Methanopyrus kandleri glutamyl-tRNA reductase.

The initial reaction of tetrapyrrole formation in archaea is catalyzed by a NADPH-dependent glutamyl-tRNA reductase (GluTR). The hemA gene encoding GluTR was cloned from the extremely thermophilic archaeon Methanopyrus kandleri and overexpressed in Escherichia coli. Purified recombinant GluTR is a tetrameric enzyme with a native M(r) = 190,000 +/- 10,000. Using a newly established enzyme assay, a specific activity of 0.75 nmol h(-1) mg(-1) at 56 degrees C with E. coli glutamyl-tRNA as substrate was measured. A temperature optimum of 90 degrees C and a pH optimum of 8.1 were determined. Neither heme cofactor, nor flavin, nor metal ions were required for GluTR catalysis. Heavy metal compounds, Zn(2+), and heme inhibited the enzyme. GluTR inhibition by the newly synthesized inhibitor glutamycin, whose structure is similar to the 3' end of the glutamyl-tRNA substrate, revealed the importance of an intact chemical bond between glutamate and tRNA(Glu) for substrate recognition. The absolute requirement for NADPH in the reaction of GluTR was demonstrated using four NADPH analogues. Chemical modification and site-directed mutagenesis studies indicated that a single cysteinyl residue and a single histidinyl residue were important for catalysis. It was concluded that during GluTR catalysis the highly reactive sulfhydryl group of Cys-48 acts as a nucleophile attacking the alpha-carbonyl group of tRNA-bound glutamate with the formation of an enzyme-localized thioester intermediate and the concomitant release of tRNA(Glu). In the presence of NADPH, direct hydride transfer to enzyme-bound glutamate, possibly facilitated by His-84, leads to glutamate-1-semialdehyde formation. In the absence of NADPH, a newly discovered esterase activity of GluTR hydrolyzes the highly reactive thioester of tRNA(Glu) to release glutamate.     

Higher specific activity of the Escherichia coli glutamyl-tRNA synthetase purified to homogeneity by a six-hour procedure.

The glutamyl-tRNA synthetase (EC 6.1.1.17) of Escherichia coli was purified to homogeneity from the overproducing strain DH5 alpha(pLQ7612) by a two-step procedure that takes only about 6 h and yields 10 mg of enzyme per gram of wet cells. The process consists of a two-phase polyethylene glycol-dextran partition, the top phase of which is diluted and directly applied to an anion-exchange FPLC MonoQ column. The purified enzyme has a specific activity about twice that of the same enzyme purified to homogeneity by the lengthy conventional procedure from either a normal strain or this overproducing strain. This difference is discussed in relation to the generation of microheterogeneity in proteins during their purification.     

Escherichia coli glutamyl-tRNA reductase. Trapping the thioester intermediate

In the first step of tetrapyrrole biosynthesis in Escherichia coli, glutamyl-tRNA reductase (GluTR, encoded by hemA) catalyzes the NADPH-dependent reduction of glutamyl-tRNA to glutamate-1-semialdehyde. Soluble homodimeric E. coli GluTR was made by co-expressing the hemA gene and the chaperone genes dnaJK and grpE. During Mg(2+)-stimulated catalysis, the reactive sulfhydryl group of Cys-50 in the E. coli enzyme attacks the alpha-carbonyl group of the tRNA-bound glutamate. The resulting thioester intermediate was trapped and detected by autoradiography. In the presence of NADPH, the end product, glutamate-1-semialdehyde, is formed. In the absence of NADPH, E. coli GluTR exhibited substrate esterase activity. The in vitro synthesized unmodified glutamyl-tRNA was an acceptable substrate for E. coli GluTR. Eight 5-aminolevulinic acid auxotrophic E. coli hemA mutants were genetically selected, and the corresponding mutations were determined. Most of the recombinant purified mutant GluTR enzymes lacked detectable activity. Based on the Methanopyrus kandleri GluTR structure, the positions of the amino acid exchanges are close to the catalytic domain (G7D, E114K, R314C, S22L/S164F, G44C/S105N/A326T, G106N, S145F). Only GluTR G191D (affected in NADPH binding) revealed esterase but no reductase activity.     

