Cloning, overexpression, purification, and characterization of S-adenosylhomocysteine hydrolase from Corynebacterium efficiens YS-314

The gene encoding S-adenosylhomocysteine hydrolase activity (SAHase: EC 3.3.1.1) from Corynebacterium efficiens (YS-314) was cloned and expressed as a fusion protein in Escherichia coli Rosetta (DE3). The analyzed nucleotide sequence of the cloned gene proved to be identical to those reported on the NCBI database. The recombinant enzyme is a tetramer, showing a molecular weight of approximately 210 kDa, as estimated by gel filtration. The K(M) values of the enzyme for S-adenosylhomocysteine (SAH), adenosine (Ado), and homocysteine (Hcy), were determined to be 1.4, 10, and 45 microM. The overexpression of the recombinant enzyme produced a high level of protein (>40 mg of protein per gram of wet cells) and revealed certain thermostability when characterized at temperatures above 40 degrees C. It also showed a high capacity for the synthesis of SAH, thermal stability, and high kinetic similarity to human SAHase, indicating a high biotechnological and pharmacological potential.     

Pyruvate:quinone oxidoreductase from Corynebacterium glutamicum: purification and biochemical characterization

Pyruvate:quinone oxidoreductase catalyzes the oxidative decarboxylation of pyruvate to acetate and CO2 with a quinone as the physiological electron acceptor. So far, this enzyme activity has been found only in Escherichia coli. Using 2,6-dichloroindophenol as an artificial electron acceptor, we detected pyruvate:quinone oxidoreductase activity in cell extracts of the amino acid producer Corynebacterium glutamicum. The activity was highest (0.055 +/- 0.005 U/mg of protein) in cells grown on complex medium and about threefold lower when the cells were grown on medium containing glucose, pyruvate, or acetate as the carbon source. From wild-type C. glutamicum, the pyruvate:quinone oxidoreductase was purified about 180-fold to homogeneity in four steps and subjected to biochemical analysis. The enzyme is a flavoprotein, has a molecular mass of about 232 kDa, and consists of four identical subunits of about 62 kDa. It was activated by Triton X-100, phosphatidylglycerol, and dipalmitoyl-phosphatidylglycerol, and the substrates were pyruvate (kcat=37.8 +/- 3 s(-1); Km=30 +/- 3 mM) and 2-oxobutyrate (kcat=33.2 +/- 3 s(-1); Km=90 +/- 8 mM). Thiamine pyrophosphate (Km=1 microM) and certain divalent metal ions such as Mg2+ (Km=29 microM), Mn2+ (Km=2 microM), and Co2+ (Km=11 microM) served as cofactors. In addition to several dyes (2,6-dichloroindophenol, p-iodonitrotetrazolium violet, and nitroblue tetrazolium), menadione (Km=106 microM) was efficiently reduced by the purified pyruvate:quinone oxidoreductase, indicating that a naphthoquinone may be the physiological electron acceptor of this enzyme in C. glutamicum.     

谷氨酸棒杆菌过氧化氢酶的异源表达、纯化以及酶学性质

过氧化氢酶能催化过氧化氢分解为水和氧气,在工业上有着较为广泛的应用。然而,纺织和造纸工业的特殊的高碱性环境,使得开发碱性过氧化氢酶有着重要的应用价值。利用大肠杆菌表达来自于谷氨酸棒杆菌的过氧化氢酶,对其表达条件进行了优化,并通过镍柱亲和层析的方法分离纯化重组蛋白,然后表征纯酶的酶学性质。最适表达条件为:诱导剂IPTG浓度0.2 mmol/L,诱导温度25℃,诱导时间11 h。过氧化氢酶比酶活达到55 266 U/mg,具有较高的催化活性。该酶具有相当宽泛的p H值适应范围,在p H 4.0–11.5范围内均具有较高的酶活性,并在p H 11.0条件下表现出最高的酶活性。将纯酶在p H 11.0的溶液中处理3 h时剩余酶活为93%,说明该酶在高碱条件下有良好的稳定性。该酶最适温度为30℃,在25–50℃热稳定性较好。其动力学参数Km为25.89mmol/L,Vmax为185.18mmol/(min?mg)。抑制剂十二烷基硫酸钠(SDS)、尿素、Na N3、β-巯基乙醇、EDTA对酶活有不同程度的抑制作用。来源于谷氨酸棒杆菌的过氧化氢酶具有较高的催化效率、良好的碱耐受性,在工业生产中有较好...     

