The control of sulphate reduction in bacteria

1. An enzyme from Escherichia coli 9723 that reduces adenosine 3′-phosphate 5′-sulphatophosphate to inorganic sulphite is described. Extracts of E. coli K₁₂ and Bacillus subtilis 1379 contain a similar enzyme. 2. This reductase and sulphite reductase (EC 1.8.1.2) of E. coli 9723, E. coli K₁₂ and of B. subtilis are repressed by growth in the presence of l-cystine. Cysteine synthase (EC 4.2.1.22) is unaffected. 3. Growth of E. coli 9723 on inorganic sulphite represses the sulphate-activating enzymes (EC 2.7.7.4 and 2.7.1.25) almost completely but has little effect on sulphite reductase. Growth on 0·042–0·056mm-l-cystine gives a similar result. 4. Such differential repression by cyst(e)ine prevents E. coli, when growing on sulphite, from synthesizing unnecessary enzymes.     

Purification and characterization of sepiapterin reductase from rat erythrocytes

Sepiapterin reductase from rat erythrocyte hemolysate was purified 2000-fold to apparent homogeneity with 30% yield. The specific activity of the purified enzyme was 18 units/mg protein, and its molecular weight was 55 000. The enzyme consists of two identical subunits, each of which has a molecular weight of 27 500. The enzyme showed a single peak by isoelectric focusing with a pI of 4.9 and partial specific volume of 0.73 cm³/g. The amino acid composition was determined. pH optimum of the enzyme was 5.5. The equilibrium constant of 2.2·10⁹ of the enzyme showed that the equilibrium lies much in favor of dihydrobiopterin formation from sepiapterin in rat erythrocytes. From steady-state kinetic measurements, ordered bi-bi mechanism was proposed to the reaction of sepiapterin reductase in which NADPH binds to free enzyme and sepiapterin binds next. NADP⁺ is released after the release of dihydrobiopterin. The K_m values for sepiapterin and NADPH were 15.4 μM and 1.7 μM, respectively, and the V_max value was 21.7 μmol/min per mg.     

Production of sepiapterin in Escherichia coli by coexpression of cyanobacterial GTP cyclohydrolase I and human 6-pyruvoyltetrahydropterin synthase

Synechocystis sp. strain PCC 6803 GTP cyclohydrolase I and human 6-pyruvoyltetrahydropterin synthase were coexpressed in Escherichia coli. The E. coli transformant produced sepiapterin, which was identified by high-performance liquid chromatography and enzymatically converted to dihydrobiopterin by sepiapterin reductase. Aldose reductase, another indispensable enzyme for sepiapterin production, may be endogenous in E. coli.     

Production of an aromatic amino acid auxotroph of a trimethoprim-resistant Escherichia coli and deuteration of the aromatic amino acids in its dihydrofolate

Interpretation of the ¹H-NMR spectra of Escherichia coli dihydrofolate reductase is complicated by the large number of overlapping resonances due to protonated aromatic amino acids. Deuteration of the aromatic protons of aromatic amino acid residues is one technique useful for simplifying the ¹H-NMR spectra. Previous attempts to label the dihydrofolate reductase from over-producing strains of Escherichia coli were not completely successful. This labeling problem was solved by transducing via P1 phage a genetic block into the de novo biosynthetic pathway of aromatic amino acids in a trimethoprim resistant strain of E. coli, MB 3746. A new strain, MB 4065, is a very high level producer of dihydrofolate reductase and requires exogenous aromatic amino acids for growth, therefore allowing efficient labeling of its dihydrofolate reductase with exogenous deuterated aromatic amino acid.     

Restoration of reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase activity of a Neurospora mutant by extracts of various chlorate-resistant mutants of Escherichia coli

Acid-treated extracts of Escherichia coli were tested for their ability to restore reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase activity to an extract of a Neurospora nit-1 mutant which produces a defective enzyme. With wild-type E. coli this complementation activity was more readily detected in the cytoplasmic fraction, although the nitrate reductase activity was found primarily in the particulate fraction. chlB mutants of E. coli appeared to have more complementation activity in the cytoplasm than was observed in the wild type, but no activity in the particulate fraction. The other chl mutants had little or no activity in either fraction. These results suggest that chlB mutants can produce a component or cofactor which is missing in the other mutants and in the Neurospora mutant, but cannot transfer it to the nitrate reductase enzyme.     

