Characterization of the Dimerization of Metabotropic Glutamate Receptors Using an N-Terminal Truncation of mGluR1α

The metabotropic glutamate receptor mGluR1alpha in membranes isolated both from rat brain and from cell lines transfected with cDNA coding for the receptor migrates as a disulphide-bonded dimer on sodium dodecyl sulphate-polyacrylamide gels. Dimerization of mGluR1alpha takes place in the endoplasmic reticulum because it is not prevented by exposing transfected human embryonic kidney (HEK) 293 cells to the drug brefeldin A, a drug that prevents egress of proteins from the endoplasmic reticulum. Dimerization was also not dependent on protein glycosylation as it was not prevented by treatment of the cells with tunicamycin. Using a mammalian expression vector containing the N-terminal domain of mGluR1alpha, truncated just before the first transmembrane domain (NT-mGluR1alpha), we show that the N-terminal domain is secreted as a soluble disulphide-bonded dimeric protein. In addition, the truncated N-terminal domain can form heterodimers with mGluR1alpha when both proteins are cotransfected into HEK 293 cells. However, mGluR1alpha and its splice variant mGluR1beta did not form heterodimers in doubly transfected HEK 293 cells. These results show that although the N-terminal domain of mGluR1alpha is sufficient for dimer formation, other domains in the molecule must regulate the process.     

Molecular and Functional Characterization of an Invertase Secreted by Ashbya gossypii

The repertoire of hydrolytic enzymes natively secreted by the filamentous fungus Ashbya (Eremothecium) gossypii has been poorly explored. Here, an invertase secreted by this flavinogenic fungus was for the first time molecularly and functionally characterized. Invertase activity was detected in A. gossypii culture supernatants and cell-associated fractions. Extracellular invertase migrated in a native polyacrylamide gel as diffuse protein bands, indicating the occurrence of at least two invertase isoforms. Hydrolytic activity toward sucrose was approximately 10 times higher than toward raffinose. Inulin and levan were not hydrolyzed. Production of invertase by A. gossypii was repressed by the presence of glucose in the culture medium. The A. gossypii invertase was demonstrated to be encoded by the AFR529W (AgSUC2) gene, which is highly homologous to the Saccharomyces cerevisiae SUC2 (ScSUC2) gene. Agsuc2 null mutants were unable to hydrolyze sucrose, proving that invertase is encoded by a single gene in A. gossypii. This mutation was functionally complemented by the ScSUC2 and AgSUC2 genes, when expressed from a 2-μm-plasmid. The signal sequences of both AgSuc2p and ScSuc2p were able to direct the secretion of invertase into the culture medium in A. gossypii.     

嗜酸菌耐酸pH平衡机制及潜在应用

嗜酸菌是一类可以在极端酸性环境下生存的微生物,在生物整治以及耐热耐酸酶的提取等领域发挥着重要作用。一些嗜中性工程菌株在发酵过程中经常遇到自身环境酸化的问题,嗜酸菌独特的耐酸能力及其耐酸模块为构建耐酸能力强的嗜中性工程菌株提供了思路。因此,从细胞膜的稳定性及低渗透性,耐酸相关的能量代谢,生物大分子的修复以及胞内缓冲作用等方面对嗜酸菌的耐酸机制进行深入探讨,并展望了嗜酸菌在耐酸工程菌株合成生物学领域的作用。     

Zoosporic Tolerance to pH Stress and Its Implications for Phytophthora Species in Aquatic Ecosystems