Overexpression, purification, and characterization of S-adenosylhomocysteine hydrolase from Leishmania donovani

The gene encoding S-adenosylhomocysteine (AdoHcy) hydrolase in Leishmania donovani was subcloned into an expression vector (pPROK-1) and expressed in Escherichia coli. Recombinant L. donovani AdoHcy hydrolase was then purified from cell-free extracts of E. coli using three chromatographic steps (DEAE-cellulose chromatofocusing, Sephacryl S-300 gel filtration, and Q-Sepharose ion exchange). The purified recombinant L. donovani enzyme exists as a tetramer with a molecular weight of approximately 48 kDa for each subunit. Unlike recombinant human AdoHcy hydrolase, the catalytic activity of the recombinant L. donovani enzyme was shown to be dependent on the concentration of NAD+ in the incubation medium. The dissociation constant (Kd) for NAD+ with the L. donovani enzyme was estimated to be 2.1 +/- 0.2 microM. The Km values for the natural substrates of the enzyme, AdoHcy, Ado, and Hcy, were determined to be 21 +/- 3, 8 +/- 2, and 82 +/- 5 microM, respectively. Several nucleosides and carbocyclic nucleosides were tested for their inhibitory effects on this parasitic enzyme, and the results suggested that L. donovani AdoHcy hydrolase has structural requirements for binding inhibitors different than those of the human enzyme. Thus, it may be possible to eventually exploit these differences to design specific inhibitors of this parasitic enzyme as potential antiparasitic agents.     

Partial purification and characterization of S-adenosylhomocysteine hydrolase isolated from Saccharomyces cerevisiae

S-Adenosylhomocysteine (SAH) hydrolase was purified 25-fold from bakers' yeast by chemical methods and column chromatography. The purified enzyme could readily synthesize SAH from adenosine and homocysteine, but could hydrolyze only negligible amounts of SAH. The purified enzyme showed no activity towards S-adenosylmethionine, methylthioadenosine, or adenosine. Several nucleotides, sulfhydryl compounds, and ribose could not replace adenosine or homocysteine in the reaction mixture. SAH could be hydrolyzed by SAH hydrolase if commercial adenosine deaminase was included in the reaction mixture. Under these conditions l-homocysteine could act as a product inhibitor. A number of compounds structurally similar to adenosine and homocysteine were found to inhibit synthesis of SAH from adenosine and homocysteine. The strongest inhibitors were adenine, adenosine-3'-monophosphate, adenosine-2'-monophosphate, adenosine diphosphate, adenosine triphosphate, and adenosine-5'-monophosphate. The biosynthetic and hydrolytic activity of SAH hydrolase in yeast cell ghosts was similar to the activity of the enzyme in vitro.     

Trypanosoma cruzi: molecular cloning and characterization of the S-adenosylhomocysteine hydrolase

S-Adenosylhomocysteine (AdoHcy) hydrolase has emerged as an attractive target for antiparasitic drug design because of its role in the regulation of all S-adenosylmethionine-dependent transmethylation reactions, including those reactions crucial for parasite replication. From a genomic DNA library of Trypanosoma cruzi, we have isolated a gene that encodes a polypeptide containing a highly conserved AdoHcy hydrolase consensus sequence. The recombinant T. cruzi enzyme was overexpressed in Escherichia coli and purified as a homotetramer. At pH 7.2 and 37 degrees C, the purified enzyme hydrolyzes AdoHcy to adenosine and homocysteine with a first-order rate constant of 1 s(-1) and synthesizes AdoHcy from adenosine and homocysteine with a pseudo-first-order rate constant of 3 s(-1) in the presence of 1 mM homocysteine. The reversible catalysis depends on the binding of NAD(+) to the enzyme. In spite of the significant structural homology between the parasitic and human AdoHcy hydrolase, the K(d) of 1.3 microM for NAD(+) binding to the T. cruzi enzyme is approximately 11-fold higher than the K(d) (0.12 microM) for NAD(+) binding to the human enzyme.     

