Biomedical subjects

J Van Beeumen

Publications and source records attributed to J Van Beeumen.

At least 55 records · Page 3Linked to original sources

PubMed2000

The dppA gene of Bacillus subtilis encodes a new D-aminopeptidase.

Different strains of Bacillus were screened for their ability to hydrolyse D-alanyl-p-nitroanilide. Activity was detected in Bacillus pumilus, Bacillus brevis, Bacillus licheniformis 749I and Bacillus subtilis 168. The last strain was the best producer and was selected for the production and purification of the enzyme. The determination of the N-terminal sequence identified the enzyme as the product of the dppA gene (previously named dciAA) belonging to the dipeptide ABC transport (dpp) operon expressed early during sporulation. Open reading frames (ORFs) encoding putative related proteins were found in the genomes of a variety of Archaea and both sporulating and non-sporulating bacteria. The enzyme behaves as a D-aminopeptidase and represents the prototype of a new peptidase family. Among the tested substrates, the highest activities were found with D-Ala-D-Ala and D-Ala-Gly-Gly. The active enzyme behaves as an octamer of identical 30 kDa subunits. It exhibits a broad pH optimum, extending between pH 9 and 11. It is reversibly inhibited in the presence of Zn2+ chelators, and the sequence comparisons highlight the conservation of potential Zn-binding residues. As it has been shown by others that null mutations in the dpp operon do not inhibit spore formation, the physiological role of DppA is probably an adaptation to nutrient deficiency.

Amino Acid Sequence↗

PubMed2000

A DNA ligase from the psychrophile Pseudoalteromonas haloplanktis gives insights into the adaptation of proteins to low temperatures.

The cloning, overexpression and characterization of a cold-adapted DNA ligase from the Antarctic sea water bacterium Pseudoalteromonas haloplanktis are described. Protein sequence analysis revealed that the cold-adapted Ph DNA ligase shows a significant level of sequence similarity to other NAD+-dependent DNA ligases and contains several previously described sequence motifs. Also, a decreased level of arginine and proline residues in Ph DNA ligase could be involved in the cold-adaptation strategy. Moreover, 3D modelling of the N-terminal domain of Ph DNA ligase clearly indicates that this domain is destabilized compared with its thermophilic homologue. The recombinant Ph DNA ligase was overexpressed in Escherichia coli and purified to homogeneity. Mass spectroscopy experiments indicated that the purified enzyme is mainly in an adenylated form with a molecular mass of 74 593 Da. Ph DNA ligase shows similar overall catalytic properties to other NAD+-dependent DNA ligases but is a cold-adapted enzyme as its catalytic efficiency (kcat/Km) at low and moderate temperatures is higher than that of its mesophilic counterpart E. coli DNA ligase. A kinetic comparison of three enzymes adapted to different temperatures (P. haloplanktis, E. coli and Thermus scotoductus DNA ligases) indicated that an increased kcat is the most important adaptive parameter for enzymatic activity at low temperatures, whereas a decreased Km for the nicked DNA substrate seems to allow T. scotoductus DNA ligase to work efficiently at high temperatures. Besides being useful for investigation of the adaptation of enzymes to extreme temperatures, P. haloplanktis DNA ligase, which is very efficient at low temperatures, offers a novel tool for biotechnology.

Adaptation, Physiological↗

PubMed2000

The microneme protein MIC3 of Toxoplasma gondii is a secretory adhesin that binds to both the surface of the host cells and the surface of the parasite.

Assay of the adhesion of cultured cells on Toxoplasma gondii tachyzoite protein Western blots identified a major adhesive protein, that migrated at 90 kDa in non-reducing gels. This band comigrated with the previously described microneme protein MIC3. Cellular binding on Western blots was abolished by MIC3-specific monoclonal and polyclonal antibodies. The MIC3 protein affinity purified from tachyzoite lysates bound to the surface of putative host cells. In addition, T. gondii tachyzoites also bound to immobilized MIC3. Immunofluorescence analysis of T. gondii tachyzoite invasion showed that MIC3 was exocytosed and relocalized to the surface of the parasite during invasion. The cDNA encoding MIC3 and the corresponding gene have been cloned, allowing the determination of the complete coding sequence. The MIC3 sequence has been confirmed by affinity purification of the native protein and N-terminal sequencing. The deduced protein sequence contains five partially overlapping EGF-like domains and a chitin binding-like domain, which can be involved in protein-protein or protein-carbohydrate interactions. Taken together, these results suggest that MIC3 is a new microneme adhesin of T. gondii.