An iron tetrahydroporphyrin prosthetic group common to both assimilatory and dissimilatory sulfite reductases

The heme² chromophore of the “assimilatory” E. coli sulfite reductase is an iron-octacarboxylic tetrahydroporphyrin of the isobacteriochlorin type (1). Although the two “dissimilatory” sulfite reductases, desulfoviridin and desulforubidin, from the sulfate reducing bacteria Desulfovibrio gigas and Desulfovibrio desulfuricans (Norway strain), have absorption spectra and reaction products which differ from those of E. coli sulfite reductase, the present studies indicate that they contain prosthetic groups with an organic structure closely similar or identical to that of the E. coli sulfite reductase heme. EPR spectra show high-spin ferriheme in all three enzymes. It is clear, however, that the prosthetic groups must reside in substantially different environments within their respective proteins.     

The Function of Cytoplasmic Flavin Reductases in the Reduction of Azo Dyes by Bacteria

A flavin reductase, which is naturally part of the ribonucleotide reductase complex of Escherichia coli, acted in cell extracts of recombinant E. coli strains under aerobic and anaerobic conditions as an “azo reductase.” The transfer of the recombinant plasmid, which resulted in the constitutive expression of high levels of activity of the flavin reductase, increased the reduction rate for different industrially relevant sulfonated azo dyes in vitro almost 100-fold. The flavin reductase gene (fre) was transferred to Sphingomonas sp. strain BN6, a bacterial strain able to degrade naphthalenesulfonates under aerobic conditions. The flavin reductase was also synthesized in significant amounts in the Sphingomonas strain. The reduction rates for the sulfonated azo compound amaranth were compared for whole cells and cell extracts from both recombinant strains, E. coli, and wild-type Sphingomonas sp. strain BN6. The whole cells showed less than 2% of the specific activities found with cell extracts. These results suggested that the cytoplasmic anaerobic “azo reductases,” which have been described repeatedly in in vitro systems, are presumably flavin reductases and that in vivo they have insignificant importance in the reduction of sulfonated azo compounds.     

Oxygen-Insensitive Nitroreductases NfsA and NfsB of Escherichia coli Function under Anaerobic Conditions as Lawsone-Dependent Azo Reductases

Quinones can function as redox mediators in the unspecific anaerobic reduction of azo compounds by various bacterial species. These quinones are enzymatically reduced by the bacteria and the resulting hydroquinones then reduce in a purely chemical redox reaction the azo compounds outside of the cells. Recently, it has been demonstrated that the addition of lawsone (2-hydroxy-1,4-naphthoquinone) to anaerobically incubated cells of Escherichia coli resulted in a pronounced increase in the reduction rates of different sulfonated and polymeric azo compounds. In the present study it was attempted to identify the enzyme system(s) responsible for the reduction of lawsone by E. coli and thus for the lawsone-dependent anaerobic azo reductase activity. An NADH-dependent lawsone reductase activity was found in the cytosolic fraction of the cells. The enzyme was purified by column chromatography and the amino-terminal amino acid sequence of the protein was determined. The sequence obtained was identical to the sequence of an oxygen-insensitive nitroreductase (NfsB) described earlier from this organism. Subsequent biochemical tests with the purified lawsone reductase activity confirmed that the lawsone reductase activity detected was identical with NfsB. In addition it was proven that also a second oxygen-insensitive nitroreductase of E. coli (NfsA) is able to reduce lawsone and thus to function under adequate conditions as quinone-dependent azo reductase.     

Heterologous biosynthesis of triterpenoid dammarenediol-II in engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To achieve heterologous biosynthesis of dammarenediol-II, which is the precursor of dammarane-type tetracyclic ginsenosides, by reconstituting the 2,3-oxidosqualene-derived triterpenoid biosynthetic pathway in Escherichia coli.

Results

By the strategy of synthetic biology, dammarenediol-II biosynthetic pathway was reconstituted in E. coli by co-expression of squalene synthase (SS), squalene epoxidase (SE), NADPH-cytochrome P450 reductase (CPR) from Saccharomyces cerevisiae, and SE from Methylococcus capsulatus (McSE), NADPH-cytochrome P450 reductase (CPR) from Arabidopsis thaliana. Sequences of transmembrane domains were truncated if necessary in each of the genes. Different sources of SE/CPR combinations were tested, during which two CPRs were detected to be new reductase partners of McSE. When the gene encoding dammarenediol-II synthase was co-expressed with the 2,3-oxidosqualene expression modules, dammarenediol-II was detected and the production was 8.63 mg l^?1 in E. coli under the shake-flask conditions.

