The complete amino-acid sequence of the rieske FeS-precursor protein from spinach chloroplasts deduced from cDNA analysis

Summary SummarySeveral cDNA clones encoding the entire Rieske FeS-precursor protein of the chloroplast cytochrome b ₆ f-complex have been isolated by high density plaque immunoscreening of a phage lambda gt11 cDNA expression library, made from poly A⁺-RNA of spinach seedlings. The identity of the cDNAs has been confirmed by N-terminal amino acid sequencing of the purified protein. The nucleotide sequence indicates a protein of 247 amino acid residues including a putative transit sequence of 68 amino acids corresponding to molecular masses of 26.3 kDa (precursor) and 18.8 kDa (mature protein; 179 amino acid residues). Alignteins of the sequence with sequences from Rieske FeS-proteins of respiratory electron transport chains, two of bacterial and three of mitochondrial origin, shows little sequence homology, but remarkable similarity in secondary structure including a putative N-terminal transmembrane segment of about 25 residues and the peptides CTHLGCV and CPCHGS in the C-terminal region of the protein that are involved in the binding of the Fe₂S₂-cluster.     

A Novel Rieske Iron-Sulfur Protein from the Hyperthermophilic Crenarchaeon Pyrobaculum aerophilum: Sequencing of the Gene, Expression in E. coli and Characterization of the Protein

The crenarchaeon Pyrobaculum aerophilum is with an optimalgrowth temperature of 100 °C one of the most thermophilic organisms knownto possess an aerobic respiratory chain. The analysis of DNA sequences fromthe Pyrobaculum genome project lead to the identification of an openreading frame potentially coding for a Rieske iron-sulfur protein. Thecomplete gene (named parR) was cloned and sequenced. The deducedamino acid sequence displays unusual amino acid exchanges and a so farunknown sequence insertion. The N-terminus shows similarities to bacterialsignal sequences. Several forms of the gene were expressed in E.coli in order to verify the classification as a Rieske protein and tofacilitate biophysical studies. Soluble, thermo-stable proteins withcorrectly inserted iron-sulfur clusters were expressed from two versions ofthe gene. The 1–23 truncated holo-protein is redox active. Itdisplays the typical spectroscopic properties of a Rieske protein. The redoxpotential was determined to be +215 mV at pH 6.5 and is pH dependentabove pH 7.5 revealing the influence of two protonation equilibria with pKavalues of 8.1 and 9.8. Phylogenetic analysis demonstrates that the parRprotein clusters together with the two other available archaeal Rieskesequences from Sulfolobus on a separate branch of the phylogenetictree apart from the proteins from thermophilic bacteria like Aquifexand Thermus.     

The cloning and sequencing of Synechococcus sp. PCC 7002 petCA operon: Implications for the cytochrome c-553 binding domain of cytochrome f

The genes encoding the Rieske iron-sulfur protein and cytochrome f from a unicellular, naturally transformable, photoheterotrophic cyanobacterium, Synechococcus sp. PCC 7002, formerly Agmenellum quadruplicatum, have been isolated and sequenced. The two genes were found to be on a single operon, petCA.The Synechococcus sp. PCC 7002 iron-sulfur protein contains 181 amino acids, the conserved putative iron-binding domains CTHLGCV, residues 108–114, and CPCHGS, residues 128–133, no presequence and has a 73% sequence identity to the Nostoc PCC 7906 iron-sulfur protein. The 325 amino acid apocytochrome f sequence contains a 42 amino acid presequence, a CANCH heme binding domain, residues 20–24 from the presumed start of the mature protein, and a predicted hydrophobic membrane-spanning domain, residues 250–269. The mature cytochrome f sequence has a 71.5% sequence identity with Nostoc PCC 7906 cytochrome f and possesses a large (-14) negative charge and low calculated pI of 4.47 compared to higher plant chloroplast sequences. Nine separate domains showing differences in charged residues among cyanobacteria and plants have been identified and the possibility that these domains are involved in the ionic interactions with plastocyanin or cytochrome c-553 is discussed.The sequences reported in this paper have been deposited in the EMBL/Genbank data base (IntelliGenetics, Mountain View, CA, and Eur. Mol. Biol. Lab., Heidelberg) (accession no. M74514).     

