Isolation of the domain containing the molybdenum, iron-sulfur I, and iron-sulfur II centers of chicken liver xanthine dehydrogenase.

Chicken liver xanthine dehydrogenase, like other xanthine-oxidizing enzymes, is a dimer of Mr = 150,000 subunits. Each subunit contains one molybdenum, one FAD, and two distinct Fe2S2 centers. Treatment with a number of proteases shows that the native enzyme subunit is cleaved at three distinct sites. However, the cleavage products can be separated only under denaturing conditions. Prolonged treatment with subtilisin at pH 10.1 has permitted the isolation of an Mr = 65,000 catalytically active fragment that is devoid of FAD but which contains the molybdenum and both types of iron-sulfur center. A model of the domain structure of the native enzyme is proposed.     

Purification,characterization, and cloning of a bifunctional molybdoenzyme with hydratase and alcohol dehydrogenase activity

A bifunctional hydratase/alcohol dehydrogenase was isolated from the cyclohexanol degrading bacterium Alicycliphilus denitrificans DSMZ 14773. The enzyme catalyzes the addition of water to α,β-unsaturated carbonyl compounds and the subsequent alcohol oxidation. The purified enzyme showed three subunits in SDS gel, and the gene sequence revealed that this enzyme belongs to the molybdopterin binding oxidoreductase family containing molybdopterins, FAD, and iron-sulfur clusters.     

8-Hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum: 1. Purification and characterization

The 8-hydroxy-5-deazaflavin (coenzyme F420) reducing hydrogenase from the obligate anaerobe Methanobacterium thermoautotrophicum delta H has been purified 41-fold to apparent homogeneity. The major active enzyme form is a high molecular weight aggregate of Mr ca. 800,000, composed of three subunits, alpha (Mr 47K), beta (Mr 31K), and gamma (Mr 26K). The hydrogenase is purified aerobically in reversibly inhibited form, and conditions for anaerobic reductive activation with H2, high salt, thiols, and electron acceptors have been defined. The minimal species transferring electrons from H2 to coenzyme F420 appears to be an alpha beta delta (Mr 115K) complex. The tightly associated redox cofactors per 115K species are 0.6-0.7 nickel atom, 0.8-0.9 flavin adenine dinucleotide (FAD), and 13-14 iron atoms in iron-sulfur centers. The subunits have been separated by denaturing gel electrophoresis, which has permitted determination of amino acid composition, subunit N-terminal sequencing, and preparation of subunit-directed antibodies. There is iron associated with the alpha-subunit, but placement of the nickel and FAD has not been established.     

Cloning, expression, and nucleotide sequence of the formate dehydrogenase genes from Methanobacterium formicicum

The genes for the two subunits of the formate dehydrogenase from Methanobacterium formicicum were cloned and their sequences determined. When expressed in Escherichia coli, two proteins were produced which had the appropriate mobility on an SDS gel for the two subunits of formate dehydrogenase and cross-reacted with antibodies raised to purified formate dehydrogenase. The genes for the two formate dehydrogenase subunits overlap by 1 base pair and are preceded by DNA sequences similar to both eubacterial and archaebacterial promoters and ribosome-binding sites. The amino acid sequences deduced from the DNA sequence were analyzed, and the arrangement of putative iron-sulfur centers is discussed.     

Bacillus subtilis mutant succinate dehydrogenase lacking covalently bound flavin: identification of the primary defect and studies on the iron-sulfur clusters in mutated and wild-type enzyme

Succinate dehydrogenase consists of two protein subunits and contains one FAD and three iron-sulfur clusters. The flavin is covalently bound to a histidine in the larger, Fp, subunit. The reduction oxidation midpoint potentials of the clusters designated S-1, S-2, and S-3 in Bacillus subtilis wild-type membrane-bound enzyme were determined as +80, -240, and -25 mV, respectively. Magnetic spin interactions between clusters S-1 and S-2 and between S-1 and S-3 were detected by using EPR spectroscopy. The point mutations of four B. subtilis mutants with defective Fp subunits were mapped. The gene of the mutant specifically lacking covalently bound flavin in the enzyme was cloned. The mutation was determined from the DNA sequence as a glycine to aspartate substitution at a conserved site seven residues downstream from the histidine that binds the flavin in wild-type enzyme. The redox midpoint potential of the iron-sulfur clusters and the magnetic spin interactions in mutated succinate dehydrogenases were indistinguishable from the those of the wild type. This shows that flavin has no role in the measured magnetic spin interactions or in the structure and stability of the iron-sulfur clusters. It is concluded from sequence and mutant studies that conserved amino acid residues around the histidyl-FAD are important for FAD binding; however, amino acids located more than 100 residues downstream from the histidyl in the Fp subunit can also effect flavinylation.     