Expression, purification, and characterization of recombinant S-adenosylhomocysteine hydrolase from the thermophilic archaeon Sulfolobus solfataricus

S-Adenosylhomocysteine hydrolase from Sulfolobus solfataricus was expressed in Escherichia coli by inserting the genomic fragment containing the gene encoding for S-adenosylhomocysteine hydrolase downstream the isopropyl-beta-d-thiogalactoside-inducible promoter of pTrc99A expression vector. An ATG positioned 25 bp upstream of the gene which is in frame with a stop codon was utilized as the initiation codon. This construct was used to transform E. coli RB791 and E. coli JM105 strains. The recombinant protein, purified by a fast and efficient two-step procedure (yield of 0.4 mg of enzyme per gram of cells), does not appear homogeneous on SDS-PAGE because of the presence of a protein contaminant corresponding to a "truncated" S-adenosylhomocysteine hydrolase subunit lacking the first 24 amino acid residues. The recombinant enzyme shows the same molecular mass, optimum temperature, and kinetic features of S-adenosylhomocysteine hydrolase isolated from S. solfataricus but it is less thermostable. To construct a vector which presents a correct distance between the ribosome-binding site and the start codon of S-adenosylhomocysteine hydrolase gene, a NcoI site was created at the translation initiation codon using site-directed mutagenesis. The expression of the homogeneous mutant S-adenosylhomocysteine hydrolase was achieved at high level (1.7 mg of mutant protein per gram of cells). The mutant S-adenosylhomocysteine hydrolase and the native one were indistinguishable in all physicochemical and kinetic properties including thermostability, indicating that the interactions involving the NH(2)-terminal sequence of the protein play a role in the thermal stability of S. solfataricus S-adenosylhomocysteine hydrolase.     

Molecular and biochemical characterization of mechanosensitive channels in Corynebacterium glutamicum

Database searches in the Corynebacterium glutamicum genome sequence revealed homologs of the mechanosensitive channels MscL and YggB of Escherichia coli. To elucidate the physiological role of these putative channels deletion mutants were constructed. Betaine efflux induced by osmotic downshock of the mscL deletion mutant was nearly identical to that of the wild-type, whereas the yggB deletion mutant showed a reduced efflux rate. Interestingly, the double deletion strain, which was expected to have an even more decreased capability of betaine excretion, had only a slightly reduced efflux rate compared to the wild-type and did not show an increased mortality after osmotic downshift. These results led to the hypothesis that C. glutamicum may possess a third type of mechanosensitive channel not related to the MscL and YggB/KefA families. Furthermore it is unlikely that an MscM-like activity is responsible for the betaine efflux, because of the high transport capacity detected in the double deletion mutant.     

Characterization and partial purification of L-asparaginase from Corynebacterium glutamicum

A high L-asparaginase (L-asparagine amidohydrolase: EC 3.5.1.1) activity was found under conditions of lysine overproduction in cultures of Corynebacterium glutamicum. L-Asparaginase was purified 98-fold by protamine sulphate precipitation. DEAE-Sephacel anion exchange, ammonium sulphate precipitation and Sephacryl S-200 gel filtration. The asparaginase protein was subjected to PAGE under non-denaturing conditions, identified by an in situ reaction and eluted from the gel in an active form. The estimated Mr from gel filtration and SDS-PAGE was 80,000. The L-asparaginase activity was inhibited by the L-asparagine analogue 5-diazo-4-oxo-L-norvaline. Neither D-asparagine nor L-glutamine was a substrate for the enzyme. L-Asparaginase was produced constitutively: its role may be that of an overflow enzyme, converting excess asparagine into aspartic acid, the direct precursor of lysine and threonine.     