A Rieske ferredoxin typifying a subtype within Rieske proteins: spectroscopic, biochemical and stability studies

A new subtype of archaeal Rieske ferredoxin (RFd) has been identified in the genome of the thermoacidophilic archaeon Acidianus ambivalens. The gene is inserted in an atypical genomic context in a gene cluster encoding a NiFe hydrogenase. Sequence and phyletic analysis showed that the protein is related to bacterial RFd but not to any of the known archaeal Rieske proteins. The recombinant 14 kDa protein isolated from Escherichia coli behaved as a dimer in solution. It contained approximately 2 Fe/mol and all visible and EPR spectroscopic features typical of Rieske centre-containing proteins. However, its redox potential (+170 mV) was significantly higher than those of canonical RFd. This difference is rationalized in terms of the protein structure environment, as discrete amino acid substitutions in key positions around the metal centre account for the higher potential.     

Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria

Summary The nucleotide sequence was determined of a 5.3 kb region of the Xanthomonas campestris pathovar campestris genome carrying a gene cluster encoding protein secretion and pathogenicity functions. A putative promoter sequence and five open reading frames (ORF) which may be part of an operon were revealed. The five predicted primary translation products comprise 531, 390, 147, 169 and 138 amino acids with M_r values of 58854, 42299, 15548, 18214 and 15108 respectively. A sixth, partial ORF is also present. Between ORF1 and ORF2 is a sequence of unknown function showing 7 by duplications. The deduced amino acid sequence of ORF1 is related to the Klebsiella pneumoniae PulE protein, to the Bacillus subtilis ComG ORF1 and to the Agrobacterium tumefaciens VirB ORF11 products. In addition, the deduced amino acid sequence of ORF2 showed homology to the Pu1F and to the ComG ORF2 products. The proteins encoded by ORF3, 4 and 5 showed amino acid homology to PulG, H and I products respectively. The proteins encoded by ORF2, 3, 4 and 5 showed significant hydrophobic domains which may represent membrane-spanning regions. By contrast the protein encoded by ORF1 was largely hydrophilic and had two putative nucleoside triphosphate binding sites.The nucleotide sequence data in this paper have been deposited in the EMBL, Genbank and DDBJ nucleotide sequence databases under the accession number X59079     

Nucleotide sequence of the staphylococcal enterotoxin C1 gene and relatedness to other pyrogenic toxins

Summary The nucleotide sequence for the structural gene entC1 encoding staphylococcal enterotoxin C1 was determined. The gene contained 801 bp and coded for a protein of 266 amino acids. Of these, 27 comprised the signal peptide. Cleavage of the signal peptide resulted in a mature protein with 239 amino acids and a calculated molecular weight of 27496. The nucleotide sequence of entC1 shared considerable homology (74% and 59%, respectively) with genes encoding enterotoxin B and streptococcal pyrogenic exotoxin A. A similar degree of amino acid homology was observed after alignment of the respective proteins. Thus, certain regions of these three toxin molecules possess structural similarities that may be responsible for shared biological properties.     

Characterization and expression in Streptomyces lividans of cefD and cefE genes from Nocardia lactamdurans: the organization of the cephamycin gene cluster differs from that in Streptomyces clavuligerus

Summary The cefD and cefE genes of Nocardia lactamdurans, which encode isopenicillin N epimerase and deacetoxycephalosporin C synthase respectively, have been located 0.63 kb upstream from the lysine-6-amino-transferase (lat) gene. cefD contains an open reading frame (ORF) of 1197 nucleotides (nt) encoding a protein of 398 amino acids with a M_r of 43 622. The deduced amino acid sequence exhibits 62.2% identity to the cefD gene product of Streptomyces clavuligerus. The sequence SXHKXL in isopenicillin N epimerase resembles the consensus sequence for pyridoxal phosphate binding found in several amino acid decarboxylases from Enterobacteria. cefE contains an ORF of 945 nt encoding a protein of 314 amino acids with a M_r of 34532, which is similar to the deacetoxycephalosporin C synthase of S. clavuligerus. Expression of both genes, cefD and cefE, in S. lividans transformants, results in deacetoxycephalosporin C synthase and isopenicillin N epimerase activities that are 10–12 times higher than those in N. lactamdurans. The cefD and cefE genes of N. lactamdurans are closely linked but the overall organization of the cephamycin gene cluster differs in N. lactamdurans and S. clavuligerus.     

