Characterization of a sodium-regulated glutaminase from cyanobacterium Synechocystis sp. PCC 6803

Glutaminase is widely distributed among microorganisms and mammals with important functions. Little is known regarding the biochemical properties and functions of the deamidating enzyme glutaminase in cyanobacteria. In this study a putative glutaminase encoded by gene slr2079 in Synechocystis sp. PCC 6803 was investigated. The slr2079 was expressed as histidine-tagged fusion protein in Escherichia coli. The purified protein possessed glutaminase activity, validating the functional assignment of the genomic annotation. The apparent K _m value of the recombinant protein for glutamine was 26.6 ± 0.9 mmol/L, which was comparable to that for some of other microbial glutaminases. Analysis of the purified protein revealed a two-fold increase in catalytic activity in the presence of 1 mol/L Na⁺. Moreover, the K _m value was decreased to 12.2 ± 1.9 mmol/L in the presence of Na⁺. These data demonstrate that the recombinant protein Slr2079 is a glutaminase which is regulated by Na⁺ through increasing its affinity for substrate glutamine. The slr2079 gene was successfully disrupted in Synechocystis by targeted mutagenesis and the Δslr2079 mutant strain was analyzed. No differences in cell growth and oxygen evolution rate were observed between Δslr2079 and the wild type under standard growth conditions, demonstrating slr2079 is not essential in Synechocystis. Under high salt stress condition, however, Δslr2079 cells grew 1.25-fold faster than wild-type cells. Moreover, the photosynthetic oxygen evolution rate of Δslr2079 cells was higher than that of the wild-type. To further characterize this phenotype, a number of salt stress-related genes were analyzed by semi-quantitative RT-PCR. Expression of gdhB and prc was enhanced and expression of desD and guaA was repressed in Δslr2079 compared to the wild type. In addition, expression of two key enzymes of ammonium assimilation in cyanobacteria, glutamine synthetase (GS) and glutamate synthase (GOGAT) was examined by semi-quantitative RT-PCR. Expression of GOGAT was enhanced in Δslr2079 compared to the wild type while GS expression was unchanged. The results indicate that slr2079 functions in the salt stress response by regulating the expression of salt stress related genes and might not play a major role in glutamine breakdown in Synechocystis. Supported by the National Natural Sciences Foundation of China (Grant No. 30500108) and Hundred Talents Program of Chinese Academy of Sciences.     

The two authentic methionine aminopeptidase genes are differentially expressed in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Background  

Two putative methionine aminopeptidase genes,map(essential) andyflG(non-essential), were identified in the genome sequence ofBacillus subtilis. We investigated whether they can function as methionine aminopeptidases and further explored possible reasons for their essentiality or dispensability inB. subtilis.     

Characterization of a sodium-regulated glutaminase from cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Glutaminase is widely distributed among microorganisms and mammals with important functions. Little is known regarding the biochemical properties and functions of the deamidating enzyme glutaminase in cyanobacteria. In this study a putative glutaminase encoded by gene slr2079 in Synechocystis sp. PCC 6803 was investigated. The slr2079 was expressed as histidine-tagged fusion protein in Escherichia coli. The purified protein possessed glutaminase activity, validating the functional assignment of the genomic annotation. The apparent K _m value of the recombinant protein for glutamine was 26.6 ± 0.9 mmol/L, which was comparable to that for some of other microbial glutaminases. Analysis of the purified protein revealed a two-fold increase in catalytic activity in the presence of 1 mol/L Na⁺. Moreover, the K _m value was decreased to 12.2 ± 1.9 mmol/L in the presence of Na⁺. These data demonstrate that the recombinant protein Slr2079 is a glutaminase which is regulated by Na⁺ through increasing its affinity for substrate glutamine. The slr2079 gene was successfully disrupted in Synechocystis by targeted mutagenesis and the Δslr2079 mutant strain was analyzed. No differences in cell growth and oxygen evolution rate were observed between Δslr2079 and the wild type under standard growth conditions, demonstrating slr2079 is not essential in Synechocystis. Under high salt stress condition, however, Δslr2079 cells grew 1.25-fold faster than wild-type cells. Moreover, the photosynthetic oxygen evolution rate of Δslr2079 cells was higher than that of the wild-type. To further characterize this phenotype, a number of salt stress-related genes were analyzed by semi-quantitative RT-PCR. Expression of gdhB and prc was enhanced and expression of desD and guaA was repressed in Δslr2079 compared to the wild type. In addition, expression of two key enzymes of ammonium assimilation in cyanobacteria, glutamine synthetase (GS) and glutamate synthase (GOGAT) was examined by semi-quantitative RT-PCR. Expression of GOGAT was enhanced in Δslr2079 compared to the wild type while GS expression was unchanged. The results indicate that slr2079 functions in the salt stress response by regulating the expression of salt stress related genes and might not play a major role in glutamine breakdown in Synechocystis.     