Phytophthora species, a group of destructive plant pathogens, are commonly referred to as water molds, but little is known about their aquatic ecology. Here we show the effect of pH on zoospore survival of seven Phytophthora species commonly isolated from irrigation reservoirs and natural waterways and dissect zoospore survival strategy. Zoospores were incubated in a basal salt liquid medium at pH 3 to 11 for up to 7 days and then plated on a selective medium to determine their survival. The optimal pHs differed among Phytophthora species, with the optimal pH for P. citricola at pH 9, the optimal pH for P. tropicalis at pH 5, and the optimal pH for the five other species, P. citrophthora, P. insolita, P. irrigata, P. megasperma, and P. nicotianae, at pH 7. The greatest number of colonies was recovered from zoospores of all species plated immediately after being exposed to different levels of pH. At pH 5 to 11, the recovery rate decreased sharply (P ≤ 0.0472) after 1-day exposure for five of the seven species. In contrast, no change occurred (P ≥ 0.1125) in the recovery of any species even after a 7-day exposure at pH 3. Overall, P. megasperma and P. citricola survived longer at higher rates in a wider range of pHs than other species did. These results are generally applicable to field conditions as indicated by additional examination of P. citrophthora and P. megasperma in irrigation water at different levels of pH. These results challenge the notion that all Phytophthora species inhabit aquatic environments as water molds and have significant implications in the management of plant diseases resulting from waterborne microbial contamination.Phytophthora species, a group of oomycetes in the kingdom of Stramenopila and well-known plant pathogens, were first listed as “water molds” by Blackwell in 1944 (5), and this notion has since been generally accepted. These species are phylogenetically close to golden-brown algae, although morphologically and physiologically, they resemble fungi. Most algae are aquatic in nature. Phytophthora species produce flagellate zoospores as their primary dispersal structure (35-37, 39). Zoospores can travel in aquatic environments actively on their own locomotion and passively through water movement (12, 13, 41).More than 20 species of Phytophthora, including P. ramorum, the sudden oak death pathogen, have been isolated from irrigation reservoirs and natural waterways (20-22, 30, 40, 43), and a number of previously unknown taxa also have been documented in aquatic environments (8, 24). These pathogens pose a threat to agricultural sustainability and natural ecosystems, as agriculture increasingly depends on recycled water for irrigation in light of rapidly spreading global water scarcity (19, 22). Recycling irrigation systems provide an efficient means of pathogen dissemination from a single point of infection to an entire farm and from a single farm to other farms sharing the same water resources (22, 24).A search of science-based solutions to this crop health issue reveals a surprising lack of information on the aquatic ecology of Phytophthora species. For instance, hydrogen ion concentration (pH) is among the most important water quality parameters which influence sporangium production and germination (1-3, 6, 32, 34, 38), survival of thick-walled chlamydospores and oospores in the soil environment, and disease development (2, 4, 33, 44). However, the effect of pH on the survival of zoospores and growth of germlings in aquatic environments is not known. As motile zoospores lack cell walls and encysted spores or cysts have thin walls, they are presumably more vulnerable to pH stress than chlamydospores and oospores are. On the other hand, the pH level is likely to fluctuate more regularly and at a greater range in aquatic systems, such as irrigation reservoirs, than in soil systems. pH can change diurnally due to respiration of aquatic plants and seasonally due to rain, oxidation of sulfide-containing sediments through the production of sulfuric acid, algal blooms, and released bases or acids from residues of fertilizer and pesticides. Thus, zoospores and aquatic systems are more prone to the influence of wide pH changes than chlamydospores/oospores in soil systems are. The aim of this study was to determine the impact of pH on zoospore survival and understand the aquatic ecology of different Phytophthora species.     