The molecular organization of the lysostaphin gene and its sequences repeated in tandem

Summary The gene encoding lysostaphin of Staphylococcus staphylolyticus was cloned in Escherichia coli and its DNA sequence was determined. The complete coding region comprises 1440 base pairs corresponding to a precursor of 480 amino acids (molecular weight 51669). It was shown by NH₂-terminal amino acid sequence analysis of the purified extracellular lysostaphin from S. staphylolyticus that the mature lysostaphin consists of 246 amino acid residues (molecular weight 26926). Polyacrylamide gel electrophoresis revealed a similar molecular weight for the most active form. By computer analysis the secondary protein structure was predicted. It revealed three distinct regions in the precursor protein: a typical signal peptide (ca. 38 aa), a hydrophilic and highly ordered protein domain with 14 repetitive sequences (296 aa) and the hydrophobic mature lysostaphin. The lysostaphin precursor protein appears to be organized as a preprolysostaphin.Abbreviations aa amino acid(s)     

Diverse dextranase genes from <Emphasis Type="Italic">Paenibacillus</Emphasis> species

Genes encoding dextranolytic enzymes were isolated from Paenibacillus strains Dex40-8 and Dex50-2. Single, similar but non-identical dex1 genes were isolated from each strain, and a more divergent dex2 gene was isolated from strain Dex50-2. The protein deduced from the Dex40-8 dex1 gene sequence had 716 amino acids, with a predicted M_r of 80.8 kDa. The proteins deduced from the Dex50-2 dex1 and dex2 gene sequences had 905 and 596 amino acids, with predicted M_r of 100.1 kDa and 68.3 kDa, respectively. The deduced amino acid sequences of all three dextranolytic proteins had similarity to family 66 glycosyl hydrolases and were predicted to possess cleavable N-terminal signal peptides. Homology searches suggest that the Dex40-8 and Dex50-2 Dex1 proteins have one and two copies, respectively, of a carbohydrate-binding module similar to CBM_4_9 (pfam02018.11). The Dex50-2 Dex2 deduced amino acid sequence had highest sequence similarity to thermotolerant dextranases from thermophilic Paenibacillus strains, while the Dex40-8 and Dex50-2 Dex1 deduced protein sequences formed a distinct sequence clade among the family 66 proteins. Examination of seven Paenibacillus strains, using a polymerase chain reaction-based assay, indicated that multiple family 66 genes are common within this genus. The three recombinant proteins expressed in Escherichia coli possessed dextranolytic activity and were able to convert ethanol-insoluble blue dextran into an ethanol-soluble product, indicating they are endodextranases (EC 3.2.1.11). The reaction catalysed by each enzyme had a distinct temperature and pH dependence.     

Cloning and nucleotide sequence of the gene braB coding for the sodium-coupled branched-chain amino acid carrier in Pseudomonas aeruginosa PAO

Summary The gene braB, encoding the Na^–-coupled carrier for branched-chain amino acids in Pseudomonas aeruginosa PAO, was cloned on cosmid pMMB34. The cosmid clones carrying the braB gene were identified as those that restored growth at low leucine concentration and Na^–-dependent leucine transport activity to P. aeruginosa PAO3536 defective in the transport of branched-chain amino acids. Determination of the nucleotide sequence of the DNA fragment shows that the braB gene comprises 1311 bp and encodes a hydrophobic protein of 437 amino acids with a calculated M_r of 45279. The hydropathy profile suggests that there exist in the carrier protein 12 hydrophobic segments long enough to traverse the membrane. The amino acid sequence shows a high degree of homology with thebrnQ product, a branched-chain amino acid carrier of Salmonella typhimurium, while no homology in the nucleotide sequences is found in the braB and brnQ genes.     