Relationship between carbon monoxide dehydrogenase and a small plasmid in Pseudomonas carboxydovorans

Abstract Isolation of plasmid DNA followed by plasmid curing was carried out to examine the relationship of plasmid to carbon monoxide dehydrogenase (CO-DH) production in carboxydobacteria. A small plasmid of almost identical size (1.52−1.76 × 10⁶) was present in Pseudomonas carboxydovorans, Azotobacter sp.1, and Azomonas sp.2. Azomonas sp.1 contained two kinds of plasmids (1.5 × 10⁶ and 2.47 × 10⁶). No plasmids were found in Pseudomonas carboxydohydrogena , JC1, and HY1. A plasmid-cured clone of P. carboxydovorans was obtained by growing the cells at 37°C. The cured cell was able to grow CO autotrophically on solid, but not in liquid, medium. CO-DH of the cured cell was active and consisted of three subunits similar to those found in the wild-type enzyme, with the exception that the β subunit of the enzyme was larger than that of the wild-type enzyme. These results suggest that the small plasmids do not carry genes encoding CO-DH but may have gene(s) for processing the β subunit of the enzyme.     

Purification and characterization of multiple forms of rat liver xanthine oxidoreductase expressed in baculovirus-insect cell system

cDNA of rat liver xanthine oxidoreductase (XOR), a molybdenum-containing iron-sulfur flavoprotein, was expressed in a baculovirus-insect cell system. The expressed XOR consisted of a heterogeneous mixture of native dimeric, demolybdo-dimeric, and monomeric forms, each of which was separated and purified to homogeneity. All the expressed forms contained flavin, of which the semiquinone form was stable during dithionite titration after dithiothreitol treatment, indicating that the flavin domains of all the expressed molecules have the intact conformations interconvertible between NAD(+)-dependent dehydrogenase (XDH) and O(2)-dependent oxidase (XO) types. The absorption spectrum and metal analyses showed that the monomeric form lacks not only molybdopterin but also one of the iron-sulfur centers. The reductive titration of the monomer with dithionite showed that the monomeric form required only three electrons for complete reduction, and the redox potential of the iron-sulfur center in the monomeric form is a lower value than that of FAD. In contrast to native or demolybdo-dimeric XDHs, the monomer showed a very slow reductive process with NADH under anaerobic conditions, although the conformation around FAD is a dehydrogenase form, suggesting the important role of the iron-sulfur center in the reductive process of FAD with the reduced pyridine nucleotide.     

Isolation, amino acid analysis and N-terminal sequence determination of the two subunits of the nickel-containing hydrogenase of Desulfovibrio gigas

The two subunits of the nickel-iron hydrogenase from Desulfovibrio gigas have been purified by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and their amino acid compositions have been determined. The N-terminal sequences for 15 residues of the large subunit (Mr 62,000) and 25 residues of the small subunit (Mr 26,000), respectively, were established. The occurrence of several cysteine residues in the small subunit is discussed in relation with their possible role in the binding of the redox centers of the enzyme.     

Photosystem II of rye. Nucleotide sequence of the psbB, psbC, psbE, psbF, psbH genes of rye and chloroplast DNA regions adjacent to them]

In order to determine structures of the barley photosystem II subunits, the following genes have been cloned: psbB, encoding 47 kDa chlorophyll-binding subunit; psbH, encoding 7.7 kDa phosphoprotein; psbE and psbF, encoding 9.3 and 4.4 kDa subunits of the cytochrome b559 apoprotein, respectively; and a fragment of psbC gene, encoding the 43 kDa chlorophyll-binding subunit. The nucleotide sequences of these genes and the deduced amino acid sequences of their products are highly homologous to the corresponding sequences for other plant species.     