Adhesins, Bacterial↗

PubMed2000

Conformational variability of the synthetic peptide 129-141 of the mouse prion protein.

The effect of solution conditions on the conformation of the peptide corresponding to residues 129-141 of the mouse prion protein has been examined by experimental and theoretical tools including circular dichroism, secondary structure predictions, and Molecular Dynamics simulations. The conformational properties of the peptide observed by CD confirm the prediction results: the peptide is chiefly random coil in water. The conformational sampling performed by Molecular Dynamics simulations in water also corroborates the flexibility of the peptide, in particular for the N-terminal part. We show, however, that the peptide samples hairpin conformations in one of several approximately 1-ns Molecular Dynamics simulations in water. Interestingly, the analysis of the CD spectra obtained in this study suggests the presence of beta-structure which, given the length of the peptide, can only consist in beta-hairpin. The peptide can also be induced to form a modest percentage of helical structure in the presence of organic cosolvents such as trifluoroethanol, or detergents such as sodium dodecyl sulfate and lysophosphatidylcholine. This result is different from that obtained for a homologous hamster fragment, which differs from the mouse sequence by the single substitution of Ile 139 to Met. Interestingly, this substitution is crucial for the barrier in the transmission of the prion disease between hamsters and mice.

Animals↗

PubMed1999

Structure and characterization of Ectothiorhodospira vacuolata cytochrome b(558), a prokaryotic homologue of cytochrome b(5).

A soluble cytochrome b(558) from the purple phototropic bacterium Ectothiorhodospira vacuolata was completely sequenced by a combination of automated Edman degradation and mass spectrometry. The protein, with a measured mass of 10,094.7 Da, contains 90 residues and binds a single protoheme. Unexpectedly, the sequence shows homology to eukaryotic cytochromes b(5). As no prokaryotic homologue had been reported so far, we developed a protocol for the expression, purification, and crystallization of recombinant cytochrome b(558). The structure was solved by molecular replacement to a resolution of 1.65 A. It shows that cytochrome b(558) is indeed the first bacterial cytochrome b(5) to be characterized and differs from its eukaryotic counterparts by the presence of a disulfide bridge and a four-residue insertion in front of the sixth ligand (histidine). Eukaryotes contain a variety of b(5) homologues, including soluble and membrane-bound multifunctional proteins as well as multidomain enzymes such as sulfite oxidase, fatty-acid desaturase, nitrate reductase, and lactate dehydrogenase. A search of the Mycobacterium tuberculosis genome showed that a previously unidentified gene encodes a fatty-acid desaturase with an N-terminal b(5) domain. Thus, it may provide another example of a bacterial b(5) homologue.

Amino Acid Sequence↗

PubMed1999

A cytochrome c peroxidase from Pseudomonas nautica 617 active at high ionic strength: expression, purification and characterization.

Cytochrome c peroxidase was expressed in cells of Pseudomonas nautica strain 617 grown under microaerophilic conditions. The 36.5 kDa dihaemic enzyme was purified to electrophoretic homogeneity in three chromatographic steps. N-terminal sequence comparison showed that the Ps. nautica enzyme exhibits a high similarity with the corresponding proteins from Paracoccus denitrificans and Pseudomonas aeruginosa. UV-visible spectra confirm calcium activation of the enzyme through spin state transition of the peroxidatic haem. Monohaemic cytochrome c(552) from Ps. nautica was identified as the physiological electron donor, with a half-saturating concentration of 122 microM and allowing a maximal catalytic centre activity of 116,000 min(-1). Using this cytochrome the enzyme retained the same activity even at high ionic strength. There are indications that the interactions between the two redox partners are mainly hydrophobic in nature.

Amino Acid Sequence↗

PubMed1999

Sequence, overproduction and purification of the family 11 endo-beta-1,4-xylanase encoded by the xyl1 gene of Streptomyces sp. S38.

