Exploration of the six tryptophan residues of Escherichia coli cystathionine β-lyase as probes of enzyme conformational change

Cystathionine β-lyase (CBL) catalyzes the hydrolysis of l-cystathionine (l-Cth), producing l-homocysteine (l-Hcys), pyruvate and ammonia, in the second step of the transsulfuration pathway of bacteria and plants. A series of 17 site-directed variants of Escherichia coli CBL (eCBL) was constructed to probe the contributions of the six tryptophan residues (W131, W188, W230, W276, W300 and W340) to the fluorescence spectrum of eCBL and to assess their mutability and utility as conformational probes. The effects of these Trp → Phe substitutions on k_cat and K_m^l-Cth are less than 2-fold, with the exception of the 8-fold increase in K_m^l-Cth observed for eCBL-W340F. The midpoint of thermal denaturation, as monitored by circular dichroism spectroscopy, is reduced 4.7 °C by the W188F substitution while the targeted replacement of the other five tryptophans alter T_m by less than 1.7 °C. The fluorescence spectrum of eCBL is dominated by W230 and the contribution of W340, situated in the active site, is minor. The observed 5-fold increase in the 336 nm fluorescence emission of W188 between 0 and 2 M urea, suggests a conformational change at the domain interface. Residues W188 and W340, conserved in proteobacterial CBL enzymes, are situated at the core of the domain interface that forms the active-site cleft. The results of this study suggest that W188 is a useful probe of subtle conformational changes at the domain interface and active site.     

Characterization of the S289A,D mutants of yeast cystathionine β-synthase

Cystathionine β-synthase (CBS) catalyzes the pyridoxal 5′-phosphate (PLP)-dependent condensation of l-serine and l-homocysteine to form l-cystathionine in the first step of the reverse transsulfuration pathway. Residue S289 of yeast CBS, predicted to form a hydrogen bond with the pyridine nitrogen of the PLP cofactor, was mutated to alanine and aspartate. The k_cat/K_m^l-Ser of the S289A mutant is reduced by a factor of ～ 800 and the β-replacement activity of the S289D mutant is undetectable. Fluorescence energy transfer between tryptophan residue(s) of the enzyme and the PLP cofactor, observed in the wild-type enzyme and diminished in the S289A mutant, is absent in S289D. These results demonstrate that residue S289 is essential in maintaining the properties and orientation of the pyridine ring of the PLP cofactor. The reduction in activity of ytCBS-S289A suggests that ytCBS catalyzes the α,β-elimination of l-Ser via an E1cB mechanism.     

Cystathionine β-synthase and cystathionine γ-lyase in tissues of the nemertean Cerebratulus marginatus Renier, 1804 (nemertea)

Using immunohistochemistry, we examined the distribution of the hydrogen sulfide (H₂S) synthesizing enzymes cystathionine β-synthase (CBS) and cystathionine Γ-lyase (CSE) in tissues of the nemertean Cerebratulus marginatus Renier, 1804 (Heteronemertea: Lineidae). The expression of both CBS and CSE was found in the body wall and in the receptor cells of the canals of the cerebral organs; the expression of CBS was found in the foregut and intestine; and CSE was found in the brain, lateral nerve cords, and cerebral organs. The peculiarities of the distribution and the possible functional role of H₂S-synthesizing elements in nemerteans are discussed.     

Expression in E. coli and purification of the fibrillogenic fusion proteins TTR-sfGFP and β2M-sfGFP

The possibility of obtaining recombinant fibrillogenic fusion proteins such as transthyretin (TTR) and β2-microglobulin (β2M) with a superfolder green fluorescent protein (sfGFP) was studied. According to the literature data, sfGFP is resistant to denaturating influences, does not aggregate during renaturation, possesses improved kinetic characteristics of folding, and folds well when fused to different polypeptides. The corresponding DNA constructs for expression in Escherichia coli were created. It could be shown that during expression of these constructs in E. coli, soluble forms of the fusion proteins are synthesized. Efficient isolation of the fusion proteins was performed with the help of nickel-affinity chromatography. For this purpose a polyhistidine sequence (6-His-tag) was incorporated into the C-terminus of the sfGFP. We could show that the purified fusion proteins contained full-size sequences of the most amyloidogenic TTR variant, TTR(L55P) and β2M, and also sfGFP possessing fluorescent properties. In the course of fibrillogenesis both fusion proteins demonstrated their ability to form fibrils that were clearly detectable by atomic force microscopy. Furthermore, with the help of confocal microscopy we were able to reveal structures (exhibiting fluorescence) that are formed during fibrillogenesis. Thus, the use of sfGFP has made it possible to avoid formation of inclusion bodies (IB) during the synthesis of recombinant fusion proteins and to obtain soluble forms of TTR(L55P) and β2M that are suitable for further studies.     