Amino acid identities in the three redox center-carrying polypeptides of cytochromebc 1/b 6 f complexes

The comparison of primary structures is extended to 22 cytochromesb orb ₆, 12 cytochromesc ₁ orf, and 8 Rieske FeS proteins. Conclusions are drawn as to their phylogenetic relationship as well as on conserved, functionally important amino acids and secondary structures. The results are in favor of two independent quinone binding sites at opposite surfaces of the membrane, topping one of the two hemes of cytochromeb each.     

Crystal structure of NirD,the small subunit of the nitrite reductase NirbD from Mycobacterium tuberculosis at 2.0 Å resolution

NirD is part of the nitrite reductase complex NirBD that catalyses the reduction of nitrite to NH₃ in nitrate assimilation and anaerobic respiration. The crystal structure analysis of NirD from Mycobacterium tuberculosis shows a double β‐sandwich fold. NirD is related in three‐dimensional structure and sequence to the Rieske proteins; however, it does not contain any Fe–S cluster or other cofactors that might be involved in electron transfer. A cysteine residue at the protein surface, conserved in NirD homologues lacking the iron–sulfur cluster might be important for the interaction with NirB and possibly stabilize one of the Fe–S centers in this subunit. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Assembly of Deletion Mutants of the Rieske Iron–Sulfur Protein into the Cytochrome bc 1 Complex of Yeast Mitochondria

The assembly of two deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc ₁ complex was investigated after import in vitro into mitochondria isolated from a strain of yeast, JPJ1, from which the iron-sulfur protein gene (RIP) had been deleted. The assembly process was investigated by immunoprecipitation of the labeled iron-sulfur protein or the two deletion mutants from detergent-solubilized mitochondria with specific antisera against either the iron-sulfur protein or the bc ₁ complex (complex III) [Fu and Beattie (1991). J. Biol. Chem. 266, 16212–16218]. The deletion mutants lacking amino acid residues 55–66 or residues 161–180 were imported into mitochondria in vitro and processed to the mature form via an intermediate form. After import in vitro, the protein lacking residues 161–180 was not assembled into the complex, suggesting that the region of the iron-sulfur protein containing these residues may be involved in the assembly of the protein into the bc ₁ complex; however, the protein lacking residues 55–66 was assembled in vitro into the bc ₁ complex as effectively as the wild type iron-sulfur protein. Moreover, this mutant protein was present in the mitochondrial membrane fraction obtained from JPJ1 cells transformed with a single-copy plasmid containing the gene for this protein lacking residues 55–66. This deletion mutant protein was also assembled into the bc ₁ complex in vivo, suggesting that the hydrophobic stretch of amino acids, residues 55–66, is not required for assembly of the iron-sulfur protein into the bc ₁ complex; however, this association did not lead to enzymatic activity of the bc ₁ complex, as the Rieske FeS cluster was not epr detectable in these mitochondria.     

The pet genes of Rhodospirillum rubrum: cloning and sequencing of the genes for the cytochrome bc1-complex

Summary A cytochrome bc ₁-complex of Rs. rubrum was isolated and the three subunits were purified to homogeneity. The N-terminal amino acid sequence of the purified subunits was determined by automatic Edman degradation. The pet genes of Rhodospirillum rubrum coding for the three subunits of the cytochrome bc ₁-complex were isolated from a genomic library of Rs. rubrum using oligonucleotides specific for conserved regions of the subunits from other organisms and a heterologous probe derived from the genes for the complex of Rb. capsulatus. The complete nucleotide sequence of a 5500 by SalI/SphI fragment is described which includes the pet genes and three additional unidentified open reading frames. The N-terminal amino acid sequence of the isolated subunits was used for the identification of the three genes. The genes encoding the subunits are organized as follows: Rieske protein, cytochrome b, cytochrome c ₁. Comparison of the N-terminal protein sequences with the protein sequences deduced from the nucleotide sequence showed that only cytochrome c ₁ is processed during transport and assembly of the three subunits of the complex. Only the N-terminal methionine of the Rieske protein is cleaved off. The similarity of the deduced amino acid sequence of the three subunits to the corresponding subunits of other organisms is described and implications for structural features of the subunits are discussed.Abbreviations BSA bovine serum albumin - SDS sodium dodecylsulphate - Rs Rhodospirillum - Rb Rhodobacter - Pc Paracoccus - Rps Rhodopseudomonas The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number X55387     