Internal Desynchronization of Circadian Rhythms and Tolerance to Shift Work

Intolerance to shift work may result from individual susceptibility to an internal desynchronization. Some shift workers (SW) who show desynchronization of their circadian rhythms (e.g., sleep‐wake, body temperature, and grip strength of both hands) exhibit symptoms of SW intolerance, such as sleep alteration, persistent fatigue, sleep medication dependence, and mood disturbances, including depression. Existing time series data previously collected from 48 male Caucasian French SW were reanalyzed specifically to test the hypothesis that internal synchronization of circadian rhythms is associated with SW intolerance and symptoms. The entry of the subjects into the study was randomized. Three groups were formed thereafter: SW with good tolerance (n=14); SW with poor tolerance, as evident by medical complaints for at least one year (n=19); and former SW (n=15) with very poor tolerance and who had been discharged from night work for at 1.5 yr span but who were symptom‐free at the time of the study. Individual and longitudinal time series of selected variables (self‐recorded sleep‐wake data using a sleep log, self‐measured grip strength of both hands using a Colin Gentile dynamometer, and oral temperature using a clinical thermometer) were gathered for at least 15 days, including during one or two night shifts. Measurements were performed 4–5 times/24 h. Power spectra that quantify the prominent period (τ) and t‐test, chi square, and correlation coefficient were used as statistical tools. The mean (±SEM) age of SW with good tolerance was greater than that of SW with poor tolerance (44.9±2.1 yrs vs. 40.1±2.6 yrs, p<.001) and of former SW discharged from night work (very poor tolerance; 33.4±1.7, p<.001). The shift-work duration (yrs) was longer in SW with good than poor tolerance (19.9±2.2 yrs vs. 15.7±2.2; p<0.002) and former SW (10.7±1.2; p<.0001). The correlation between subject age and shift-work duration was stronger in tolerant SW (r=0.97, p<.0001) than in non‐tolerant SW (r=0.80, p<0.001) and greater than that of former SW (r=0.72, p<.01). The mean sleep‐wake rhythm τ was 24 h for all 48 subjects. The number of desynchronized circadian rhythms (τ differing from 24 h) was greater in non‐tolerant than in tolerant SW (chi square=38.9, p<.0001). In Former SW (i.e., 15 individuals assessed in follow‐up studies done 1.5 to 20 yrs after return to day work), both symptoms of intolerance and internal desynchronization were reduced or absent. The results suggest that non‐tolerant SW are particularly sensitive to the internal desynchronization of their circadian time organization.     

Determination of CGTase from Bacillus ohbensis and Its Optimum pH Using HPLC

Glucose is widely known to be required during superoxide generation in phagocytic cells. However, when an specific chemiluminescence probe with the Cypridina luciferin analog 2-methyl-6-(p-methoxyphenyl)-3, 7 -dihydroimidazo[ 1,2-a]pyrazin-3-one (MCLA) was used, about 60% of the chemiluminescence remained in stimulated macrophages in the presence of the glycolytic inhibitor 2-deoxyglucose. -nonspecific luminol-dependent chemiluminescence disappeared when the same drug was added. These results clearly demonstrate that the generation of by macrophages is not completely glucose-dependent, and strongly suggest that macrophages have both glucoseindependent NADPH-supplying pathway(s) and glucose dependent pathway(s) which generate reactive oxygen species other than .     

Purification and Partial Characterization of Potato (Solanum tuberosum) Invertase and Its Endogenous Proteinaceous Inhibitor

Invertase plays an important role in the hydrolysis of sucrose in higher plants, especially in the storage organs. In potato (Solanum tuberosum) tubers, and in some other plant tissues, the enzyme seems to be controlled by interaction with an endogenous proteinaceous inhibitor. An acid invertase from potato tubers (variety russet) was purified 1560-fold to electrophoretic homogeneity by consecutive use of concanvalin A-Sepharose 4B affinity chromatography, DEAE-Sephadex A-50-120 chromatography, Sephadex G-150 chromatography, and DEAE-Sephadex A-50-120 chromatography. The enzyme contained 10.9% carbohydrate, had an apparent molecular weight of 60,000 by gel filtration, and was composed of two identical molecular weight subunits (M_r 30,000). The enzyme had a K_m for sucrose of 16 millimolar at pH 4.70 and was most stable and had maximum activity around pH 5. The endogenous inhibitor was purified 610-fold to homogeneity by consecutive treatment at pH 1 to 1.5 at 37°C for 1 hour, (NH₄)₂SO₄ fractionation, Sephadex G-100 chromatography, DEAE-Sephadex G-50-120 chromatography, and hydroxylapatite chromatography. The inhibitor appears to be a single polypeptide (M_r 17,000) without glyco groups. The purified inhibitor was stable over the pH range of 2 to 7 when incubated at 37°C for 1 hour.     