Integration, stability and expression of the E. coli phytase transgene in the Cassie line of Yorkshire Enviropig™

The genomic structure and generational stability of the transgene carried by the Cassie (CA) line of the transgenic Enviropig?, a prospective food animal, are reported here. This transgene is composed of the Escherichia coli phytase coding sequence regulated by the mouse parotid secretory protein promoter to direct secretion of phytase in the saliva. In the CA line the transgene integrated in chromosome 4 is present as a concatemer of three copies, two in a head to tail orientation and the third in a reverse orientation 3′ to the other copies with a 6 kbp deletion in the 5′ promoter region. The overall size of the integrated transgene complex is 46 kbp. During integration a 66 kbp segment of the chromosome was deleted, but a BLAST search of the segment from a GenBank clone did not reveal any essential genes. The transgene integration site was stable through 9 generations analyzed. Phytase activity in the saliva was similar among 11 day old hemizygous boars and gilts and remained relatively constant through nine generations of hemizygous pigs. However, as the pigs grew there generally was a gradual decrease in activity that stabilized when pigs reached the finisher phase of growth (4–6 months old). Homozygous pigs exhibited 1.5 fold higher phytase activity (P < 0.0001) than that of hemizygous littermates. Moreover, no differential salivary phytase activity was seen in hemizygotes arising from CA-Yorkshire and CA-Duroc breed outcrosses, suggesting that expression of the transgene is unaffected by genetic background. This data demonstrates that an exogenous phytase gene can be stably transmitted and expressed in the salivary glands of a domestic food animal.     

Nitroxide spin labels as EPR reporters of the relaxation and magnetic properties of the heme–copper site in cytochrome bo 3, E. coli

A nitroxide spin label (SL) has been used to probe the electron spin relaxation times and the magnetic states of the oxygen-binding heme–copper dinuclear site in Escherichia coli cytochrome bo _3, a quinol oxidase (QO), in different oxidation states. The spin lattice relaxation times, T ₁, of the SL are enhanced by the paramagnetic metal sites in QO and hence show a strong dependence on the oxidation state of the latter. A new, general form of equations and a computer simulation program have been developed for the calculation of relaxation enhancement by an arbitrary fast relaxing spin system of S ≥ 1/2. This has allowed us to obtain an accurate estimate of the transverse relaxation time, T ₂, of the dinuclear coupled pair Fe(III)–Cu_B(II) in the oxidized form of QO that is too short to measure directly. In the case of the F′ state, the relaxation properties of the heme–copper center have been shown to be consistent with a ferryl [Fe(IV)=O] heme and Cu_B(II) coupled by approximately 1.5–3 cm⁻¹ to a radical. The magnitude suggests that the coupling arises from a radical form of the covalently linked tyrosine–histidine ligand to Cu(II) with unpaired spin density primarily on the tyrosine component. This work demonstrates that nitroxide SLs are potentially valuable tools to probe both the relaxation and the magnetic properties of multinuclear high-spin paramagnetic active sites in proteins that are otherwise not accessible from direct EPR measurements.     

Molecular Characterization of CTX-M β-Lactamase and Associated Addiction Systems in Escherichia coli Circulating among Cattle,Farm Workers,and the Farm Environment