地衣芽孢杆菌E7氨肽酶的异源表达及其在高效蛋白水解中的应用

【目的】将地衣芽孢杆菌(Bacilluslicheniformis)E7氨肽酶基因pepN克隆到大肠杆菌(Escherichia coli) BL21中,实现氨肽酶Ec PepN的异源表达,研究重组酶的酶学性质及其与碱性蛋白酶协同作用,高效水解大豆蛋白和酪蛋白,产生小分子活性肽和游离氨基酸。【方法】以地衣芽孢杆菌E7基因组DNA为模板,将氨肽酶基因pepN克隆到载体pET28a中,构建重组表达载体pET28-pepN,转化到大肠杆菌BL21感受态细胞中,经DNA测序验证,获得重组菌E. coli BL21/pET28-pepN。利用镍离子亲和层析柱对重组酶进行分离纯化,研究纯酶的pH和温度稳定性、半衰期和NaCl的耐受性等酶学性质。以商品化氨肽酶与碱性蛋白酶协同作用为对照,重组酶Ec PepN与碱性蛋白酶协同水解大豆蛋白和酪蛋白,测定水解产物中小分子活性肽和游离氨基酸的组成。【结果】Ec PepN在大肠杆菌BL21中可溶性表达,SDS-PAGE分析表明纯化的重组酶在52kDa左右显示单一条带。在7种测定底物中,Ec PepN的最适底物为Ala-pNA。在最适条件(pH 9.0和50°C...     

Analyzing the binding of Co(II)-specific inhibitors to the methionyl aminopeptidases from Escherichia coli and Pyrococcus furiosus

Methionine aminopeptidases (MetAPs) represent a unique class of protease that is capable of the hydrolytic removal of an N-terminal methionine residue from nascent polypeptide chains. MetAPs are physiologically important enzymes; hence, there is considerable interest in developing inhibitors that can be used as antiangiogenic and antimicrobial agents. A detailed kinetic and spectroscopic study has been performed to probe the binding of a triazole-based inhibitor and a bestatin-based inhibitor to both Mn(II)- and Co(II)-loaded type-I (Escherichia coli) and type-II (Pyrococcus furiosus) MetAPs. Both inhibitors were found to be moderate competitive inhibitors. The triazole-type inhibitor was found to interact with both active-site metal ions, while the bestatin-type inhibitor was capable of switching its mode of binding depending on the metal in the active site and the type of MetAP enzyme.     

Ethylene formation by cell-free extracts of Escherichia coli

The pathway leading to the formation of ethylene as a secondary metabolite from methionine by Escherichia coli strain B SPAO has been investigated. Methionine was converted to 2-oxo-4-methylthiobutyric acid (KMBA) by a soluble transaminase enzyme. 2-Hydroxy-4-methylthiobutyric acid (HMBA) was also a product, but is probably not an intermediate in the ethylene-forming pathway. KMBA was converted to ethylene, methanethiol and probably carbon dioxide by a soluble enzyme system requiring the presence of NAD(P)H, Fe³⁺ chelated to EDTA, and oxygen. In the absence of added NAD(P)H, ethylene formation by cell-free extracts from KMBA was stimulated by glucose. The transaminase enzyme may allow the amino group to be salvaged from methionine as a source of nitrogen for growth. As in the plant system, ethylene produced by E. coli was derived from the C-3 and C-4 atoms of methionine, but the pathway of formation was different. It seems possible that ethylene production by bacteria might generally occur via the route seen in E. coli.Abbreviations EDTA ethylenediaminetetraacetic acid - HMBA 2-hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - HSS high speed supernatant - KMBA 2-oxo-4-methylthiobutyric acid - PCS phase combining system     

Colicin Ia and Ib binding to Escherichia coli envelopes and partially purified cell walls

The specific binding of ¹²⁵ Iodine labelled colicin Ia and Ib to Escherichia coli cell envelopes and partially purified cell walls is demonstrated. Neither partially purified cytoplasmic membranes isolated from a wild type sensitive strain nor envelopes or cell walls prepared from an E. coli mutant known to be defective in the colicin I receptor could bind the colicins. Competition studies suggest that colicins Ia and Ib have a common bacterial receptor which resides in the bacterial cell wall.     