Soil Bacterial Biomass, Activity, Phospholipid Fatty Acid Pattern, and pH Tolerance in an Area Polluted with Alkaline Dust Deposition

Soil bacterial biomass, phospholipid fatty acid pattern, pH tolerance, and growth rate were studied in a forest area in Finland that is polluted with alkaline dust from an iron and steel works. The pollution raised the pH of the humus layer from 4.1 to 6.6. Total bacterial numbers and the total amounts of bacterial phospholipid fatty acids in the humus layer did not differ between the unpolluted control sites and the polluted ones. The number of CFU increased by a factor of 6.4 in the polluted sites compared with the controls, while the bacterial growth rate, measured by the thymidine incorporation technique, increased about 1.8-fold in the polluted sites. A shift in the pattern of phospholipid fatty acids indicated a shift in the bacterial species composition. The largest proportional increase was found for the fatty acid 10Me18:0, which indicated an increase in the number of actinomycetes in the polluted sites. The levels of the fatty acids i14:0, 16:1ω5, cy17:0, 18:1ω7, and 19:1 also increased in the polluted sites while those of fatty acids 15:0, i15:0, 10Me16:0, 16:1ω7t, 18:1ω9, and cy19:0 decreased compared with the unpolluted sites. An altered pH tolerance of the bacterial assemblage was detected either as a decrease in acid-tolerant CFU in the polluted sites or as altered bacterial growth rates at different pHs. The latter was estimated by measuring the thymidine incorporation rate of bacteria extracted from soil by homogenization-centrifugation at different pHs.     

Bactericidal Activity of Photocatalytic TiO2 Reaction: toward an Understanding of Its Killing Mechanism

When titanium dioxide (TiO₂) is irradiated with near-UV light, this semiconductor exhibits strong bactericidal activity. In this paper, we present the first evidence that the lipid peroxidation reaction is the underlying mechanism of death of Escherichia coli K-12 cells that are irradiated in the presence of the TiO₂ photocatalyst. Using production of malondialdehyde (MDA) as an index to assess cell membrane damage by lipid peroxidation, we observed that there was an exponential increase in the production of MDA, whose concentration reached 1.1 to 2.4 nmol · mg (dry weight) of cells⁻¹ after 30 min of illumination, and that the kinetics of this process paralleled cell death. Under these conditions, concomitant losses of 77 to 93% of the cell respiratory activity were also detected, as measured by both oxygen uptake and reduction of 2,3,5-triphenyltetrazolium chloride from succinate as the electron donor. The occurrence of lipid peroxidation and the simultaneous losses of both membrane-dependent respiratory activity and cell viability depended strictly on the presence of both light and TiO₂. We concluded that TiO₂ photocatalysis promoted peroxidation of the polyunsaturated phospholipid component of the lipid membrane initially and induced major disorder in the E. coli cell membrane. Subsequently, essential functions that rely on intact cell membrane architecture, such as respiratory activity, were lost, and cell death was inevitable.     

Ethylene Signaling Causing Tolerance of Arabidopsis thaliana Roots to Low pH Stress is Linked to Class III Peroxidase Activity

Journal of Plant Growth Regulation - One irreversible consequence of acidic pH for roots is cell death. Growing evidence suggests the role of hormones and cell wall-related enzymes in response to...     

Structural Characterization of an Alkali-Soluble Polysaccharide from Angelica sinensis and Its Antitumor Activity in Vivo