近平滑假丝酵母(R)-羰基还原酶在大肠杆菌中的外泌表达及高效转化(R)-苯基乙二醇

克隆了近平滑假丝酵母(Candida parapsilosis)(R)-羰基还原酶基因rcr,构建胞外表达工程茵Escherichia coli BL21(DE3)/pET20b-rcr,实现了(R)-羰基还原酶在大肠杆菌中高效外泌表达,周质空间和发酵液酶的比活力分别达0.68 U/mg和0.26 U/mg,与大肠杆菌的胞内体系重组酶相比,酶的比活力提高了近两倍。为了更好地促进该重组酶的外分泌于大肠杆菌细胞外,通过添加温和型化学渗透剂甘氨酸,改善细胞壁的透性,(R)-羰基还原酶的活力提高至1.99 U,与添加甘氨酸前相比,酶活力提高了12.4倍,比活提高了4.3倍。浓缩后的发酵液催化2-羟基苯乙酮,产生(R)-苯基乙二醇,产率为88.1%,e.e.值为93.9%。与胞内重组酶相比,产率和光学纯度分别提高了44.4%和15.9%。本研究通过构建(R)-羰基还原酶的大肠杆菌分泌表达体系,大幅度提高了(R)-羰基还原酶的比活和生物转化手性醇的效率。     

Characterization of the rhodobacter sphaeroides 5-aminolaevulinic acid synthase isoenzymes, HemA and HemT, isolated from recombinant Escherichia coli.

The hemA and hemT genes encoding 5-aminolaevulinic acid synthase (ALAS) from the photosynthetic bacterium Rhodobacter sphaeroides, were cloned to allow high expression in Escherichia coli. Both HemA and HemT appeared to be active in vivo as plasmids carrying the respective genes complemented an E. coli hemA strain (glutamyl-tRNA reductase deficient). The over-expressed isoenzymes were isolated and purified to homogeneity. Isolated HemA was soluble and catalytically active whereas HemT was largely insoluble and failed to show any activity ex vivo. Pure HemA was recovered in yields of 5-7 mg x L-1 of starting bacterial culture and pure HemT at 10 mg x L-1 x HemA has a final specific activity of 13 U x mg-1 with 1 unit defined as 1 micromol of 5-aminolaevulinic acid formed per hour at 37 degrees C. The Km values for HemA are 1.9 mM for glycine and 17 microM for succinyl-CoA, with the enzyme showing a turnover number of 430 h-1. In common with other ALASs the recombinant R. sphaeroides HemA requires pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor resulted in inactive apo-ALAS. Similarly, reduction of the HemA-PLP complex using sodium borohydride led to > 90% inactivation of the enzyme. Ultraviolet-visible spectroscopy with HemA suggested the presence of an aldimine linkage between the enzyme and pyridoxal 5'-phosphate that was not observed when HemT was incubated with the cofactor. HemA was found to be sensitive to reagents that modify histidine, arginine and cysteine amino acid residues and the enzyme was also highly sensitive to tryptic cleavage between Arg151 and Ser152 in the presence or absence of PLP and substrates. Antibodies were raised to both HemA and HemT but the respective antisera were not only found to bind both enzymes but also to cross-react with mouse ALAS, indicating that all of the proteins have conserved epitopes.     