Cloning and expression of the genes of two fumarate reductase subunits from Wolinella succinogenes

The fumarate reductase complex of the anaerobic bacterium Wolinella succinogenes catalyzes the electron transfer from menaquinol to fumarate. Two structural genes coding for subunits of the enzyme have been cloned in Escherichia coli. The genes were isolated from a lambda EMBL3 phage gene bank by immunological screening and subcloned in an expression vector. The genes frdA and frdB, which encode the FAD protein (Frd A, Mr 79,000) and the iron-sulfur protein (Frd B, Mr 31,000) of the fumarate reductase complex, were cloned together with a W. succinogenes promoter. The gene order was promoter-frdA-frdB. The FAD protein and the iron-sulfur protein were expressed in the correct molar mass in E. coli from the clones. The identity of the frdA gene and the suggested polarity were confirmed by comparing the amino-terminal sequence of the Frd A protein with that predicted from the 5'-terminal nucleotide sequence of frdA. The frdA and frdB genes are present only once in the genome. A region downstream of frdB, possibly a gene encoding cytochrome b of the fumarate reductase complex, hybridizes with a second site in the genome.     

Microbial metabolism of quinoline and related compounds. XII. Isolation and characterization of the quinoline oxidoreductase from Rhodococcus spec. B1 compared with the quinoline oxidoreductase from Pseudomonas putida 86.

Quinoline oxidoreductase from Rhodococcus spec. B1 was purified 39-fold to apparent homogeneity in a 5-step procedure with a recovery of 26%. The Mr of the native enzyme as determined by gel chromatography was 300,000. SDS polyacrylamide gel electrophoresis of the enzyme revealed 3 protein bands corresponding to Mr 82,000, 32,000, and 18,000. The enzyme contains 1.3 atoms of molybdenum, 8 atoms of iron, 8 atoms of acid-labile sulphur, 2 molecules of FAD and 2 molecules of molybdopterin cytosine dinucleotide. Cyanide, 4-hydroxymercuribenzoate and methanol were effective as inhibitors. The amino-terminal protein sequences of the 3 subunits of quinoline oxidoreductase from Rhodococcus B1 compared to those of quinoline oxidoreductase from Pseudomonas putida 86 revealed no difference among 71 amino acids examined.     

Nucleotide sequence encoding the flavoprotein and iron-sulfur protein subunits of the Bacillus subtilis PY79 succinate dehydrogenase complex.

The nucleotide sequence of a 2.7-kilobase segment of DNA containing the sdhA and sdhB genes encoding the flavoprotein (Fp, sdhA) and iron-sulfur protein (Ip, sdhB) subunits of the succinate dehydrogenase of Bacillus subtilis was determined. This sequence extends the previously reported sequence encoding the cytochrome b558 subunit (sdhC) and completes the sequence of the sdh operon, sdhCAB. The predicted molecular weights for the Fp and Ip subunits, 65,186 (585 amino acids) and 28,285 (252 amino acids), agreed with the values determined independently for the labeled Fp and Ip antigens, although it appeared that the B. subtilis Fp was not functional after expression of the sdhA gene in Escherichia coli. Both subunits closely resembled the corresponding Fp and Ip subunits of the succinate dehydrogenase (SDH) and fumarate reductase of E. coli in size, composition, and amino acid sequence. The sequence homologies further indicated that the B. subtilis SDH subunits are equally related to the SDH and fumarate reductase subunits of E. coli but are less closely related than are the corresponding pairs of E. coli subunits. The regions of highest sequence conservation were identifiable as the catalytically significant flavin adenine dinucleotide-binding sites and cysteine clusters of the iron-sulfur centers.     