A novel alkali-soluble polysaccharide (AASP) was isolated from Angelica sinensis (Oliv.) Diels under aqueous alkali treatment, and its structural characterization and antitumor activity in Vivo were evaluated in present study. Results of HPGPC and IC revealed that AASP was a neutral polysaccharide containing Ara, Gal and Glc in the mole ratio of 1.00 : 2.26 : 24.43, with the average molecular weight of 4.7 kDa. Periodate oxidation, Smith degradation, methylation, FT-IR, and NMR analyses further demonstrated that a preliminary structure of AASP was proposed as follows: (1→3)-linked arabinose, (1→6)-linked galactose, and (1→), (1→4), (1→6), (1→3,6)-linked glucose with α- and β-configuration. In Vivo antitumor assays, AASP exhibited prominent antitumor effects on H22 hepatoma cells with an inhibitory ratio of 48.57 % and effectively protected thymuses and spleens of tumor-bearing mice. Besides, AASP displayed a proliferation stimulating activity of immunocytes (splenocytes, peritoneal macrophages and natural killer cells), and an auxo-action for cytokines release (TNF-α, IL-2 and IFN-γ), leading to the apoptosis of H22 solid tumors cells via G0/G1 phase arrested. The above data demonstrated that AASP holds great application potential to be a safe and effective antitumor supplement in the future.     

Identification and Characterization of Proteins Associated with Plant Tolerance to Heat Stress

Heat stress is a major abiotic stress limiting plant growth and productivity in many areas of the world. Understanding mechanisms of plant adaptation to heat stress would facilitate the development of heat-tolerant cultivars for improving productivity in warm climatic regions. Protein metabolism involving protein synthesis and degradation is one of the most sensitive processes to heat stress. Changes in the level and expression pattern of some proteins may play an important role in plant adaptation to heat stress. The identification of stress-responsive proteins and pathways has been facilitated by an increasing number of tools and resources, including two-dimensional electrophoresis and mass spectrometry, and the rapidly expanding nucleotide and amino acid sequence databases. Heat stress may induce or enhance protein expression or cause protein degradation. The induction of heat-responsive proteins, particularly heat shock proteins (HSPs), plays a key role in plant tolerance to heat stress. Protein degradation involving various proteases is also important in regulating plant responses to heat stress. This review provides an overview of recent research on proteomic profiling for the identification of heat-responsive proteins associated with heat tolerance, heat induction and characteristics of HSPs, and protein degradation in relation to plant responses to heat stress.     

Synthesis and the Intestinal Glucosidase Inhibitory Activity of 2-Aminoresorcinol Derivatives toward an Investigation of Its Binding Site

2-Aminoresorcinol is a potent and selective intestinal glucosidase inhibitor. Unlike the majority of glucosidase inhibitors, it shows an uncompetitive mode of inhibition. In this study, we tested the intestinal glucosidase inhibitory activity of various 2-aminoresorcinol derivatives. We found that structural changes, in amino and two phenolic hydroxyl groups had a negative impact on inhibitory activity, but methylation of the phenolic hydroxyl group was found to maintain its activity and replacement of the aromatic ring with an acyl or alkoxy carbonyl group at the 4th position also retained its activity. This enable us to design a molecular probe for further study of the inhibition mechanism of 2-aminoresorcinol.     

Identification and Characterization of Proteins Associated with Plant Tolerance to Heat Stress

Heat stress is a major abiotic stress limiting plant growth and productivity in many areas of the world. Understanding mechanisms of plant adaptation to heat stress would facilitate the development of heat-tolerant cultivars for improving productivity in warm climatic regions. Protein metabolism involving protein synthesis and degradation is one of the most sensitive processes to heat stress. Changes in the level and expression pattern of some proteins may play an important role in plant adaptation to heat stress. The identification of stress-responsive proteins and pathways has been facilitated by an increasing number of tools and resources, including two-dimensional electrophoresis and mass spectrometry, and the rapidly expanding nucleotide and amino acid sequence databases. Heat stress may induce or enhance protein expression or cause protein degradation. The induction of heat-responsive proteins, particularly heat shock proteins (HSPs), plays a key role in plant tolerance to heat stress. Protein degradation involving various proteases is also important in regulating plant responses to heat stress. This review provides an overview of recent research on proteomic profiling for the identification of heat-responsive proteins associated with heat tolerance, heat induction and characteristics of HSPs, and protein degradation in relation to plant responses to heat stress.     