Efficient production of active Vibrio proteolyticus aminopeptidase in Escherichia coli by co-expression with engineered vibriolysin

The Vibrio proteolyticus aminopeptidase is synthesized as a preproprotein and then converted into an active enzyme by cleavage of the N-terminal propeptide. In recombinant Escherichia coli, however, the aminopeptidase is not processed correctly and the less-active form that has the N-terminal propeptide accumulates in the culture medium. Recently, we isolated a novel vibriolysin that was expressed as an active form in E. coli by random mutagenesis; this enzyme shows potential as a candidate enzyme for the processing of aminopeptidase. The E. coli cells were engineered to co-express the novel vibriolysin along with aminopeptidase. Co-expression of vibriolysin resulted in an approximately 13-fold increase in aminopeptidase activity, and a further increase was observed in the form lacking its C-terminal propeptide. The active aminopeptidase was purified from the culture supernatant including the recombinant vibriolysin by heat treatment and ion exchange and hydroxyapatite chromatography with high purity and 35% recovery rate. This purified aminopeptidase effectively converted methionyl-human growth hormone (Met-hGH) to hGH. Thus, this co-expression system provides an efficient method for producing active recombinant V. proteolyticus aminopeptidase.     

Deficiency in methionine adenosyltransferase resulting in limited repressibility of methionine biosynthetic enzymes in Aspergillus nidulans

Summary In Aspergillus nidulans methionine can be metabolized to cysteine. Mutants blocked in this pathway were selected and divided into three groups representing three separate loci: mecA, mecB and mecC. mecC13 mutant possesses a low level of methionine adenosyltransferase and shows a limited extent of methionine-caused repression of three enzymes of the methionine biosynthetic pathway: sulfate permease, sulfite reductase and 0-acetylhomoserine sulfhydrylase. Intracellular pools of methionine do not differ markedly in the mutant and in wild type, while the S-adenosylmethionine (SAM) pool is decreased in the mutant. Methionine adenosyltransferase was found to be inducible by methionine, SAM is postulated to be involved in regulation of methionine biosynthetic enzymes in A. nidulans. Differences in regulation of methionine biosynthesis in A. nidulans, Escherichia coli and Saccharomyces cerevisiae are discussed.     

Cloning of a New Bacillus thuringiensis cry1I-Type CrystalProtein Gene

A new cry1I-type gene, cry1Id1, was cloned from a B. thuringiensis isolate, and its nucleotide sequence was determined. The deduced amino acid sequence of Cry1Id1 is 89.7%, 87.2%, and 83.4% identical to the Cry1Ia, Cry1Ib, and Cry1Ic proteins, respectively. The upstream sequence of the cry1Id1 structural gene was not functional as promoter in B. subtilis. The Cry1Id1 protein, purified from recombinant E. coli cells, had a toxicity comparable to that of Cry1Ia against Plutella xylostella, but it was significantly less active than Cry1Ia against Bombyx mori. Cry1Id1 was not active against the coleopteran insect, Agelastica coerulea. Received: 19 January 2000 / Accepted: 22 February 2000     

Identification of the 7,8-didemethyl-8-hydroxy-5-deazariboflavin synthase required for coenzyme F<Subscript>420</Subscript> biosynthesis

The hydride carrier coenzyme F₄₂₀ contains the unusual chromophore 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO). Microbes that generate F₄₂₀ produce this FO moiety using a pyrimidine intermediate from riboflavin biosynthesis and the 4-hydroxyphenylpyruvate precursor of tyrosine. The fbiC gene, cloned from Mycobacterium smegmatis, encodes the bifunctional FO synthase. Expression of this protein in Escherichia coli caused the host cells to produce FO during growth, and activated cell-free extracts catalyze FO biosynthesis in vitro. FO synthase in the methanogenic euryarchaeon Methanocaldococcus jannaschii comprises two proteins encoded by cofG (MJ0446) and cofH (MJ1431). Both subunits were required for FO biosynthesis in vivo and in vitro. Cyanobacterial genomes encode homologs of both genes, which are used to produce the coenzyme for FO-dependent DNA photolyases. A molecular phylogeny of the paralogous cofG and cofH genes is consistent with the genes being vertically inherited within the euryarchaeal, cyanobacterial, and actinomycetal lineages. Ancestors of the cyanobacteria and actinomycetes must have acquired the two genes, which subsequently fused in actinomycetes. Both CofG and CofH have putative radical S-adenosylmethionine binding motifs, and pre-incubation with S-adenosylmethionine, Fe²⁺, sulfide, and dithionite stimulates FO production. Therefore a radical reaction mechanism is proposed for the biosynthesis of FO.Abbreviations AdoMet (SAM) S-adenosyl-l-methionine - Compound 6 5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione - FO 7,8-Didemethyl-8-hydroxy-5-deazariboflavin - HPP 4-Hydroxyphenylpyruvate     

