indicated that I-FABP did not bind sterols. Thus, L-FABP appears able to bind 1 mol of cholesterol/mol of L-FABP, the L-FABP sterol binding site is equivalent to one of the two fatty acid binding sites, and L-FABP stimulates acyl-CoA:cholesterol acyltransferase by transfer of cholesterol.     

Clinical applications of recombinant human colony-stimulating factors.

The differentiation and maturation of hematopoietic progenitor cells are regulated by certain growth factors. Several of these glycoproteins have been characterized, and their amino acid sequences have been delineated. Modern DNA technology provides sufficient quantities of these hormones for testing in clinical trials. Erythropoietin (EPO) has been shown to increase the hemoglobin level and hematocrit in patients with end-stage renal disease. Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF) can increase the numbers of neutrophils and monocytes, in a dose-dependent fashion. The function of granulocytes and monocytes is also enhanced. Clinical studies of the toxicity and activity of G-CSF and GM-CSF have been conducted in patients with acquired immune deficiency syndrome, aplastic anemia, myelodysplastic syndromes, and neutropenia due to cancer and chemotherapy. In almost all patients the neutrophil count increased within 24 hours after the start of treatment. Side effects of G-CSF and GM-CSF are infrequent and usually mild. Combinations of CSFs may be even more effective.     

The neutral lipid composition and size of recombinant high density lipoproteins regulates lecithin:cholesterol acyltransferase activity

Recombinant high density lipoprotein (rHDL) particles were prepared from purified lipids and human apoproteins, and the ability of these complexes to act as substrates for purified lecithin:cholesterol acyltransferase (LCAT) was determined. Increasing the triacylglycerol content relative to cholesteryl ester in rHDL markedly decreased the maximum catalytic potential of LCAT. Kinetic analysis showed that the Vmax of the LCAT reaction was significantly and negatively correlated to the triacylglycerol content. The apparent Km was not directly affected by relative neutral lipid content, but was significantly related to protein and surface lipid content as well as to particle size. These results suggest that while particulate size may regulate the interaction between LCAT and HDL, the relative neutral lipid content of the particle may play a major role in regulating the catalytic potential of the enzyme, particularly with HDL from hypertriglyceridemic patients.     

The distribution and characteristics of nitrate reductase and glutamate dehydrogenase in the maize seedling

In a study on 3-day maize (Zea mays) seedlings, grown on nitrate, requirements were established for the maximum extraction and optimum stabilization of nitrate reductase in vitro. With the primary root, 5 mm cysteine were required in the extraction medium, but for the scutellum, which has a high level of endogenous thiol, the use of additional thiol resulted in a reduced yield of a more labile enzyme. Activity of the root and scutella nitrate reductase was obtained with either NADH or NADPH, but that of the root enzyme with NADPH was only demonstrated in the absence of phosphate.Before leaf expansion, the nitrate reductase in the maize seedling was mainly in the scutellum. The enzyme present in the primary root was predominantly in the apical region (0-2 mm). In contrast, glutamate dehydrogenase was concentrated in the mature basal region of the root (30-60 mm). A high level of nitrate (approximately 100 mm) was required to saturate the induction of nitrate reductase in the root tip, mature root, and scutellum. The concentration of nitrate required to give half the maximum level of enzyme induced was the same for each region (29 mm).After leaf expansion, more than 90% of the nitrate reductase was in the shoot, mainly in the leaf blade, and a marked decrease occurred in the level of the enzyme in the scutellum. A large proportion of the glutamate dehydrogenase was still found in the root.     

Evaluation of recombinant phenylalanine dehydrogenase behavior in aqueous two-phase partitioning

Recombinant Bacillus sphaericus phenylalanine dehydrogenase (PheDH) partitioning was studied in polyethylene glycol (PEG) and ammonium sulfate aqueous two-phase systems (ATPS). The objectives of this work were to investigate influences; varying the molecular mass and concentration of PEG, pH, phase volume ratio (V_R), tie-line length (TLL) and concentration of (NH₄)₂SO₄ on the partition behavior of PheDH. It was revealed that the partitioning was not affected by V_R, while PEG molecular mass and concentration and (NH₄)₂SO₄ concentration had significant effects on enzyme partitioning. Longer TLL and higher pH resulted in better partitioning into the top phase. Under the most favorable partition conditions with 8.5% (w/w) PEG-6000, 17.5% (w/w) (NH₄)₂SO₄ and V_R = 0.25 at pH 8.0, partition coefficient (K_E), recovery (R%), yield (Y%) and TLL were achieved 58.7%, 135%, 94.42% and 39.89% (w/w), respectively. Overall, the promising results obtained in this research indicated that the ATPS partitioning can be provided an efficient and powerful tool for recovery and purification of recombinant PheDH.     

High-level expression of recombinant glucose dehydrogenase and its application in NADPH regeneration

Two glucose dehydrogenase (E.C. 1.1.1.47) genes, gdh223 and gdh151, were cloned from Bacillus megaterium AS1.223 and AS1.151, and were inserted into pQE30 to construct the expression vectors, pQE30-gdh223 and pQE30-gdh151, respectively. The transformant Escherichia coli M15 with pQE30-gdh223 gave a much higher glucose dehydrogenase activity than that with the plasmid pQE30-gdh151. Thus it was used to optimize the expression of glucose dehydrogenase. An proximately tenfold increase in GDH activity was achieved by the optimization of culture and induction conditions, and the highest productivity of glucose dehydrogenase (58.7 U/ml) was attained. The recombinant glucose dehydrogenase produced by E. coli M15 (pQE30-gdh223) was then used to regenerate NADPH. NADPH was efficiently regenerated in vivo and in vitro when 0.1 M glucose was supplemented concomitantly in the reaction system. Finally, this coenzyme-regenerating system was coupled with a NADPH-dependent bioreduction for efficient synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate from ethyl 4-chloro-3-oxobutanoate.