Growth and Sucrose Metabolism of Carrot Callus Strains with Normal and Low Acid Invertase Activity

The properties of two strains of carrot (Daucus carota) callus are presented. One has a very low acid invertase activity which is accompanied by differences in morphology and metabolic rate, but not in growth rate. We conclude that one of the main functions of plant acid invertases is in controlling the levels of sugars which, by interaction with hormones, affect differentiation, both morphological and biochemical. The effect of tris on sucrose metabolizing enzymes, and the cause of the “sucrose effect” are considered.     

人精子中性鞘糖脂的纯化与分析及其抗体的制备与鉴定

人精子中性鞘糖脂的纯化与分析及其抗体的制备与鉴定刘建军,崔肇春,朱正美（大连医科大学生化教研室,大连１１６０２３）抗精子抗体（ＡｓＡｂ）可导致免疫不孕已被证实［１］。有研究表明鞘糖脂（ＧＳＬ）在精卵识别中可能具有重要作用［２］。已知ＧＳＬ具有抗原性。...     

Characterization of a Family GH5 Xylanase with Activity on Neutral Oligosaccharides and Evaluation as a Pulp Bleaching Aid

A new bacterial xylanase belonging to family 5 of glycosyl hydrolases was identified and characterized. The xylanase, Xyn5B from Bacillus sp. strain BP-7, was active on neutral, nonsubstituted xylooligosaccharides, showing a clear difference from other GH5 xylanases characterized to date that show a requirement for methyl-glucuronic acid side chains for catalysis. The enzyme was evaluated on Eucalyptus kraft pulp, showing its effectiveness as a bleaching aid.The catabolic breakdown of xylan is a critical step in the recycling of carbon in nature and has been targeted as a subject of intense research as a renewable energy resource as well as for bioconversion of plant biomass into high-added-value products (21, 29, 37, 40). Biodegradation of xylan is a complex process that requires the coordinate action of several enzymes, among which xylanases (1,4-β-D-xylan xylanohydrolase; EC 3.2.1.8), cleaving internal linkages on the β-1,4-xylose backbone, play a key role.Most known xylanases are grouped into glycoside hydrolase (GH) families 10 and 11 (CAZy [Carbohydrate-Active enZYmes] database) (17), although a few xylanases have recently been ascribed to glycoside hydrolase families 5, 7, 8, and 43 (8, 9, 24). Among xylanases not grouped in the typical families GH10 and GH11, only two xylanases belonging to family GH5 have been biochemically characterized in detail. The enzymes XynA from Erwinia chrysanthemi (18, 39) and XynC from Bacillus subtilis (32) hydrolyze glucuronoxylan to branched xylooligosaccharides. The activity of Erwinia chrysanthemi XynA has also been evaluated on other substrates containing xylose, showing an absolute requirement for methyl-glucuronic substitutions. In this way, only methyl-glucuronic acid-branched oligosaccharides can be cleaved by XynA, whereas linear xylooligosaccharides or arabinoxylans are not cleaved by this enzyme (39). This type of xylanases must play an important role in complementing the action of GH10 and GH11 enzymes during depolymerization of glucuronoxylans in lignocellulosic fibers.Xylanases are widely used in the pulp industry to enhance the effectiveness of bleaching agents, thereby reducing the generation of toxic wastes (adsorbable organic halogens; AOX) (1, 38). Several reports have evaluated the effectiveness of family GH10 and GH11 xylanases on the bleaching process, showing that GH11 xylanases usually display better performance (7, 12), although there are many other factors that contribute to the bleach-boosting effect of a xylanase, such as the source of the pulp and the pulping process itself (6, 11). Besides their contribution to the increase in brightness, an innovative aspect of the application of xylanases is their contribution to the removal of hexenuronic acids (HexA) produced during the kraft cooking process, which can accelerate the brightness reversion (yellowing tendency) of paper (35). However, it remains to be known if all xylanases are capable of removing HexAs and/or enhancing bleachability.Bacillus sp. strain BP-7 is a xylanolytic strain isolated from agricultural soils (25). It shows a multiple enzymatic system for xylan degradation, including a GH11 xylanase cloned and characterized previously (13). In this work, we describe the identification and cloning of a second xylanase from the strain, belonging to the GH5 family. The enzyme hydrolyzes linear xylooligosaccharides, clearly differing from the two GH5 xylanases characterized up to date. The new enzyme has been tested on Eucalyptus pulps, showing good performance as a bleaching aid. The results obtained suggest an important role for the enzyme in xylan degradation and indicate the potential of this xylanase for biotechnological applications in the bioconversion of glucuronoxylan-containing biomass.     