A total of 84 extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates from cattle, farm workers, and the farm environment isolated from February to September 2008 in the Republic of Korea were investigated. All 84 ESBL-producing isolates carried bla_CTX-M genes that belonged to the CTX-M-1 (n = 35) or CTX-M-9 (n = 49) family. The most predominant CTX-M type identified was CTX-M-14 (n = 49), followed by CTX-M-32 (n = 26). The bla_CTX-M genes were identified most commonly in E. coli isolates from feces (n = 29), teats (n = 25), and milk (n = 14). A bla_CTX-M-14 gene was also detected in an E. coli isolate from a farmer''s hand. Transfer of the bla_CTX-M gene from 60 bla_CTX-M-positive E. coli isolates to the recipient E. coli J53 strain by conjugation was demonstrated. Plasmid isolation from bla_CTX-M-positive transconjugants revealed a large (95- to 140-kb) conjugative plasmid. Almost all (82/84) bla_CTX-M genes possessed an insertion sequence, ISEcp1, upstream of the bla_CTX-M gene. Only in the case of the CTX-M-14 genes was IS903 downstream of the gene. The bla_CTX-M genes were associated with seven kinds of addiction systems. Among them, pndAC, hok-sok, and srnBC were the most frequently identified addiction systems in both wild strains and transconjugants. The spread of bla_CTX-M genes was attributed to both clonal expansion and horizontal dissemination. Our data suggest that a combination of multiple addiction systems in plasmids carrying bla_CTX-M genes could contribute to their maintenance in the host cells. To our knowledge, the bla_CTX-M-32 gene has not previously been reported in animal isolates from the Republic of Korea.     

Expression in E. coli and tissue distribution of the human homologue of the mouse Ke 6 gene, 17β-hydroxysteroid dehydrogenase type 8

Expression of the human Ke 6 gene, 17β-hydroxysteroid dehydrogenase type 8, in E. coli and the substrate specificity of the expressed protein were examined. The tissue distribution of mRNA expression of the human Ke 6 gene was also studied using real-time PCR. Human Ke 6 gene was expressed as an enzymatically-active His-tag fusion protein, whose molecular weight was estimated to be 32.5 kDa by SDS-polyacrylamide gel electrophoresis. Expressed human Ke 6 gene effectively catalyzed the conversion of estradiol into estrone. Testosterone, 5α-dihydrotestosterone, and 5-androstene-3β,17β-diol were also catalyzed into the corresponding 17-ketosteroid at 2.4–5.9% that of estradiol oxidation. Furthermore, expressed enzyme catalyzed the reduction of estrone to estradiol, but the rate was a mere 2.3%. Human Ke 6 gene mRNA was expressed in the various tissues examined, such as brain, cerebellum, heart, lung, kidney, liver, small intestine, ovary, testis, adrenals, placenta, prostate, and stomach. Expression of human Ke 6 gene mRNA was especially abundant in prostate, placenta, and kidney. The levels in prostate and placenta were higher than that in kidney, where it is known to be expressed in large quantities.     

Use of Lead(II) to Probe the Structure of Large RNA's. Conformation of the 3′ Terminal Domain of E. coli 16S rRNA and its Involvement in Building the tRNA Binding Sites

Abstract

The present work shows that lead(II) can be used as a convenient structure probe to map the conformation of large RNA's and to follow discrete conformational changes at different functional states. We have investigated the conformation of the 3′ domain of the E. coli 16S rRNA (nucleotides 1295–1542) in its naked form, in the 30S subunit and in the 70S ribosome. Our study clearly shows a preferential affinity of Pb(II) for interhelical and loop regions and suggests a high sensitivity for dynamic and flexible regions. Within 30S subunits, some cleavages are strongly decreased as the result of protein-induced protection, while others are enhanced suggesting local conformational ajustments. These rearrangements occur at functionally strategic regions of the RNA centered around nucleotides 1337,1400,1500 and near the 3′ end of the RNA The association of 30S and 50S subunits causes further protections at several nucleotides and some enhanced reactivities that can be interpreted in terms of subunits interface and allosteric transitions. The binding of E. coli tRNA-Phe to the 70S ribosome results in message-independent (positions 1337 and 1397) and message-dependent (1399–1400, 1491–1492 and 1505) protections. Athird class ofprotection(1344–1345,1393–1395,1403–1409,1412–1414, 1504, 1506–1507 and 1517–1519) is observed in message-directed 30S subunits, which are induced by both tRNA binding and 50S subunit association. This extensive reduction of reactivity most probably reflects an allosteric transition rather than a direct shielding.     