The xyl1 gene encoding the Xyl1 xylanase of Streptomyces sp. strain S38 was cloned by screening an enriched DNA library with a specific DNA probe and sequenced. Three short 5 bp -CGAAA- sequences are located upstream of the Streptomyces sp. S38 xyl1 gene 105, 115 and 250 bp before the start codon. These sequences, named boxes 1, 2 and 3, are conserved upstream of the Actinomycetales xylanase genes and are specifically recognized by a DNA-binding protein (Giannotta et al., 1994. FEMS Microbiol. Lett. 142, 91-97) and could be probably involved in the regulation of xylanase production. The Xyl1 ORF encodes a 228 residue polypeptide and the Xyl1 preprotein contains a 38 residue signal peptide whose cleavage yields a 190 residue mature protein of calculated M(r) = 20,585 and basic pI value of 9.12. The molecular mass of the produced and purified mature protein determined by mass spectrometry (20,586 +/- 1 Da) and its pI (9.8) agree with these calculated values. Its N-terminal amino-acid sequence confirmed the proposed cleavage site between the signal peptide and the mature protein. Comparisons between Xyl1 and the 62 other xylanases belonging to family 11 allowed the construction of a phylogenetic tree and revealed its close relationship with Actinomycetales enzymes. Moreover, nine residues were found to be strictly conserved among the 63 xylanases.

Amino Acid Sequence↗

PubMed1999

Production of recombinant rat proopiomelanocortin1-74 and characterization of its mitogenic action on pituitary lactotrophs.

We report the production of biologically active recombinant rat Gly-2-Ser-1-POMC1-74 (rrPOMC1-74) in a prokaryotic expression system. The polypeptide was produced as a fusion protein with glutathione-S-transferase (GST), using the pGEX-4T-1 vector and subsequently cleaved by thrombin. Amino acid sequencing, up to residue 45, showed a correct primary structure including the two additional amino acids at the N-terminus, Gly and Ser, derived from the thrombin cleavage site. Electrospray ionization mass spectrometry showed a Mr of 8358.5 Da which was 14-16 Da heavier (oxidation or methylation) than the calculated mass. Combined digestion with trypsin and endoproteinase Glu-C followed by MALDI-TOF mass spectrometry and N-terminal sequencing of the separated fragments showed a correct disulphide bridge configuration. In reaggregate cell cultures of immature rat pituitary, rrPOMC1-74 displayed biological activity similar to that of natural human (h) POMC1-76 or rat POMC1-74: it stimulated DNA replication in lactotrophs but not in other pituitary cell types. However, its efficacy was significantly lower than that of the natural product. Gamma3-MSH, a peptide that can be generated from POMC1-74 and a typical ligand of the melanocortin-3 (MC-3) receptor, also stimulated DNA replication in lactotrophs and, in contrast to rrPOMC1-74, also in somatotrophs and thyrotrophs. rrPOMC1-74 increased cAMP levels in 293HEK cells stably transfected with the MC-3 receptor with an intrinsic activity and potency similar to that of gamma3-MSH. However, natural hPOMC1-76 was inactive in the latter test system. These data show that rrPOMC1-74 mimics the selective mitogenic action of natural POMC1-74 on lactotrophs. Since natural POMC1-74 is N- and O-glycosylated and rrPOMC1-74 is not, glycosylation does not seem to determine the selectivity for lactotrophs. In spite of the feature that rrPOMC1-74 is an agonist at the MC-3 receptor and the reported evidence that the MC-3 receptor is expressed in the anterior pituitary, the mitogenic action of rrPOMC1-74 on lactotrophs does not seem to be mediated by the MC-3 receptor.

Animals↗

PubMed1999

The DmpA aminopeptidase from Ochrobactrum anthropi LMG7991 is the prototype of a new terminal nucleophile hydrolase family.