Conclusions

Two E. coli chassis for production of dammarenediol-II were established which could be potentially applied in other triterpenoid production in E. coli when different oxidosqualene cyclases (OSCs) introduced into the system.

     

Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

Bacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly in Escherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the mimABCD gene expression in E. coli revealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction of E. coli cells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformed E. coli cells inactive. We found that the following factors are important for functional expression of MimABCD in E. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble in E. coli cells, and the optimization of the mimD nucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed in E. coli. The strategy described here should be generally applicable to the E. coli expression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes.     

The dihydrolipoamide dehydrogenase of Aeromonas caviae ST exhibits NADH-dependent tellurite reductase activity

Potassium tellurite (K₂TeO₃) is extremely toxic for most forms of life and only a limited number of organisms are naturally resistant to the toxic effects of this compound. Crude extracts prepared from the environmental isolate Aeromonas caviae ST catalize the in vitro reduction of in a NADH-dependent reaction. Upon fractionation by ionic exchange column chromatography three major polypeptides identified as the E1, E2, and E3 components of the pyruvate dehydrogenase (PDH) complex were identified in fractions exhibiting tellurite-reducing activity. Tellurite reductase and pyruvate dehydrogenase activities co-eluted from a Sephadex gel filtration column. To determine which component(s) of the PDH complex has tellurite reductase activity, the A. caviae ST structural genes encoding for E1 (aceE), E2 (aceF), and E3 (lpdA) were independently cloned and expressed in Escherichia coli and their gene products purified. Results indicated that tellurite reductase activity lies almost exclusively in the E3 component, dihydrolipoamide dehydrogenase. The E3 component of the PDH complex from E. coli, Zymomonas mobilis, Streptococcus pneumoniae, and Geobacillus stearothermophilus also showed NADH-dependent tellurite reductase in vitro suggesting that this enzymatic activity is widely distributed among microorganisms.     

Biosynthesis of tetrahydrobiopterin: possible involvement of tetrahydropterin intermediates

The biosynthetic pathway of tetrahydrobiopterin (BH₄) from dihydroneopterin triphosphate (NH₂P₃) was studied in fresh as well as heat-treated human liver extracts. The question of NAD(P)H dependency for the formation of sepiapterin was examined. NH₂P₃ was converted by fresh extracts to sepiapterin in low quantities (2% conversion) in the absence of exogenously added NADPH as well as under conditions that ensured the destruction of endogenous, free NAD(P)H. The addition of NADPH to the fresh liver extracts stimulated the synthesis of BH₄ to a much higher yield (17% conversion), and the amount of sepiapterin formed was reduced to barely detectable levels. In contrast, the heat-treated extract (enzyme A2 fraction) formed sepiapterin (1.3% conversion) only in the presence and not in the absence of NADPH. These results indicate that sepiapterin may not be an intermediate on the pathway leading to BH₄ biosynthesis under normal $\text{in vivo}$ conditions. Rather, sepiapterin may result from the breakdown of an as yet unidentified intermediate that is actually on the pathway. It is speculated that NH₂P₃ may be converted to a diketo-tetrahydropterin intermediate (or an equivalent tautomeric structure) by a mechanism involving an intramolecular oxidoreduction reaction. A diketo-tetrahydropterin intermediate could be converted to 5,6-dihydrosepiapterin, which also has a tetrahydropterin ring system and can be converted directly to BH₄ by sepiapterin reductase. This proposed pathway can explain ho the tetrahydropterin ring system can be formed without sepiapterin, dihydrobiopterin, or dihydrofolate reductase being involved in BH₄ biosynthesis $\text{in vivo}$.     