Expression,purification, and crystallization of meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum

The gene encoding the meso-diaminopimelate dehydrogenase (DAPDH) from Corynebacterium glutamicum was overexpressed and purified to homogeneity. Crystals of the binary DAPDH-NADP⁺ complex were obtained from solutions of polyethylene glycol 8000, 100 mM sodium cacodylate, pH 6.5, and 150–300 mM Mg(OAc)₂. The crystals diffract to 2.2 Å, belong to the orthorhombic space group P2₁, and contain two molecules per asymmetric unit. © 1996 Wiley-Liss, Inc.     

Temperature-sensitive cloning vector for Corynebacterium glutamicum

We constructed a temperature-sensitive form of the Corynebacterium glutamicum ATCC13869 cryptic plasmid, pBL1. The C. glutamicum/Escherichia coli shuttle vector pSFK6, which is composed of pBL1 and the E. coli cloning vector pK1, was mutagenized in vitro by treatment with hydroxylamine, and introduced into C. glutamicum cells. A mutant plasmid, which was stably maintained at 25 degrees C but not at 34 degrees C, was isolated from the cells. Sequencing the plasmid, which was named p48K, revealed four substitutions in the Rep protein coding region. Moreover, site-directed single-nucleotide substitutions showed that a G to A transition at position 2,920, which resulted in a Pro-47 to Ser substitution in the Rep protein, was responsible for its temperature-sensitive replication. Pro-47 is conserved among the Rep proteins of the pIJ101/pJV1 family of plasmids. This temperature-sensitive cloning vector will be useful for disrupting genes in this industrially important bacterium.     

Purification and characterization of fumarase from Corynebacterium glutamicum

Fumarase (EC 4.2.1.2) from Corynebacterium glutamicum (Brevibacterium flavum) ATCC 14067 was purified to homogeneity. Its amino-terminal sequence (residues 1 to 30) corresponded to the sequence (residues 6 to 35) of the deduced product of the fumarase gene of C. glutamicum (GenBank accession no. BAB98403). The molecular mass of the native enzyme was 200 kDa. The protein was a homotetramer, with a 50-kDa subunit molecular mass. The homotetrameric and stable properties indicated that the enzyme belongs to a family of Class II fumarase. Equilibrium constants (K(eq)) for the enzyme reaction were determined at pH 6.0, 7.0, and 8.0, resulting in K(eq)=6.4, 6.1, and 4.6 respectively in phosphate buffer and in 16, 19, and 17 in non-phosphate buffers. Among the amino acids and nucleotides tested, ATP inhibited the enzyme competitively, or in mixed-type, depending on the buffer. Substrate analogs, meso-tartrate, D-tartrate, and pyromellitate, inhibited the enzyme competitively, and D-malate in mixed-type.     

Genetic and biochemical analysis of the aspartokinase from Corynebacterium glutamicum

The lysC/asd gene cluster of Corynebacterium glutamicum ATCC 13032 was cloned and sequenced. The lysC locus coding for aspartokinase consists of two in-frame overlapping genes, lysC alpha encoding a protein of 421 amino acids (Mr 44,300) and lysC beta encoding a protein of 172 amino acids (Mr 18,600). The C. glutamicum aspartokinase was purified and found to contain two proteins of Mr 47,000 and Mr 18,000. A C. glutamicum mutant expressing a feedback-resistant aspartokinase was shown to be changed in a single base pair of the lysC beta gene, leading to an amino acid exchange in the beta-subunit of the aspartokinase. In addition, the identified mutation was found to be responsible for the enhanced expression of the asd gene located downstream of lysC.     

