Chemoenzymatic synthesis of glutamic acid analogues: substrate specificity and synthetic applications of branched chain aminotransferase from Escherichia coli

A new route to alpha-keto acids is described, based on the ozonolysis of enol acetates obtained from alpha-substituted beta-keto esters. Escherichia coli branched chain aminotransferase (BCAT) activity toward a variety of substituted 2-oxoglutaric acids was demonstrated analytically. BCAT was shown to have a broad substrate spectrum, complementary to that of aspartate aminotransferase, and to offer access to a variety of glutamic acid analogues. The usefulness of BCAT was demonstrated through the synthesis of several 3- and 4-substituted derivatives.     

Studies on the substrate specificity of Escherichia coli galactokinase

In vitro glycorandomization (IVG) technology is dependent upon the ability to rapidly synthesize sugar phosphates. Compared with chemical synthesis, enzymatic (kinase) routes to sugar phosphates would be attractive for this application. This work focuses upon the development of a high-throughput colorimetric galactokinase (GalK) assay and its application toward probing the substrate specificity and kinetic parameters of Escherichia coli GalK. The demonstrated dinitrosalicylic assay should also be generally applicable to a variety of sugar-processing enzymes. [reaction: see text]     

Solid-state NMR spectroscopy detects interactions between tryptophan residues of the E. coli sugar transporter GalP and the alpha-anomer of the D-glucose substrate

An experimental approach is described in which high resolution 13C solid-state NMR (SSNMR) spectroscopy has been used to detect interactions between specific residues of membrane-embedded transport proteins and weakly binding noncovalent ligands. This procedure has provided insight into the binding site for the substrate D-glucose in the Escherichia coli sugar transport protein GalP. Cross-polarization magic-angle spinning (CP-MAS) SSNMR spectra of GalP in its natural membrane at 4 degrees C indicated that the alpha- and beta-anomers of D-[1-(13)C]glucose were bound by GalP with equal affinity and underwent fast exchange between the free and bound environments. Further experiments confirmed that by lowering the measurement temperature to -10 degrees C, peaks could be detected selectively from the substrate when restrained within the binding site. Dipolar-assisted rotational resonance (DARR) SSNMR experiments at -10 degrees C showed a selective interaction between the alpha-anomer of D-[1-(13)C]glucose and 13C-labels within [13C]tryptophan-labeled GalP, which places the carbon atom at C-1 in the alpha-anomer of D-glucose to within 6 A of the carbonyl carbon of one or more tryptophan residues in the protein. No interaction was detected for the beta-isomer. The role of tryptophan residues in substrate binding was investigated further in CP-MAS experiments to detect D-[1-(13)C]glucose binding to the GalP mutants W371F and W395F before and after the addition of the inhibitor forskolin. The results suggest that both mutants bind D-glucose with similar affinities, but have different affinities for forskolin. This work highlights a useful general experimental strategy for probing the binding sites of membrane proteins, using methodology which overcomes the problems associated with the unfavorable dynamics of weak ligands.     

Analysis of E. coli K5 capsular polysaccharide heparosan

Heparosan is the key precursor for the preparation of bioengineered heparin, a potential replacement for porcine intestinal heparin, an important anticoagulant drug. The molecular weight (MW) distribution of heparosan produced by the fermentation of E. coli K5 was investigated. Large-slab isocratic and mini-slab gradient polyacrylamide gel electrophoresis (PAGE) were used to analyze the MW and polydispersity of heparosan. A preparative method that allowed fractionation by continuous-elution PAGE was used to obtain heparosan MW standards. The MWs of the heparosan standards were determined by electrospray ionization Fourier-transform mass spectrometry (ESI-FT-MS). A ladder of the standards was then used to determine the MW properties of polydisperse heparosan samples. Unbleached and bleached heparosan produced by fermentation of E. coli K5 had similar number-averaged MWs (M_N), weight-averaged MWs (M_W), and MW ranges of 3,000 to 150,000?Da.     