Challenges Associated with Heterologous Expression of <Emphasis Type="Italic">Flavobacterium psychrophilum</Emphasis> Proteins in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A two-parameter statistical model was used to predict the solubility of 96 putative virulence-associated proteins of Flavobacterium psychrophilum (CSF259-93) upon over expression in Escherichia coli. This analysis indicated that 88.5% of the F. psychrophilum proteins would be expressed as insoluble aggregates (inclusion bodies). These solubility predictions were verified experimentally by colony filtration blot for six different F. psychrophilum proteins. A comprehensive analysis of codon usage identified over a dozen codons that are used frequently in F. psychrophilum, but that are rarely used in E. coli. Expression of F. psychrophilum proteins in E. coli was often associated with production of minor molecular weight products, presumably because of the codon usage bias between these two organisms. Expression of recombinant protein in the presence of rare tRNA genes resulted in marginal improvements in the expressed products. Consequently, Vibrio parahaemolyticus was developed as an alternative expression host because its codon usage is similar to F. psychrophilum. A full-length recombinant F. psychrophilum hemolysin was successfully expressed and purified from V. parahaemolyticus in soluble form, whereas this protein was insoluble upon expression in E. coli. We show that V. parahaemolyticus can be used as an alternate heterologous expression system that can remedy challenges associated with expression and production of F. psychrophilum recombinant proteins.     

Characterization,cloning, sequencing,and expression of an aminopeptidase N from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. TH-4

The aminopeptidase N (TH-4AP) of Streptomyces sp. TH-4 was purified from a culture supernatant. The purified enzyme had a molecular mass of 95 kDa. The gene encoding TH-4AP was cloned and sequenced. The primary structure of the protein possessed the PepN-conserved motif GxMEN and the zinc-binding motif HExxHx₁₈E, and showed 88% identity with that of PepN from Streptomyces lividans strain 66. We succeeded in overproducing a His-tagged recombinant enzyme using Escherichia coli. The enzyme had a 1.5-fold higher activity in the presence of cobalt ions than in their absence. To evaluate the possible application of TH-4AP to decrease the content of bitter peptides, we investigated the ability of Streptomyces aminopeptidases to hydrolyze synthetic peptides by a coupling method using l-amino acid oxidase and peroxidase. The substrate specificity of TH-4AP toward synthetic peptides was significantly different from that toward aminoacyl-p-nitroanilide derivatives.     

N-terminus deletion affecting the preparation of soluble cyanobacterial glutaredoxin in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Glutaredoxin has been implicated in maintenance of a normal cellular thiol/disufide ratio and the regeneration of oxidatively damaged proteins. In order to obtain more information about these important regulatory proteins in cyanobacteria, we have previously cloned and expressed the first cyanobacterial glutaredoxin gene ssr2061 in Escherichia coli. In this work, the second glutaredoxin gene slr1562 was studied. About 90% of Grx2061 coded by ssr2061 was produced in a soluble form while 90% of Grx1562 coded by slr1562 was found in inclusion bodies. To improve the production of soluble Grx1562, we constructed two mutants: Grx1562NC with cysteines in conserved site substituted by serines, and Grx1562M with N-terminus hydrophobic region deletion. Only the latter mutant was successfully expressed in soluble form with increased glutaredoxin activity and showed less sensitivity in oxidative stress. Spectroscopic analysis shows that the structure of Grx1562M with less hydrophobic nature could give more opportunity for protein solubility and could improve the substrate catalytic efficiency. These results suggest that hydrophobic N-terminus determines the insolubility of Grx1562 and may provide another strategy for increasing expression level of soluble heterologous proteins in E. coli. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 3, pp. 383–391.     