Separation and partial characterization of enzymes catalyzing delta-aminolevulinic acid formation in Synechocystis sp. PCC 6803

Formation of the universal tetrapyrrole precursor, delta-aminolevulinic acid (ALA), from glutamate via the five-carbon pathway requires three enzymes: glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde (GSA) aminotransferase. All three enzymes were separated from extracts of the unicellular cyanobacterium Synechocystis sp. PCC 6803, and two of them, glutamyl-tRNA synthetase and GSA aminotransferase, were partially characterized. After an initial high speed centrifugation and differentiatial ammonium sulfate fractionation of cell extract, the enzymes were separated by successive affinity chromatography on Reactive Blue 2-Sepharose and 2',5'-ADP-agarose. All three enzyme fractions were required to reconstitute ALA formation from glutamate. The apparent native molecular masses of glutamyl-tRNA synthetase and GSA aminotransferase were determined by gel filtration chromatography to be 63 and 98 kDa, respectively. Neither glutamyl-tRNA synthetase nor GSA aminotransferase activity was affected by hemin concentrations up to 10 and 30 microM, respectively, and neither activity was affected by protochlorophyllide concentrations up to 2 microM. GSA aminotransferase was inhibited 50% by 0.5 microM gabaculine. The gabaculine inhibition was reversible for up to 1 h after its addition, if the gabaculine was removed by gel filtration before the enzyme was incubated with substrate. However, irreversible inactivation was obtained by preincubating the enzyme at 30 degrees C either for several hours with gabaculine alone or for a few minutes with both gabaculine and GSA. Neither pyridoxal phosphate nor pyridoxamine phosphate significantly affected the activity of GSA aminotransferase at physiologically relevant concentrations, and neither of these compounds reactivated the gabaculine-inactivated enzyme. It was noted that the presence of pyridoxamine phosphate in the ALA assay mixture produced a false positive color reaction even in the absence of enzyme.     

Glutamate recognition and hydride transfer by Escherichia coli glutamyl-tRNA reductase

The initial step of tetrapyrrole biosynthesis in Escherichia coli involves the NADPH-dependent reduction by glutamyl-tRNA reductase (GluTR) of tRNA-bound glutamate to glutamate-1-semialdehyde. We evaluated the contribution of the glutamate moiety of glutamyl-tRNA to substrate specificity in vitro using a range of substrates and enzyme variants. Unexpectedly, we found that tRNA(Glu) mischarged with glutamine was a substrate for purified recombinant GluTR. Similarly unexpectedly, the substitution of amino acid residues involved in glutamate side chain binding (S109A, T49V, R52K) or in stabilizing the arginine 52 glutamate interaction (glutamate 54 and histidine 99) did not abrogate enzyme activity. Replacing glutamine 116 and glutamate 114, involved in glutamate-enzyme interaction near the aminoacyl bond to tRNA(Glu), by leucine and lysine, respectively, however, did abolish reductase activity. We thus propose that the ester bond between glutamate and tRNA(Glu) represents the crucial determinant for substrate recognition by GluTR, whereas the necessity for product release by a 'back door' exit allows for a degree of structural variability in the recognition of the amino acid moiety. Analyzing the esterase activity, which occured in the absence of NADPH, of GluTR variants using the substrate 4-nitrophenyl acetate confirmed the crucial role of cysteine 50 for thioester formation. Finally, the GluTR variant Q116L was observed to lack reductase activity whereas esterase activity was retained. Structure-based molecular modeling indicated that glutamine 116 may be crucial in positioning the nicotinamide group of NADPH to allow for productive hydride transfer to the substrate. Our data thus provide new information about the distinct function of active site residues of GluTR from E. coli.     

Identification of a new class of 5'-adenylylsulfate (APS) reductases from sulfate-assimilating bacteria

A gene was cloned from Burkholderia cepacia DBO1 that is homologous with Escherichia coli cysH encoding 3'-phosphoadenylylsulfate (PAPS) reductase. The B. cepacia gene is the most recent addition to a growing list of cysH homologs from a diverse group of sulfate-assimilating bacteria whose products show greater homology to plant 5'-adenylylsulfate (APS) reductase than they do to E. coli CysH. The evidence reported here shows that the cysH from one of the species, Pseudomonas aeruginosa, encodes APS reductase. It is able to complement an E. coli cysH mutant and a cysC mutant, indicating that the enzyme is able to bypass PAPS, synthesized by the cysC product. Insertional knockout mutation of P. aeruginosa cysH produced cysteine auxotrophy, indicating its role in sulfate assimilation. Purified P. aeruginosa CysH expressed as a His-tagged recombinant protein is able to reduce APS, but not PAPS. The enzyme has a specific activity of 5.8 micromol. min(-1). mg of protein(-1) at pH 8.5 and 30 degrees C with thioredoxin supplied as an electron donor. APS reductase activity was detected in several bacterial species from which the novel type of cysH has been cloned, indicating that this enzyme may be widespread. Although an APS reductase from dissimilatory sulfate-reducing bacteria is known, it shows no structural or sequence homology with the assimilatory-type APS reductase reported here. The results suggest that the dissimilatory and assimilatory APS reductases evolved convergently.     