Nucleotide sequence of the pntA and pntB genes encoding the pyridine nucleotide transhydrogenase of Escherichia coli

A 3240-base-pair DNA fragment spanning the pyridine nucleotide transhydrogenase (pnt) genes of Escherichia coli has been sequenced. The sequence contains two open-reading frames, pntA and pntB of 1506 and 1386 base pairs, coding for the transhydrogenase alpha and beta subunits, respectively. The coding sequences are preceded by a promoter-like structure and are most likely co-transcribed. Each coding sequence is preceded by a Shine-Dalgarno sequence. The amino-terminal amino acid sequences were determined from the purified alpha and beta subunits of the transhydrogenase. These sequences agree with those predicted from the nucleotide sequences of the pntA and pntB genes. The predicted relative molecular masses of 53906 (alpha) and 48667 (beta) are close to the values obtained by analysis of the subunits by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Several hydrophobic regions large enough to span the cytoplasmic membrane were observed in each subunit. These results indicate that transhydrogenase is an intrinsic membrane protein.     

Structural analysis and molybdenum-dependent expression of the pAO1-encoded nicotine dehydrogenase genes of Arthrobacter nicotinovorans

The genes of nicotine dehydrogenase (NDH) were identified, cloned and sequenced from the catabolic plasmid pA01 of Arthrobacter nicotinovorans. In immediate proximity to this gene cluster is the beginning of the 6-hydroxy-L-niotine oxidase (6-HLNO) gene. NDH is composed of three subunits (A, B and C) of M_r 30011, 14924 and 87677. It belongs to a family of bacterial hydroxylases with a similar subunit structure; they have molybdopterin dinucleotide, FAD and Fe-S clusters as cofactors. Here the first complete primary structure of a bacterial hydroxylase is provided. Sequence alignments of each of the NDH subunits show similarities to the sequences of eukaryotic xanthine dehydrogenase (XDH) but not to other known molybdenum-containing bacterial enzymes. Based on alignment with XDH it is inferred that the smallest subunit (NDHB) carries an iron-sulphur cluster, that the middle-sized subunit (NDHA) binds FAD, and that the largest NDH subunit (NDHC) corresponds to the molybdopterin-binding domain of XDH. Expression of both the ndh and the 6-hlno genes required the presence of nicotine and molybdenum in the culture medium. Tungsten inhibited enzyme activity but not the synthesis of the enzyme protein. The enzyme was found in A. nicotinovorans cells in a soluble form and in a membrane-associated form. In the presence of tungsten the fraction of membrane-associated NDH increased.     

Cloning and sequencing of the genes encoding the large and small subunits of the periplasmic (NiFeSe) hydrogenase of Desulfovibrio baculatus.

The genes coding for the large and small subunits of the periplasmic hydrogenase from Desulfovibrio baculatus have been cloned and sequenced. The genes are arranged in an operon with the small subunit gene preceding the large subunit gene. The small subunit gene codes for a 32 amino acid leader sequence supporting the periplasmic localization of the protein, however no ferredoxin-like or other characteristic iron-sulfur coordination sites were observed. The periplasmic hydrogenases from D. baculatus (an NiFeSe protein) and D. vulgaris (an Fe protein) exhibit no homology suggesting that they are structurally different, unrelated entities.     

Functional properties of recombinant Azospirillum brasilense glutamate synthase, a complex iron-sulfur flavoprotein.