A thermostable leucine aminopeptidase from<Emphasis Type="Italic"> Bacillus kaustophilus</Emphasis> CCRC 11223

Two degenerate primers established from the consensus sequences of bacterial leucine aminopeptidases (LAP) were used to amplify a 360-bp gene fragment from the chromosomal DNA of thermophilic Bacillus kaustophilus CCRC 11223 and the amplified fragment was successfully used as a probe to clone a leucine aminopeptidase (lap) gene from a genomic library of the strain. The gene consists of an open reading frame (ORF) of 1,494 bp and encodes a protein of 497 amino acid residues with a calculated molecular mass of 53.7 kDa. The complete amino acid sequence of the cloned enzyme showed greater than 30% identity with prokaryotic and eukaryotic LAPs. Phylogenetic analysis showed that B. kaustophilus LAP is closely related to the enzyme from Bacillus subtilis and is grouped with the M17 family. His₆-tagged LAP was generated in Escherichia coli by cloning the coding region into pQE-30 and the recombinant enzyme was purified by nickel-chelate chromatography. The pH and temperature optima for the purified enzyme were 8 and 65°C, respectively, and 50% of its activity remained after incubation at 60°C for 32 min. The enzyme preferentially hydrolyzed l-leucine-p-nitroanilide (l-Leu-p-NA) followed by Cys derivative.Communicated by G. Antranikian     

Major outer membrane proteins of E. coli K12 serve as receptors for the phages T2 (protein Ia) and 434 (protein Ib).

Summary Mutants of E. coli resistant to bacteriophage T2 have lowered amounts of protein Ia in their outer membrane. Bacteriophage T2 was inactivated by a mixture of protein Ia-lipopolysaccharide. Protein Ia or lipopolysaccharide alone had no neutralizing activity. However, only protein Ia was required to inactivate a T2 host range mutant. In the presence of polymyxin B T2 receptor activity of protein Ia — lipopolysaccharide mixtures could not be restored. E. coli strains missing protein Ib were resistant against the lambdoid phage 434. Purified protein Ib inactivated 434 and virh ⁴³⁴. Addition of lipopolysaccharide did not enhance the neutralizing activity of protein Ib, indicating that lipopolysaccharide may not be necessary for the inactivation of the phage.     

Polyamines reduce paraquat-induced soxS and its regulon expression in Escherichia coli

Polyamines, ubiquitous polycationic compounds, are involved in many cellular responses and relieve paraquat-induced cytotoxicity inEscherichia coli. We constructed a newE. coli mutant strain, JIL528, which is deficient in the biosynthesis of both putrescine and spermidine, to examine the physiological role of polyamines under oxidative stress caused by paraquat. Putrescine and spermidine downregulate the expression ofsoxS induced by paraquat in a concentration-dependent manner. The product of SoxS is a key regulator governing cellular responses against oxidative stress inE. coli. The downregulation ofsoxS expression by polyamines was not shown in thesoxR mutant background. Glucose-6-phosphate dehydrogenase (G6PDH; encoded byzwf) and manganese-containing superoxide dismutase (Mn-SOD; encoded bysodA) activities induced by paraquat were decreased by exogenous polyamines. The induction of thezwf expression by paraquat was also decreased by exogenous polyamines. The polyamine-deficient mutant strain JIL528 showed a highersoxS expression than its parent polyamine-proficient wild type BW1157, on exogenous supplementation of paraquat concentrations below 1 mol/L. While the growth rate of the mutant was decreased,soxS expression was increased in a concentration-dependent manner above 0.01 mol/L of paraquat. In contrast, growth inhibition of the mutant by paraquat was relieved, andsoxS was no longer induced by exogenous putrescine (1 mmol/L). In conclusion, polyamines protect against paraquat-induced toxicity but downregulatesoxS expression, suggesting that the protective role of polyamines against oxidative damage induced by paraquat results insoxS downregulation.     

Cloning, sequencing, and expression of a β-1,4-endoxylanase gene from Indonesian Bacillus licheniformis strain I5 in Escherichia coli

The gene encoding an endo-β-1,4-xylanase from an Indonesian indigenous Bacillus licheniformis strain I5 was amplified using PCR, cloned, and expressed in Escherichia coli. The nucleotide sequence of a 642 bp DNA fragment was determined, revealing one open reading frame that encoded a xylanase. Based on the nucleotide sequence, calculated molecular mass of the enzyme was 23 kDa. This xylanase has a predicted typical putative signal peptide; however, in E. coli, the active protein was located mainly in intracellular form. Neither culture supernatant of recombinant E. coli nor periplasmic fraction has significantly detectable xylanase activity. The deduced amino acid of the gene has 91% identity with that of Bacillus subtilis endoxylanase. Optimal activity of the recombinant enzyme was at pH 7 and 50°C     

Precise physical mapping of theEscherichia coli rnb gene, encoding ribonuclease II

Ribonuclease II (encoded byrnb) is one of the two main exonucleases involved in mRNA degradation inEscherichia coli. We report the precise physical mapping ofrnb to 29 min on the chromosomal map in the vicinity ofpyrF, and clarify the genetic and physical maps of thisE. coli chromosomal region. The results were confirmed by the construction of a strain partially deleted forrnb.