Coproduction of acetaldehyde and hydrogen during glucose fermentation by Escherichia coli

Escherichia coli K-12 strain MG1655 was engineered to coproduce acetaldehyde and hydrogen during glucose fermentation by the use of exogenous acetyl-coenzyme A (acetyl-CoA) reductase (for the conversion of acetyl-CoA to acetaldehyde) and the native formate hydrogen lyase. A putative acetaldehyde dehydrogenase/acetyl-CoA reductase from Salmonella enterica (SeEutE) was cloned, produced at high levels, and purified by nickel affinity chromatography. In vitro assays showed that this enzyme had both acetaldehyde dehydrogenase activity (68.07 ± 1.63 μmol min(-1) mg(-1)) and the desired acetyl-CoA reductase activity (49.23 ± 2.88 μmol min(-1) mg(-1)). The eutE gene was engineered into an E. coli mutant lacking native glucose fermentation pathways (ΔadhE, ΔackA-pta, ΔldhA, and ΔfrdC). The engineered strain (ZH88) produced 4.91 ± 0.29 mM acetaldehyde while consuming 11.05 mM glucose but also produced 6.44 ± 0.26 mM ethanol. Studies showed that ethanol was produced by an unknown alcohol dehydrogenase(s) that converted the acetaldehyde produced by SeEutE to ethanol. Allyl alcohol was used to select for mutants with reduced alcohol dehydrogenase activity. Three allyl alcohol-resistant mutants were isolated; all produced more acetaldehyde and less ethanol than ZH88. It was also found that modifying the growth medium by adding 1 g of yeast extract/liter and lowering the pH to 6.0 further increased the coproduction of acetaldehyde and hydrogen. Under optimal conditions, strain ZH136 converted glucose to acetaldehyde and hydrogen in a 1:1 ratio with a specific acetaldehyde production rate of 0.68 ± 0.20 g h(-1) g(-1) dry cell weight and at 86% of the maximum theoretical yield. This specific production rate is the highest reported thus far and is promising for industrial application. The possibility of a more efficient "no-distill" ethanol fermentation procedure based on the coproduction of acetaldehyde and hydrogen is discussed.     

Purification of glutamyl-tRNA reductase from Synechocystis sp. PCC 6803

delta-Aminolevulinic acid is the universal precursor for all tetrapyrroles including hemes, chlorophylls, and bilins. In plants, algae, cyanobacteria, and many other bacteria, delta-aminolevulinic acid is synthesized from glutamate in a reaction sequence that requires three enzymes, ATP, NADPH, and tRNA(Glu). The three enzymes have been characterized as glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde aminotransferase. All three enzymes have been separated and partially characterized from plants and algae. In prokaryotic phototrophs, only the glutamyl-tRNA synthetase and glutamate-1-semialdehyde aminotransferase have been decribed. We report here the purification and some properties of the glutamyl-tRNA reductase from extracts of the unicellular cyanobacterium, Synechocystis sp. PCC 6803. The glutamyl-tRNA reductase has been purified over 370-fold to apparent homogeneity. Its native molecular mass was determined to be 350 kDa by glycerol density gradient centrifugation, and its subunit size was estimated to be 39 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was determined for 42 residues. Much higher activity occurred with NADPH than with NADH as the reduced pyridine nucleotide substrate. Half-maximal rates occurred at 5 microM NADPH, whereas saturation was not reached even at 10 mM NADH. Purified Synechocystis glutamyl-tRNA reductase was inhibited 50% by 5 microM heme. Activity was unaffected by 10 microM 3-amino-2,3-dihydrobenzoic acid. No flavin, pyridine nucleotide, or other light-absorbing prosthetic group was detected on the purified enzyme. The catalytic turnover number of purified Synechocystis glutamyl-tRNA reductase is comparable to those of prokaryotic and plastidic glutamyl-tRNA synthetases.     