Geranylgeranyl Reductase and Ferredoxin from Methanosarcina acetivorans Are Required for the Synthesis of Fully Reduced Archaeal Membrane Lipid in Escherichia coli Cells

Archaea produce membrane lipids that typically possess fully saturated isoprenoid hydrocarbon chains attached to the glycerol moiety via ether bonds. They are functionally similar to, but structurally and biosynthetically distinct from, the fatty acid-based membrane lipids of bacteria and eukaryotes. It is believed that the characteristic lipid structure helps archaea survive under severe conditions such as extremely low or high pH, high salt concentrations, and/or high temperatures. We detail here the first successful production of an intact archaeal membrane lipid, which has fully saturated isoprenoid chains, in bacterial cells. The introduction of six phospholipid biosynthetic genes from a methanogenic archaeon, Methanosarcina acetivorans, in Escherichia coli enabled the host bacterium to synthesize the archaeal lipid, i.e., diphytanylglyceryl phosphoglycerol, while a glycerol modification of the phosphate group was probably catalyzed by endogenous E. coli enzymes. Reduction of the isoprenoid chains occurred only when archaeal ferredoxin was expressed with geranylgeranyl reductase, suggesting the role of ferredoxin as a specific electron donor for the reductase. This report is the first identification of a physiological reducer for archaeal geranylgeranyl reductase. On the other hand, geranylgeranyl reductase from the thermoacidophilic archaeon Sulfolobus acidocaldarius could, by itself, replace both its orthologue and ferredoxin from M. acetivorans, which indicated that an endogenous redox system of E. coli reduced the enzyme.     

Fatty alcohol production in engineered E. coli expressing Marinobacter fatty acyl-CoA reductases

Although successful production of fatty alcohols in metabolically engineered Escherichia coli with heterologous expression of fatty acyl-CoA reductase has been reported, low biosynthetic efficiency is still a hurdle to be overcome. In this study, we examined the characteristics of two fatty acyl-CoA reductases encoded by Maqu_2220 and Maqu_2507 genes from Marinobacter aquaeolei VT8 on fatty alcohol production in E. coli. Fatty alcohols with diversified carbon chain length were obtained by co-expressing Maqu_2220 with different carbon chain length-specific acyl-ACP thioesterases. Both fatty acyl-CoA reductases displayed broad substrate specificities for C12–C18 fatty acyl chains in vivo. The optimized mutant strain of E. coli carrying the modified tesA gene and fadD gene from E. coli and Maqu_2220 gene from Marinobacter aquaeolei VT8 produced fatty alcohols at a remarkable level of 1.725 g/L under the fermentation condition.     

Sepiapterin Reductase Mediates Chemical Redox Cycling in Lung Epithelial Cells

In the lung, chemical redox cycling generates highly toxic reactive oxygen species that can cause alveolar inflammation and damage to the epithelium, as well as fibrosis. In this study, we identified a cytosolic NADPH-dependent redox cycling activity in mouse lung epithelial cells as sepiapterin reductase (SPR), an enzyme important for the biosynthesis of tetrahydrobiopterin. Human SPR was cloned and characterized. In addition to reducing sepiapterin, SPR mediated chemical redox cycling of bipyridinium herbicides and various quinones; this activity was greatest for 1,2-naphthoquinone followed by 9,10-phenanthrenequinone, 1,4-naphthoquinone, menadione, and 2,3-dimethyl-1,4-naphthoquinone. Whereas redox cycling chemicals inhibited sepiapterin reduction, sepiapterin had no effect on redox cycling. Additionally, inhibitors such as dicoumarol, N-acetylserotonin, and indomethacin blocked sepiapterin reduction, with no effect on redox cycling. Non-redox cycling quinones, including benzoquinone and phenylquinone, were competitive inhibitors of sepiapterin reduction but noncompetitive redox cycling inhibitors. Site-directed mutagenesis of the SPR C-terminal substrate-binding site (D257H) completely inhibited sepiapterin reduction but had minimal effects on redox cycling. These data indicate that SPR-mediated reduction of sepiapterin and redox cycling occur by distinct mechanisms. The identification of SPR as a key enzyme mediating chemical redox cycling suggests that it may be important in generating cytotoxic reactive oxygen species in the lung. This activity, together with inhibition of sepiapterin reduction by redox-active chemicals and consequent deficiencies in tetrahydrobiopterin, may contribute to tissue injury.     