S-adenosylhomocysteine hydrolase from the archaeon Pyrococcus furiosus: biochemical characterization and analysis of protein structure by comparative molecular modeling

S-adenosylhomocysteine hydrolase (AdoHcyHD) is an ubiquitous enzyme that catalyzes the breakdown of S-adenosylhomocysteine, a powerful inhibitor of most transmethylation reactions, to adenosine and L-homocysteine. AdoHcyHD from the hyperthermophilic archaeon Pyrococcus furiosus (PfAdoHcyHD) was cloned, expressed in Escherichia coli, and purified. The enzyme is thermoactive with an optimum temperature of 95 degrees C, and thermostable retaining 100% residual activity after 1 h at 90 degrees C and showing an apparent melting temperature of 98 degrees C. The enzyme is a homotetramer of 190 kDa and contains four cysteine residues per subunit. Thiol groups are not involved in the catalytic process whereas disulfide bond(s) could be present since incubation with 0.8 M dithiothreitol reduces enzyme activity. Multiple sequence alignment of hyperthermophilic AdoHcyHD reveals the presence of two cysteine residues in the N-terminus of the enzyme conserved only in members of Pyrococcus species, and shows that hyperthermophilic AdoHcyHD lack eight C-terminal residues, thought to be important for structural and functional properties of the eukaryotic enzyme. The homology-modeled structure of PfAdoHcyHD shows that Trp220, Tyr181, Tyr184, and Leu185 of each subunit and Ile244 from a different subunit form a network of hydrophobic and aromatic interactions in the central channel formed at the subunits interface. These contacts partially replace the interactions of the C-terminal tail of the eukaryotic enzyme required for tetramer stability. Moreover, Cys221 and Lys245 substitute for Thr430 and Lys426, respectively, of the human enzyme in NAD-binding. Interestingly, all these residues are fairly well conserved in hyperthermophilic AdoHcyHDs but not in mesophilic ones, thus suggesting a common adaptation mechanism at high temperatures.     

Molecular cloning, overexpression, purification, and characterization of an aerobic FMN-dependent azoreductase from Enterococcus faecalis

Azo dyes represent a major class of synthetic colorants that are ubiquitous in foods and consumer products. Enterococcus faecalis is a predominant member of the human gastrointestinal microflora. Strain ATCC 19433 grew in the presence of azo dyes and metabolized them to colorless products. A gene encoding a putative FMN-dependent aerobic azoreductase that shares 34% identity with azoreductase (AcpD) of Escherichia coli has been identified in this strain. The gene in E. faecalis, designated as azoA, encoded a protein of 208 amino acids with a calculated isoelectric point of 4.8. AzoA was heterologously overexpressed in E. coli with a strong band of 23 kDa on SDS-PAGE. The purified recombinant enzyme was a homodimer with a molecular weight of 43 kDa, probably containing one molecule of FMN per dimer. AzoA required FMN and NADH, but not NADPH, as a preferred electron donor for its activity. The apparent Km values for both NADH and 2-[4-(dimethylamino)phenylazo]benzoic acid (Methyl red) substrates were 0.14 and 0.024 mM, respectively. The apparent Vmax was 86.2 microM/min/mg protein. The enzyme was not only able to decolorize Methyl red, but was also able to convert sulfonated azo dyes Orange II, Amaranth, Ponceau BS, and Ponceau S. AzoA is the first aerobic azoreductase to be identified and characterized from human intestinal gram-positive bacteria.     

Human granulocyte colony stimulating factor (hG-CSF): cloning,overexpression, purification and characterization

Background  

Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells.     

Gene overexpression and biochemical characterization of the biotechnologically relevant chlorogenic acid hydrolase from Aspergillus niger

The full-length gene that encodes the chlorogenic acid hydrolase from Aspergillus niger CIRM BRFM 131 was cloned by PCR based on the genome of the strain A. niger CBS 513.88. The complete gene consists of 1,715 bp and codes for a deduced protein of 512 amino acids with a molecular mass of 55,264 Da and an acidic pI of 4.6. The gene was successfully cloned and overexpressed in A. niger to yield 1.25 g liter(-1), i.e., 330-fold higher than the production of wild-type strain A. niger CIRM BRFM131. The histidine-tagged recombinant ChlE protein was purified to homogeneity via a single chromatography step, and its main biochemical properties were characterized. The molecular size of the protein checked by mass spectroscopy was 74,553 Da, suggesting the presence of glycosylation. ChlE is assembled in a tetrameric form with several acidic isoforms with pIs of around 4.55 and 5.2. Other characteristics, such as optimal pH and temperature, were found to be similar to those determined for the previously characterized chlorogenic acid hydrolase of A. niger CIRM BRFM 131. However, there was a significant temperature stability difference in favor of the recombinant protein. ChlE exhibits a catalytic efficiency of 12.5 x 10(6) M(-1) s(-1) toward chlorogenic acid (CGA), and its ability to release caffeic acid from CGA present in agricultural by-products such as apple marc and coffee pulp was clearly demonstrated, confirming the high potential of this enzyme.     