A heme-deficient strain of Escherichia coli has a three-base pair deletion in a "hotspot" in hemA

The key regulatory step in heme biosynthesis in Escherichia coli is at the level of glutamyl-tRNA reductase (GTR), an enzyme which is encoded by hemA. A strain, HU227, with a spontaneous in-frame mutation in hemA has no GTR activity. The mutation is shown to be a three-base deletion at a "hotspot" in the gene. The amino acid sequence in this region is highly conserved.     

Complex formation between glutamyl-tRNA synthetase and glutamyl-tRNA reductase during the tRNA-dependent synthesis of 5-aminolevulinic acid in Chlamydomonas reinhardtii.

The formation of a stable complex between glutamyl-tRNA synthetase and the first enzyme of chlorophyll biosynthesis glutamyl-tRNA reductase was investigated in the green alga Chlamydomonas reinhardtii. Apparently homogenous enzymes, purified after previously established purification protocols were incubated in various combinations with ATP, glutamate, tRNA(Glu) and NADPH and formed complexes were isolated via glycerol gradient centrifugation. Stable complexes were detected only after the preincubation of glutamyl-tRNA synthetase, glutamyl-tRNA reductase with either glutamyl-tRNA or free tRNA(Glu), ATP and glutamate, indicating the obligatory requirement of aminoacylated tRNA(Glu) for complex formation. The further addition of NADPH resulting in the reduction of the tRNA-bound glutamate to glutamate 1-semialdehyde led to the dissociation of the complex. Once complexed to the two enzymes tRNA(Glu) was found to be partially protected from ribonuclease digestion. Escherichia coli, Bacillus subtilis and Synechocystis 6803 tRNA(Glu) were efficiently incorporated into the protein-RNA complex. The detected complexes provide the chloroplast with a potential channeling mechanism for Glu-tRNA(Glu) into chlorophyll synthesis in order to compete with the chloroplastic protein synthesis machinery.     

Identification of the enzymatic basis for delta-aminolevulinic acid auxotrophy in a hemA mutant of Escherichia coli.

The hemA mutation of Escherichia coli K-12 confers a requirement for delta-aminolevulinic acid (ALA). Cell extract prepared from the hemA strain SASX41B was incapable of producing ALA from either glutamate or glutamyl-tRNA, whereas extract of the hem+ strain HB101 formed colorimetrically detectable amounts of ALA and transferred label from 1-[14C]glutamate and 3,4-[3H]glutamyl-tRNA to ALA. Extracts of both strains converted glutamate-1-semialdehyde to ALA and were capable of aminoacylating tRNAGlu. Glutamyl-tRNA formed by extracts of both strains could be converted to ALA by the extract of hem+ cells. The extract of hemA cells did not convert glutamyl-tRNA formed by either strain to ALA. However, the hemA cell extract, when supplemented in vitro with glutamyl-tRNA dehydrogenase isolated from Chlorella vulgaris cells, formed about as much ALA as did the unsupplemented hem+ cell extract. We conclude from these observations that the enzyme activity that is lacking in the ALA auxotrophic strain carrying the hemA mutation is that of glutamyl-tRNA dehydrogenase.     

Bovine liver dihydrofolate reductase: purification and properties of the enzyme.