Azospirillum brasilense glutamate synthase is a complex iron-sulfur flavoprotein that catalyses the NADPH-dependent reductive transfer of glutamine amide group to the C(2) carbon of 2-oxoglutarate to yield L-glutamate. Its catalytically active alphabeta protomer is composed of two dissimilar subunits (alpha subunit, 164.2 kDa; beta subunit, 52.3 kDa) and contains one FAD (at Site 1, the pyridine nucleotide site within the beta subunit), one FMN (at Site 2, the 2-oxoglutarate/L-glutamate site in the alpha subunit) and three different iron-sulfur clusters (one 3Fe-4S center on the alpha subunit and two 4Fe-4S clusters of unknown location). A plasmid harboring the gltD and gltB genes, the genes encoding the glutamate synthase beta and alpha subunits, respectively, each one under the control of the T7/lac promoter of pET11a was found to be suitable for the overproduction of glutamate synthase holoenzyme in Escherichia coli BL21(DE3) cells. Recombinant A. brasilense glutamate synthase could be purified to homogeneity from overproducing E. coli cells by ion exchange chromatography, gel filtration and affinity chromatography on a 2',5' ADP-Sepharose 4B column. The purified enzyme was indistinguishable from that prepared from Azospirillum cells with respect to cofactor content, N-terminal sequence of the subunits, aggregation state, kinetic and spectroscopic properties. The study of the recombinant holoenzyme allowed us to establish that the tendency of glutamate synthase to form a stable (alphabeta)4 tetramer at high protein concentrations is a property unique to the holoenzyme, as the isolated beta subunit does not oligomerize, while the isolated glutamate synthase alpha subunit only forms dimers at high protein concentrations. Furthermore, the steady-state kinetic analysis of the glutamate synthase reaction was extended to the study of the effect of adenosine-containing nucleotides. Compounds such as cAMP, AMP, ADP and ATP have no effect on the enzyme activity, while the 2'-phosphorylated analogs of AMP and NADP(H) analogs act as inhibitors of the reaction, competitive with NADPH. Thus, it can be ruled out that glutamate synthase reaction is subjected to allosteric modulation by adenosine containing (di)nucleotides, which may bind to the putative ADP-binding site at the C-terminus of the alpha subunit. At the same time, the strict requirement of a 2'-phosphate group in the pyridine nucleotide for binding to glutamate synthase (GltS) was established. Finally, by comparing the inhibition constants exhibited by a series of NADP+ analogs, the contribution to the binding energy of the various parts of the pyridine nucleotide has been determined along with the effect of substituents on the 3 position of the pyridine ring. With the exception of thio-NADP+, which binds the tightest to GltS, it appears that the size of the substituent is the factor that affects the most the interaction between the NADP(H) analog and the enzyme.     

Nucleotide sequences of the genes for the alpha,beta and epsilon subunits of wheat chloroplast ATP synthase

Summary The nucleotide sequences of the chloroplast genes for the alpha, beta and epsilon subunits of wheat chloroplast ATP synthase have been determined. Open reading frames of 1512 bp, 1494 bp and 411 bp are deduced to code for polypeptides of molecular weights 55201, 53796 and 15200, identified as the alpha, beta and epsilon subunits respectively by homology with the subunits from other sources and by amino acid sequencing of the epsilon subunit. The genes for the beta and epsilon subunits overlap by 4 bp. The gene for methionine tRNA is located 118 bp downstream from the epsilon subunit gene. Comparisons of the deduced amino acid sequences of the alpha and beta subunits with those from other species suggest regions of the proteins involved in adenine nucleotide binding.     

Beta-conglycinin from soybean proteins. Isolation and immunological and physicochemical properties of the monomeric forms

Beta-conglycinin consisting of six major isomers (designated B1- to B6-conglycinin) was dissociated and fractionated on columns of DEAE- and CM-Sephadex in buffers containing 6 M urea. Three major (alpha, alpha' and beta) and one minor (gamma) subunits were isolated and further characterized by gel electrophoresis and gel electrofocusing. Gel electrophoresis in urea and in sodium dodecyl sulfate, and gel filtration in 6 M guanidine hydrochloride gave a molecular weight of 57 000 for alpha, alpha' subunits; and 42 000 for beta and gamma subunits. The isoelectric points of the isolated subunits, measured by disc gel electrofocusing, were as follows: alpha, 4.90; alpha', 5.18; beta, 5.66-6.00. On gel electrofocusing, beta subunit showed four microheterogeneous components; three of them comprised 95% of the total beta subunit. Leucine and valine were the N-terminal amino acids of beta and alpha alpha' subunits, respectively. The isolated subunits contained mannose and glucosamine in varying quantities. Two carbohydrate moieties were calculated for one mole of alpha, alpha' subunits; and one carbohydrate moiety for the beta subunit. Considerable similarity in the amino acid composition of alpha and alpha' subunits was observed. The beta subunit was devoid of cysteine and methionine; and in comparison with alpha, alpha' subunits, had a higher content of hydrophobic amino acids. The isolated subunits exhibited antigen-antibody reaction with antisera to the native beta-conglycinin. Each of them was partglycinins. The alpha and alpha' subunits were in addition identical with each other and with B5-, B6-conglycinins. They were immunologically unrelated with beta subunit. The recovery of immuno-properties from the individual subunits may be attributed to the reconstruction of the three-dimensional structure upon removal of denaturing reagents.