Microbial deracemization of alpha-substituted carboxylic acids: substrate specificity and mechanistic investigation

A new enzymatic method for the preparation of optically active alpha-substituted carboxylic acids is reported. This technique is called deracemization reaction, which provides us with a route to obtain the enantiomerically pure compounds, theoretically in 100% yield starting from the racemic mixture. This means that the synthesis of a racemate is almost equal to the synthesis of the optically active compound, and this concept is entirely different from the commonly accepted one in the asymmetric synthesis. Using the growing cell system of Nocardia diaphanozonaria JCM3208, racemates of 2-aryl- and 2-aryloxypropanoic acid are deracemized smoothly and (R)-form-enriched products are recovered in high chemical yield (>50%). In addition, using optically active starting compounds and deuterated derivatives as well as inhibitors, we have disclosed the fact that a new type of enzyme takes part in this biotransformation, and that the reaction proceeds probably via the same mechanism as that in rat liver.     

Exploring the active site of amine:pyruvate aminotransferase on the basis of the substrate structure-reactivity relationship: how the enzyme controls substrate specificity and stereoselectivity

An active site model of the amine:pyruvate aminotransferase (APA) from Vibrio fluvialis JS17 was constructed on the basis of the relationship between substrate structure and reactivity. Due to the broad substrate specificity of the APA, various amino donors (chiral and achiral amine, amino acid, and amino acid derivative) and amino acceptors (keto acid, keto ester, aldehyde, and ketone) were used to explore the active site structure. The result suggested a two-binding site model consisting of two pockets, one large (L) and the other small (S). The difference in the size of each binding pocket and strong repulsion for a carboxylate in the S pocket were key determinants to control its substrate specificity and stereoselectivity. The L pocket showed dual recognition mode for both hydrophobic and carboxyl groups as observed in the side-chain pockets of aspartate aminotransferase and aromatic aminotransferase. Comparison of the model with those of other aminotransferases revealed that the L and S pockets corresponded to carboxylate trap and side-chain pocket, respectively. The active site model successfully explains the observed substrate specificity as well as the stereoselectivity of the APA.     

Characterization of G-quadruplex/hemin peroxidase: substrate specificity and inactivation kinetics

Recently, G-quadruplex/hemin (G4/hemin) complexes have been found to exhibit peroxidase activity, and this feature has been extensively exploited for colorimetric detection of various targets. To further understand and characterize this important DNAzyme, its substrate specificity, inactivation mechanism, and kinetics have been examined by comparison with horseradish peroxidase (HRP). G4/hemin DNAzyme exhibits broader substrate specificity and much higher inactivation rate than HRP because of the exposure of the catalytic hemin center. The inactivation of G4/hemin DNAzyme is mainly attributed to the degradation of hemin by H(2)O(2) rather than the destruction of G4. Both the inactivation rate and catalytic oxidation rate of G4/hemin DNAzyme depend on the concentration of H(2)O(2), which suggests that active intermediates formed by G4/hemin and H(2)O(2) are the branch point of catalysis and inactivation. Reducing substrates greatly inhibit the inactivation of G4/hemin DNAzyme by rapidly reacting with the active intermediates. A possible catalytic and inactivation process of G4/hemin has been proposed. These results imply a potential cause for the hemin-mediated cellular injury and provide insightful information for the future application of G4/hemin DNAzyme.     

Characterization of S‐thiolation on secreted proteins from E. coli by mass spectrometry

S‐thiolation is a reversible post‐translational modification in which thiol metabolites of low molecular masses are linked to protein sulfhydryl groups through disulfide bonds. This modification is commonly observed in recombinant proteins secreted from E. coli cells. Since it can alter protein functions and introduce molecular heterogeneity, S‐thiolation is undesirable for recombinant protein production. To date, few published studies have characterized thiol modifiers or investigated the mechanism of S‐thiolation in recombinant proteins. In this work, reversed‐phase liquid chromatography and mass spectrometry were used to characterize four of the most abundant thiol modifiers on recombinant proteins secreted from E. coli BL21 (DE3) strain. These thiol modifiers have been identified as glutathione, 4‐phosphopantetheine, gluconoylated glutathione, and dephosphorylated coenzyme A. S‐thiolation by these thiol modifiers increases protein mass by 305, 356, 483, and 685 Da, respectively. These specific mass increases can be used as markers for identifying S‐thiolation in recombinant proteins. Copyright © 2009 John Wiley & Sons, Ltd.     

The molecular mechanism of dicarboxylic acid transport in Escherichia coli K 12.