A purification procedure is reported for obtaining bovine liver dihydrofolate reductase in high yield and amounts of 100-200 mg. A key step in the procedure is the use of an affinity gel prepared by coupling pteroyl-L-lysine to Sepharose. The purified reductase has a specific activity of about 100 units/mg and is homogeneous as judged by analytical ultracentrifugation, polyacrylamide gel electrophoresis, and titration with methotrexate. The products of the first step of Edman degradation indicated a minimum purity of 79%. The reductase has a molecular weight of about 21500 on the basis of amino acid composition and 22100 +/- 300 from equilibrium sedimentation. It is not inhibited by antiserum to the Streptococcus faecium reductase (isoenzyme 2). Unlike the reductase of many other vertebrate tissues, the bovine enzyme is inhibited by mercurials rather than activated and it has a single pH optimum at both low and high ionic strength. However, the position of the pH optimum is shifted and the activity increased by increasing ionic strength. Automatic Edman degradation has been used to determine 34 of the amino-terminal 37 amino acid residues. Considerable homology exists between this region and the corresponding regions of the reductase from S. faecium and from Escherichia coli. This strengthens the idea that this region contributes to the structure of the binding site for dihydrofolate.     

R plasmid dihydrofolate reductase with a dimeric subunit structure

Dihydrofolate reductase specified by plasmid R483 from a trimethoprim-resistant strain of Escherichia coli has been purified 2,000-fold to homogeneity using dye-ligand chromatography, gel filtration, and polyacrylamide gel electrophoresis. The protein migrated as a single band on nondenaturing polyacrylamide gel electrophoresis and had a specific activity of 250 mumol/mg min(-1). The molecular weight was estimated to be 32,000 by gel filtration and 39,000 by Ferguson analysis of polyacrylamide gel electrophoresis. When subjected to electrophoresis in the presence of sodium dodecyl sulfate, the protein migrated as a single 19,000-molecular weight species, a fact that suggests that the native enzyme is a dimer of similar or identical subunits. Antibody specific for R483-encoded dihydrofolate reductase did not cross-react with dihydrofolate reductase encoded by plasmid R67, T4 phage, E. coli RT500, or mouse L1210 leukemia cells. The amino acid sequence of the first 34 NH2-terminal residues suggests that the R483 plasmid dihydrofolate reductase is more closely related to the chromosomal dihydrofolate reductase than is the enzyme coded by plasmid R67.     

Cloning, expression, and characterization of Mycobacterium tuberculosis dihydrofolate reductase

The gene for dihydrofolate reductase of Mycobacterium tuberculosis was amplified by polymerase chain reaction (PCR) from M. tuberculosis H37Rv strain genomic DNA. The protein was expressed in inclusion bodies in high yield in Escherichia coli under the control of the T7 promoter. Active enzyme was obtained by refolding from guanidine HCl and after a single chromatography step the sample was > 99% homogeneous with a specific activity of approximately 15.5 micromol min(-1) mg(-1). Mass spectrometry analysis confirmed the expected mass of 17.6 kDa. Gel filtration of the enzyme indicated that it was a monomer. Steady-state kinetic parameters were determined and the effect of pH and KCl on the enzyme examined. Methotrexate and trimethoprim inhibited the enzyme.     

Enzymatic basis of thiol-stimulated secretion of porphyrins by Escherichia coli.

1-Thioglycerol (TG) stimulates the synthesis of porphyrin in aerobically growing Escherichia coli. Here the levels of delta-aminolevulinate biosynthetic enzymes in untreated and TG-treated E. coli THU and PUC2 (a mutant of THU which overproduces porphyrins in the presence of thiols) cells were determined. TG treatment elevated the activity of glutamyl-tRNA reductase in both strains. The increased activity was not caused by activation of preexisting enzymes by thiols or by oxidizing agents but was dependent on new protein synthesis.     