Molecular Cloning and Characterization of the Myostatin Gene in Croceine Croaker, Pseudosciaena crocea

Myostatin (MSTN) is a negative regulator of skeletal muscle mass and has a potential application in aquaculture. We reported the characterization of the myostatin gene and its expression in the croceine croaker, Pseudosciaena crocea. The myostatin gene had three exons encoding 376 amino acids. The cDNA was 1,906 bp long with a 5′-UTR and 3′-UTR of 108 bp and 667 bp, respectively. A microsatellite sequence, CA₃₀ and CA₂₆ separated by TA, existed in the 3′-UTR. Intron I and II were 343 bp and 758 bp in length, respectively. The deduced amino acid sequence was highly conserved, and had more than 90% identical to shi drum, gilthead seabream, striped sea-bass, white perch, and white bass proteins. The myostatin of croceine croaker had a putative amino terminal signal sequence (residues 1–22), a transforming growth factor-beta (TGF-β) propeptide domain (residues 41–256), a RXXR proteolytic processing site (RARR, residues 264–267, matching the RXXR consensus site), and a TGF-β domain (residues 282–376). There were 13 conserved cysteine residues in croceine croaker myostatin, nine of which are common to all TGF-β superfamily members. The most conserved region of vertebrate myostatins is the TGF-β domain, which was the mature bioactive domain of the myostatin protein. The myostatin gene was expressed not only in the skeletal muscle, but also in the other tissues.     

Cloning and characterization of a novel human TEKTIN1 gene

Tektins comprise a family of filament-forming proteins that are known to be coassembled with tubulins to form ciliary and flagellar microtubules. A new member of the tektin gene family was cloned from the human fetal brain cDNA library. We hence named it the human TEKTIN1 gene. TEKTIN1 cDNA consists of 1375 bp and has a putative open reading frame encoding 418 amino acids. The predicted protein is 48.3 kDa in size, and its amino acid sequence is 82% identical to that of the mouse, rat, and dog. One conserved peptide RPNVELCRD was observed at position number 323–331 of the amino acid sequence, which is a prominent feature of tektins and is likely to represent a functionally important protein domain. TEKTIN1 gene was mapped to the human chromosome 17 by BLAST search, and at least eight exons were found. Northern blot analysis indicated that TEKTIN1 was predominantly expressed in testis. By in-situ hybridization analysis, TEKTIN1 mRNA was localized to spermatocytes and round spermatids in the seminiferous tubules of the mouse testis, indicating that it may play a role in spermatogenesis.     

Escherichia coli and other species of the enterobacteriaceae encode a protein similar to the family of Mip-like FK506-binding proteins

A newly identified gene in Escherichia coli, fkpA, encodes a protein with extensive similarity to the macrophage infectivity potentiator (Mip) proteins of Legionella pneumophila and Chlamydia trachomatis. The FkpA protein may be a new member of the family of FK506-binding proteins (FKBPs) because its carboxyl domain includes a sequence that matches the consensus FK506-binding motif in 40 of 48 positions. including those amino acids at the active site that form hydrogen bonds with the drug FK506. The amino acid sequence of the 29kDa FkpA protein is 30–35% identical to the Mip proteins of L. pneumophila, L. micdadei, and C. trachomatis. Of the 270 amino acids of FkpA, 113 (42%) are identical to the sequence of one or another of these Mip proteins. Overexpression of FkpA or deletion of fkpA from the E. coli chromosome had no detrimental effect on bacterial growth, indicating that fkpA is not an essential gene. Hybridization of fkpA-specific DNA probes to genomic blots révealed that similar genes exist in several representatives of the Enterobacteriaceae. Thus, mip-like genes are not found exelusively in bacteria having a predominately intracellular life style, but instead appear to be a new FKBP subfamily that is a common constituent of many bacteria.