It is the purpose of this communication to review the properties of the dicarboxylic acid transport system in Escherichia coli K 12, in particular the role of various dicarboxylate transport proteins, and the disposition of these components in the cytoplasmic membrane. The dicarboxylate transport system is an active process and is responsible for the uptake of succinate, fumarate, and malate. Membrane vesicles prepared from the EDTA, lysozyme, and osmotic shock treatment take up the dicarboxylic acids in the presence of an electron donor. Genetic analysis of various transport mutants indicates that there is only one dicarboxylic acid transport system present in Escherichia coli K 12, and that at least 3 genes, designated cbt, dct A, and dct B, are involved in this transport system. The products corresponding to the 3 genes are: a periplasmic binding protein (PBP) specified by cbt, and 2 membrane integral proteins, SBP 1 and SBP 2, specified by dct B and dct A, respectively. Components SBP 1 and SBP 2 appear to be exposed on both the inner and outer surfaces of the membrane, and lie in close proximity to each other. The substrate recognition sites of SBP 2 and SBP 1 are exposed on the outer and inner surfaces of the membrane respectively. The data presently available suggest that dicarboxylic acids may be translocated across the membrane via a transport channel. A tentative working model on the mechanism of translocation of dicarboxylic acids across the cell envelope by the periplasmic binding protein, and the 2 membrane carrier proteins is presented.     

Antigenic specificity of Escherichia coli alkaline phosphatase studied with monoclonal antibodies: Immunological characterization of E. coli and Shigella strains

Monoclonal antibodies (MoAb) to the alkaline phosphatase of Escherichia coli were produced from spleen cells of BALB/c mice primed with purified alkaline phosphatase of E. coli and SP₂O/Ag-14 myeloma cells. Five stable clones were established. They all produced antibodies which reacted by enzyme-linked immunosorbent assay (ELISA) with alkaline phosphatase of all E. coli (25 strains) independently of their origin (drinking water, saline water, surface water, faecal or clinical origin), and with that of four Shigella species (7 strains) tested. Four of these MoAb gave a positive reaction with 52 % (MoAb 4G10), 73 % (MoAb 4F8, MoAb 4G6) and 89 % (MoAb 3C8) of 14 other bacterial species (30 strains) studied, while one (MoAb 2E5) did not react with alkaline phosphatase of these unrelated bacterial strains and thus appeared specific for E. coli and Shigella species. This MoAb was still detectable in ascitic fluids at 1/500,000 in ELISA, and detected all E. coli strains in an indirect immunofluorescence assay at 1/100. It could therefore be used as a reagent for routine detection of E. coli in drinking water, food or clinical specimens.     

微量热法测定Vitex negundo Linn对大肠杆菌抑制作用的活性部位

开发药用植物首先必须确定其活性部位. 我们采用微量热法鉴定了一种植物Vitex negundo Linn的四种不同提取物: 正己烷提取物、氯仿提取物、乙酸乙酯提取物和甲醇提取物对大肠杆菌(E. coli)的抑制作用. 结果表明甲醇提取物对大肠杆菌生长的代谢热谱抑制作用最强. 通过计算该提取物作用下大肠杆菌的生长速率常数, 表明该提取物的抑制作用随浓度的升高而增强.     

Centrifugal separation in the recovery of intracellular protein from E. coli

A 1000 l fermenter has been used to produce a feed of E. coli containing high levels of β-galactosidase. The individual unit operations have been investigated for the primary recovery of the enzyme, i.e. cell harvesting, cell disruption and cell debris removal. The cell separation yield was found to be in excess of 99%. Four passes through a high-pressure homogenizer released 95% of the enzyme. Approximately 87% of the β-galactosidase could be recovered after cell debris removal. The performance of the centrifuge during solid-liquid separation could be modelled using simple curve-fitting techniques.     

Physicochemical properties and substrate specificity of protease C from dormant cotton seeds

The substrate specificity of a proteolytic enzyme — protease C — isolated from cotton seeds has been studied. The activity of protease C is suppressed completely under the action of diisopropyl phosphorofluoridate. Protein inhibitors — duck ovomucoid, soybean inhibitor, and also TPCK — suppressed the activity of protease C to different degrees. On the basis of results obtained in the hydrolysis of the cottonseed reserve proteins, 7S and 11S globulins, and the B chain of insulin, protease C has been assigned to the serine group of endopeptidases. The optimum conditions — pH, time, and temperature — at which protease C exhibits its maximum activity has been determined.Institute of Chemistry of Plant Substances, Uzbek SSR Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 744–746, September–October, 1988.     