Characterization of sulfate assimilation in marine algae focusing on the enzyme 5'-adenylylsulfate reductase

5'-Adenylylsulfate (APS) reductase was characterized in diverse marine algae. A cDNA encoding APS reductase from Enteromorpha intestinalis (EAPR) was cloned by functional complementation of an Escherichia coli cysH mutant. The deduced amino acid sequence shows high homology with APS reductase (APR) from flowering plants. Based on the probable transit peptide cleavage site the mature protein is 45.7 kD. EAPR expressed as a His-tagged recombinant protein catalyzes reduced glutathione-dependent reduction of APS to sulfite, exhibiting a specific activity of approximately 40 micromol min(-1) mg protein(-1) and Michealis-Menten kinetic constants of approximately 1.4 mM for reduced glutathione and approximately 6.5 microM for APS. APR activity and expression were studied in relation to the production of 3-dimethylsulfoniopropionate (DMSP), a sulfonium compound produced by many marine algae. A diverse group of DMSP-producing species showed extremely high enzyme activity (up to 400 times that found in flowering plants). Antibodies raised against a conserved peptide of APR strongly cross-reacted with a protein of 45 kD in several chlorophytes but insignificantly with chromophytes. In the chlorophyte Tetraselmis sp., APR activity varies significantly during the culture cycle and does not follow the changes in cellular DMSP content. However, a positive correlation was found between cell-based APR activity and specific growth rate.     

Purification and properties of nitrite reductase from Escherichia coli K12.

NADH-nitrite oxidoreductase (EC 1.6.4) was purified to better than 95% homogeneity from batch cultures of Escherichia coli strain OR75Ch15, which is partially constitutive for nitrite reductase synthesis. Yields of purified enzyme were low, mainly because of a large loss of activity during chromatography on DEAE-cellulose. The quantitative separation of cytochrome c-552 from nitrite reductase activity resulted in an increase in the specific activity of the enzyme: this cytochrome is not therefore an integral part of nitrite reductase. The subunit molecular weights of nitrite reductase and of a haemoprotein contaminant, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, were 88000 and 80000 respectively. The sedimentation coefficient was calculated to be in the range 8.5-9.5S, consistent with a mol.wt. of 190000. It is suggested therefore that the native enzyme is a dimer with two identical or similar-sized subunits. Purest samples contained 0.4 mol of flavin/mol of enzyme, but no detectable haem. Catalytic activity was totally inhibited by 20 micron-p-chloromercuribenzoate and 1 mM-cyanide, slightly inhibited by 1 micron-sulphite and 10mM-arsenite, but insensitive to 1 mM-2,2'-bipyridine, 4mM-1,10-phenanthroline and 10mM-NaN3. Three molecules of NADH were oxidized for each NO2-ion reduced: the product of the reaction is therefore assumed to be NH4+. The specific activity of hydroxylamine reductase increased at each step in the purification of nitrite reductase, and the elution profiles for these two activities during chromatography on DEAE-Sephadex were coincident. It is likely that a single enzyme is responsible for both activities.     

V-shaped structure of glutamyl-tRNA reductase, the first enzyme of tRNA-dependent tetrapyrrole biosynthesis.

Processes vital to life such as respiration and photosynthesis critically depend on the availability of tetrapyrroles including hemes and chlorophylls. tRNA-dependent catalysis generally is associated with protein biosynthesis. An exception is the reduction of glutamyl-tRNA to glutamate-1-semialdehyde by the enzyme glutamyl-tRNA reductase. This reaction is the indispensable initiating step of tetrapyrrole biosynthesis in plants and most prokaryotes. The crystal structure of glutamyl-tRNA reductase from the archaeon Methanopyrus kandleri in complex with the substrate-like inhibitor glutamycin at 1.9 A resolution reveals an extended yet planar V-shaped dimer. The well defined interactions of the inhibitor with the active site support a thioester-mediated reduction process. Modeling the glutamyl-tRNA onto each monomer reveals an extensive protein-tRNA interface. We furthermore propose a model whereby the large void of glutamyl-tRNA reductase is occupied by glutamate-1-semialdehyde-1,2-mutase, the subsequent enzyme of this pathway, allowing for the efficient synthesis of 5-aminolevulinic acid, the common precursor of all tetrapyrroles.