Nitrogen regulation in Corynebacterium glutamicum: isolation of genes involved and biochemical characterization of corresponding proteins

The regulation of nitrogen assimilation was investigated in the Gram-positive actinomycete Corynebacterium glutamicum. Biochemical studies and site-directed mutagenesis revealed that glutamine synthetase activity is regulated via adenylylation in this organism. The genes encoding the central signal transduction protein PH (glnB) and the primary nitrogen sensor uridylyltransferase (glnD) were isolated and sequenced. Additionally, genes putatively involved in the degradation of ornithine (ocd) and sarcosine (soxA), ammonium uptake (amtP) and protein secretion (ftsY, srp) were identified in C. glutamicum. Based on these observations, the mechanism of N regulation in C. glutamicum is similar to that of the Gram-negative Escherichia coli. As deduced from data base searches, the described regulation may also hold true for the important pathogen Mycobacterium glutamicum.     

Polyphosphate/ATP-dependent NAD kinase of Corynebacterium glutamicum: biochemical properties and impact of ppnK overexpression on lysine production

Nicotinamide adenine dinucleotide phosphate (NADP) is synthesized by phosphorylation of either oxidized or reduced nicotinamide adenine dinucleotide (NAD/NADH). Here, the cg1601/ppnK gene product from Corynebacterium glutamicum genome was purified from recombinant Escherichia coli and enzymatic characterization revealed its activity as a polyphosphate (PolyP)/ATP-dependent NAD kinase (PPNK). PPNK from C. glutamicum was shown to be active as homotetramer accepting PolyP, ATP, and even ADP for phosphorylation of NAD. The catalytic efficiency with ATP as phosphate donor for phosphorylation of NAD was higher than with PolyP. With respect to the chain length of PolyP, PPNK was active with short-chain PolyPs. PPNK activity was independent of bivalent cations when using ATP, but was enhanced by manganese and in particular by magnesium ions. When using PolyP, PPNK required bivalent cations, preferably manganese ions, for activity. PPNK was inhibited by NADP and NADH at concentrations below millimolar. Overexpression of ppnK in C. glutamicum wild type slightly reduced growth and ppnK overexpression in the lysine producing strain DM1729 resulted in a lysine product yield on glucose of 0.136 ± 0.006 mol lysine (mol glucose)⁻¹, which was 12% higher than that of the empty vector control strain.     

Tabtoxin-resistant protein: overexpression, purification, and characterization

One of the self-protection mechanisms in Pseudomonas syringae pv. tabaci, a pathogen of tobacco wildfire, is thought to be due to its tabtoxin-resistance gene (ttr). In this study, the ttr gene was inserted into an expression vector, pQE30, and successfully expressed in Escherichia coli M15 at high levels. The purified recombinant tabtoxin-resistant protein (TTR) had an apparent molecular mass of about 21 kDa on SDS-PAGE as well as by mass spectroscopy and had a pI of 6.6 on isoelectric focusing-PAGE. Spectral analysis showed that TTR possesses a maximum fluorescence wavelength (lambda(max)) of 325 nm upon excitation at 282 nm and a positive band with a maximum at 195 nm and a broad negative band with a minimum at 215 nm in the far-UV CD spectrum. The spectrophotometric assay demonstrated the strong detoxification activity of TTR. These results are the first report of the characterization of the purified tabtoxin-resistant protein. Its capacity to detoxify tabtoxinine-beta-lactam shows that it must be one of the self-protection mechanisms in pv. tabaci.