Insights into the role of Val45 and Gln182 of Escherichia coli MutY in DNA substrate binding and specificity

Background  

Escherichia coli MutY (EcMutY) reduces mutagenesis by removing adenines paired with guanines or 7,8-dihydro-8-oxo-guanines (8-oxoG). V45 and Q182 of EcMutY are considered to be the key determinants of adenine specificity. Both residues are spatially close to each other in the active site and are conserved in MutY family proteins but not in Methanobacterium thermoautotrophicum Mig.MthI T/G mismatch DNA glycosylase (A50 and L187 at the corresponding respective positions).     

Broad substrate specificity of the amide synthase in S. hygroscopicus--new 20-membered macrolactones derived from geldanamycin

The amide synthase of the geldanamycin producer, Streptomyces hygroscopicus, shows a broader chemoselectivity than the corresponding amide synthase present in Actinosynnema pretiosum, the producer of the highly cytotoxic ansamycin antibiotics, the ansamitocins. This was demonstrated when blocked mutants of both strains incapable of biosynthesizing 3-amino-5-hydroxybenzoic acid (AHBA), the polyketide synthase starter unit of both natural products, were supplemented with 3-amino-5-hydroxymethylbenzoic acid instead. Unlike the ansamitocin producer A. pretiosum, S. hygroscopicus processed this modified starter unit not only to the expected 19-membered macrolactams but also to ring enlarged 20-membered macrolactones. The former mutaproducts revealed the sequence of transformations catalyzed by the post-PKS tailoring enzymes in geldanamycin biosynthesis. The unprecedented formation of the macrolactones together with molecular modeling studies shed light on the mode of action of the amide synthase responsible for macrocyclization. Obviously, the 3-hydroxymethyl substituent shows similar reactivity and accessibility toward C-1 of the seco-acid as the arylamino group, while phenolic hydroxyl groups lack this propensity to act as nucleophiles in the macrocyclization. The promiscuity of the amide synthase of S. hygroscopicus was further demonstrated by successful feeding of four other m-hydroxymethylbenzoic acids, leading to formation of the expected 20-membered macrocycles. Good to moderate antiproliferative activities were encountered for three of the five new geldanamycin derivatives, which matched well with a competition assay for Hsp90α.     

Expression of Shiga Toxin B Subunit at Cell Surface in E. coli K-12

The three parts(Stx17B, Stx27B and StxB) of Shiga toxin B subunit have been fused into a cell surface exposed loop of the LamB protein at a BamH I site between residues 153 and 154. Western blotting revealed that the three parts of Shiga toxin B subunit could be expressed as the Lamb fusion proteins in E. coli. Indirect immunofluorescence and immunoelectron microscopy analyses showed fusion proteins LamB/Stx17B and LamB/Stx27B could be expressed at cell surface in E. coli, but fusion protein LamB/StxB could not be expressed at cell surface; it was aggregated in cytoplasm and was toxic to host. This expression system provided a new way to construct an oral live vaccine against Shigella dysenteriae 1.     

The substrate specificity of the heat-stable stereospecific amidase from Klebsiella oxytoca

The substrate specificity of the heat-stable stereospecific amidase from Klebsiella oxytoca was investigated. In addition to the original substrate, 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide, the amidase accepted 2-hydroxy-2-(trifluoromethyl)-butanamide and 3,3,3-trifluoro-2-amino-2-methylpropanamide as substrates. Compounds with larger side chains and compounds where the hydroxyl group was substituted with a methoxy group, or in which the CF₃ group was substituted by CCl₃, were not accepted. The biotransformation is a new synthetic route to (R)-(+)-3,3,3-trifluoro-2-amino-2-methylpropanoic acid, and its related (S)-(−)-amide, and to (R)-(+)-2-hydroxy-2-(trifluoromethyl)-butanoic acid and its related (S)-(−)-amide.