Discovery of an Escherichia coli Esterase with High Activity and Enantioselectivity toward 1,2-O-Isopropylideneglycerol Esters

Escherichia coli has been widely used as an expression host for the identification of desired biocatalysts through screening or selection assays. We have previously used E. coli in growth selection and screening assays for identification of Bacillus subtilis lipase variants (located in the periplasm) with improved activity and enantioselectivity toward 1,2-O-isopropylideneglycerol (IPG) esters. In the course of these studies, we discovered that E. coli itself exhibits significant cytoplasmic esterase activity toward IPG esters. In order to identify the enzyme (or enzymes) responsible for this esterase activity, we analyzed eight E. coli knockout strains, in which single esterase genes were deleted, for their ability to hydrolyze IPG butyrate. This approach led to the identification of esterase YbfF as the major E. coli enzyme responsible for the hydrolytic activity toward IPG esters. The gene coding for YbfF was cloned and overexpressed in E. coli, and the corresponding protein was purified and characterized for its biocatalytic performance. YbfF displays a high level of activity toward IPG butyrate and IPG caprylate and prefers the R-enantiomer of these substrates, producing the S-enantiomer of the IPG product with high enantiomeric excess (72 to 94% ee). The enantioselectivity of YbfF for IPG caprylate (E = 40) could be significantly enhanced when using dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) as cosolvents in kinetic resolution experiments. The enzyme also shows high enantioselectivity toward 1-phenylethyl acetate (E ≥ 200), giving the chiral product (R)-1-phenylethanol with >99% ee. The high activity and enantioselectivity of YbfF make it an attractive enzyme for organic synthesis.     

Mutation of His465 Alters the pH-dependent Spectroscopic Properties of Escherichia coli Glutamate Decarboxylase and Broadens the Range of Its Activity toward More Alkaline pH

Glutamate decarboxylase (GadB) from Escherichia coli is a hexameric, pyridoxal 5′-phosphate-dependent enzyme catalyzing CO₂ release from the α-carboxyl group of l-glutamate to yield γ-aminobutyrate. GadB exhibits an acidic pH optimum and undergoes a spectroscopically detectable and strongly cooperative pH-dependent conformational change involving at least six protons. Crystallographic studies showed that at mildly alkaline pH GadB is inactive because all active sites are locked by the C termini and that the 340 nm absorbance is an aldamine formed by the pyridoxal 5′-phosphate-Lys²⁷⁶ Schiff base with the distal nitrogen of His⁴⁶⁵, the penultimate residue in the GadB sequence. Herein we show that His⁴⁶⁵ has a massive influence on the equilibrium between active and inactive forms, the former being favored when this residue is absent. His⁴⁶⁵ contributes with n ≈ 2.5 to the overall cooperativity of the system. The residual cooperativity (n ≈ 3) is associated with the conformational changes still occurring at the N-terminal ends regardless of the mutation. His⁴⁶⁵, dispensable for the cooperativity that affects enzyme activity, is essential to include the conformational change of the N termini into the cooperativity of the whole system. In the absence of His⁴⁶⁵, a 330-nm absorbing species appears, with fluorescence emission spectra more complex than model compounds and consisting of two maxima at 390 and 510 nm. Because His⁴⁶⁵ mutants are active at pH well above 5.7, they appear to be suitable for biotechnological applications.