Molecular cloning,nucleotide sequence and expression in Escherichia coli of the β-cyclodextrin glycosyltransferase gene from Bacillus circulans strain no. 8

Summary The -cyclodextrin glycosyltransferase (-CGTase) gene was isolated from a -library prepared from Bacillus circulans strain no. 8. It was subcloned into plasmid pTZ and expressed by its endogenous regulatory sequences in Escherichia coli JM 103. The structural gene was sequenced and showed an open reading frame for a polypeptide of 718 amino acid residues. The recombinant -CGTase had the same enzymatic properties as the extracellular CGTase (684 amino acid residues, corresponding to a mol. wt. of 74416) produced by B. circulans strain no. 8. The amino acid sequence showed the highest homology (74.6% identical amino acids) with the CGTase of B. circulans strain F-2, which had been erroneously described as an amylase. The homology with the enzyme from the alkalophilic Bacillus sp. strain no. 1011 was 71.4%. The amino acid sequence derived will be used for elucidating the three-dimensional structure of the enzyme. Offprint requests to: H. Bender     

Bryophyte flora of the Sierras de Filabres,Cabrera, Alhamilla and Cabo de Gata (Almería,S.E. Spain)

Abstract

The bryophyte flora of the Sierras de Filabres, Cabrera, Alhamilla and Cabo de Gata of Almería province (S.E. Spain) includes 280 taxa (236 mosses and 44 liverworts) of which 74 are new to the province of Almería, 31 to the southeastern part of the Iberian Peninsula and 4 to the Iberian Peninsula. Data about their biogeography and ecology are given.     

Characterization of a new 4BS.7HL wheat-barley translocation line using GISH, FISH, and SSR markers and its effect on the β-glucan content of wheat

A spontaneous interspecific Robertsonian translocation was revealed by genomic in situ hybridization (GISH) in the progenies of a monosomic 7H addition line originating from a new wheat 'Asakaze komugi' × barley 'Manas' hybrid. Fluorescence in situ hybridization (FISH) with repetitive DNA sequences (Afa family, pSc119.2, and pTa71) allowed identification of all wheat chromosomes, including wheat chromosome arm 4BS involved in the translocation. FISH using barley telomere- and centromere-specific repetitive DNA probes (HvT01 and (AGGGAG)(n)) confirmed that one of the arms of barley chromosome 7H was involved in the translocation. Simple sequence repeat (SSR) markers specific to the long (L) and short (S) arms of barley chromosome 7H identified the translocated chromosome segment as 7HL. Further analysis of the translocation chromosome clarified the physical position of genetically mapped SSRs within 7H, with a special focus on its centromeric region. The presence of the HvCslF6 gene, responsible for (1,3;1,4)-β-D-glucan production, was revealed in the centromeric region of 7HL. An increased (1,3;1,4)-β-D-glucan level was also detected in the translocation line, demonstrating that the HvCslF6 gene is of potential relevance for the manipulation of wheat (1,3;1,4)-β-D-glucan levels.     

Identification and Characterization of a Mucilaginibacter sp. Strain QM49 β-Glucosidase and Its Use in the Production of the Pharmaceutically Active Minor Ginsenosides (S)-Rh1 and (S)-Rg2

Here, we isolated and characterized a new ginsenoside-transforming β-glucosidase (BglQM) from Mucilaginibacter sp. strain QM49 that shows biotransformation activity for various major ginsenosides. The gene responsible for this activity, bglQM, consists of 2,346 bp and is predicted to encode 781 amino acid residues. This enzyme has a molecular mass of 85.6 kDa. Sequence analysis of BglQM revealed that it could be classified into glycoside hydrolase family 3. The enzyme was overexpressed in Escherichia coli BL21(DE3) using a maltose binding protein (MBP)-fused pMAL-c2x vector system containing the tobacco etch virus (TEV) proteolytic cleavage site. Overexpressed recombinant BglQM could efficiently transform the protopanaxatriol-type ginsenosides Re and Rg₁ into (S)-Rg₂ and (S)-Rh₁, respectively, by hydrolyzing one glucose moiety attached to the C-20 position at pH 8.0 and 30°C. The K_m values for p-nitrophenyl-β-d-glucopyranoside, Re, and Rg₁ were 37.0 ± 0.4 μM and 3.22 ± 0.15 and 1.48 ± 0.09 mM, respectively, and the V_max values were 33.4 ± 0.6 μmol min⁻¹ mg⁻¹ of protein and 19.2 ± 0.2 and 28.8 ± 0.27 nmol min⁻¹ mg⁻¹ of protein, respectively. A crude protopanaxatriol-type ginsenoside mixture (PPTGM) was treated with BglQM, followed by silica column purification, to produce (S)-Rh₁ and (S)-Rg₂ at chromatographic purities of 98% ± 0.5% and 97% ± 1.2%, respectively. This is the first report of gram-scale production of (S)-Rh₁ and (S)-Rg₂ from PPTGM using a novel ginsenoside-transforming β-glucosidase of glycoside hydrolase family 3.