过表达亚硝酸盐还原酶的重组大肠杆菌辅助污水短程硝化

【目的】为了体现并突出亚硝酸盐还原酶在污水脱氮以及短程硝化中的重要性,对过表达亚硝酸盐还原酶的大肠杆菌进行了污水脱氮的研究。【方法】通过转化带有亚硝酸盐还原酶基因的重组质粒,将亚硝酸盐还原酶在大肠杆菌中过表达,通过分析重组大肠杆菌的产物研究了该酶的表达及还原亚硝酸盐的情况,通过将该重组菌与已报道的硝化-反硝化细菌或生活污水进行混合培养,研究重组菌用于辅助氨氮去除的短程硝化能力。【结果】重组大肠杆菌能正确表达亚硝酸盐还原酶,OD600=2.0的菌悬液在2 h内还原约1 mmol/L的亚硝酸盐,并产生几乎等量的一氧化氮;重组大肠杆菌与Acinetobacter sp.YF14菌株等比例混合时,12 h能够提高氨氮脱氮效率约(36.0±7.4)%,且在4 h时,最大亚硝酸盐的积累量减少37%;重组大肠杆菌(OD600=1.0)12 h内能够提高污水厂活性污泥的脱氮效率约(31.0±5.7)%,且未检测到亚硝酸盐和硝酸盐的积累;溶氧水平对于亚硝酸盐还原酶重组菌辅助脱氮具有明显的影响,中等溶氧量[(6.4?0.7)mg/L]时脱氮效果最好。【结论】过表达亚硝酸盐还原酶的大肠杆菌可以提高污水脱氮的短程硝化能力。     

Enhanced Heterologous Expression of Two Streptomyces griseolus Cytochrome P450s and Streptomyces coelicolor Ferredoxin Reductase as Potentially Efficient Hydroxylation Catalysts

The herbicide-inducible, soluble cytochrome P450s CYP105A1 and CYP105B1 and their adjacent ferredoxins, Fd1 and Fd2, of Streptomyces griseolus were expressed in Escherichia coli to high levels. Conditions for high-level expression of active enzyme able to catalyze hydroxylation have been developed. Analysis of the expression levels of the P450 proteins in several different E. coli expression hosts identified E. coli BL21 Star(DE3)pLysS as the optimal host cell to express CYP105B1 as judged by CO difference spectra. Examination of the codons used in the CYP1051A1 sequence indicated that it contains a number of codons corresponding to rare E. coli tRNA species. The level of its expression was improved in the modified forms of E. coli BL21(DE3), which contain extra copies of rare codon E. coli tRNA genes. The activity of correctly folded cytochrome P450s was further enhanced by cloning a ferredoxin reductase from Streptomyces coelicolor downstream of CYP105A1 and CYP105B1 and their adjacent ferredoxins. Expression of CYP105A1 and CYP105B1 was also achieved in Streptomyces lividans 1326 by cloning the P450 genes and their ferredoxins into the expression vector pBW160. S. lividans 1326 cells containing CYP105A1 or CYP105B1 were able efficiently to dealkylate 7-ethoxycoumarin.     

Refolding of recombinant Pasteurella haemolytica A1 glycoprotease expressed in an Escherichia coli thioredoxin gene fusion system

Pasteurella haemolytica A1 secretes an O-sialoglycoprotein endopeptidase (EC. 3.4.24.57) (glycoprotease: Gcp) which is specific for O-linked sialoglycoproteins. When the cloned gene is expressed in Escherichia coli, the recombinant glycoprotease (rGcp) is secreted to the peripalsm where it is present as a disulfide-linked aggregate which lacks enzymatic activity. In vitro refolding and activation of rGcp by mammalian protein disulfide isomerase (PDI) or by the E. Coli chaperones (DnaK, DnaJ and GrpE) indicate that the redox environment of rGcp is critical in restoring biological activity. A fusion protein, rTrx-Gcp, was constructed to investigate the role of thioredoxin (E. coli TrxA) in the production of enzymatically active rGcp. This 47 kDa protein was expressed at a high level, in a soluble, monomeric form, in the cytoplasm of E. coli. Cleavage of the fusion protein by enterokinase released the rGcp fragment (35 kDa) with glycoprotease activity. A higher recombinant glycoprotease activity was recoveref after anion exchange chromatography of lystates of E. coli expressing rTrx-Gcp. Thus when E. coli TrxA is combined in a recombinant fusion protein with P. haemolytica A1 Gcp, productive folding of the glycoprotease can occur as a result of the chaperone action of the protein disulfide reductase coupled with its ability to retain the fusion gene product in the E. coli cytopalsm.