The DmpA (d-aminopeptidase A) protein produced by Ochrobactrum anthropi hydrolyses p-nitroanilide derivatives of glycine and d-alanine more efficiently than that of l-alanine. When regular peptides are utilized as substrates, the enzyme behaves as an aminopeptidase with a preference for N-terminal residues in an l configuration, thus exemplifying an interesting case of stereospecificity reversal. The best-hydrolysed substrate is l-Ala-Gly-Gly, but tetra- and penta-peptides are also efficiently hydrolysed. The gene encodes a 375-residue precursor, but the active enzyme contains two polypeptides corresponding to residues 2-249 (alpha-subunit) and 250-375 (beta-subunit) of the precursor. Residues 249 and 250 are a Gly and a Ser respectively, and various substitutions performed by site-directed mutagenesis result in the production of an uncleaved and inactive protein. The N-terminal Ser residue of the beta-subunit is followed by a hydrophobic peptide, which is predicted to form a beta-strand structure. All these properties strongly suggest that DmpA is an N-terminal amidohydrolase. An exploration of the databases highlights the presence of a number of open reading frames encoding related proteins in various bacterial genomes. Thus DmpA is very probably the prototype of an original family of N-terminal hydrolases.

Amidohydrolases↗

PubMed1999

Two new aminopeptidases from Ochrobactrum anthropi active on D-alanyl-p-nitroanilide.

Two new enzymes which hydrolyse D-alanyl-p-nitroanilide have been detected in Ochrobactrum anthropi LMG7991 extracts. The first enzyme, DmpB, was purified to homogeneity and found to be homologous to the Dap protein produced by O. anthropi SCRC C1-38 (ATCC49237). The second enzyme, DmpA, exhibits a similar substrate profile when tested on p-nitroanilide derivatives of glycine and L/D-alanine, but the amounts produced by the Ochrobactrum strain were not sufficient to allow complete purification. Interestingly, the DmpA preparation also exhibited an L-aminopeptidase activity on the tripeptide L-Ala-Gly-Gly but it was not possible to be certain that the same protein was responsible for both p-nitroanilide and peptide hydrolysing activities. The gene encoding the DmpA protein was cloned and sequenced. The deduced protein sequence exhibits varying degrees of similarity with those corresponding to several open reading frames found in the genomes of other prokaryotic organisms, including Mycobacteria. None of these gene products has been isolated or characterised, but a tentative relationship can be proposed with the NylC amidase from Flavobacterium sp. K172.

Amino Acid Sequence↗

PubMed1999

The catalytic, glycosyl transferase and acyl transferase modules of the cell wall peptidoglycan-polymerizing penicillin-binding protein 1b of Escherichia coli.

The penicillin-binding protein (PBP) 1b of Escherichia coli catalyses the assembly of lipid-transported N-acetyl glucosaminyl-beta-1, 4-N-acetylmuramoyl-L-alanyl-gamma-D-glutamyl-(L)-meso-diaminopimelyl+ ++- (L)-D-alanyl-D-alanine disaccharide pentapeptide units into polymeric peptidoglycan. These units are phosphodiester linked, at C1 of muramic acid, to a C55 undecaprenyl carrier. PBP1b has been purified in the form of His tag (M46-N844) PBP1bgamma. This derivative provides the host cell in which it is produced with a functional wall peptidoglycan. His tag (M46-N844) PBP1bgamma possesses an amino-terminal hydrophobic segment, which serves as transmembrane spanner of the native PBP. This segment is linked, via an congruent with 100-amino-acid insert, to a D198-G435 glycosyl transferase module that possesses the five motifs characteristic of the PBPs of class A. In in vitro assays, the glycosyl transferase of the PBP catalyses the synthesis of linear glycan chains from the lipid carrier with an efficiency of congruent with 39 000 M-1 s-1. Glu-233, of motif 1, is central to the catalysed reaction. It is proposed that the Glu-233 gamma-COOH donates its proton to the oxygen atom of the scissile phosphoester bond of the lipid carrier, leading to the formation of an oxocarbonium cation, which then undergoes attack by the 4-OH group of a nucleophile N-acetylglucosamine. Asp-234 of motif 1 or Glu-290 of motif 3 could be involved in the stabilization of the oxocarbonium cation and the activation of the 4-OH group of the N-acetylglucosamine. In turn, Tyr-310 of motif 4 is an important component of the amino acid sequence-folding information. The glycosyl transferase module of PBP1b, the lysozymes and the lytic transglycosylase Slt70 have much the same catalytic machinery. They might be members of the same superfamily. The glycosyl transferase module is linked, via a short junction site, to the amino end of a Q447-N844 acyl transferase module, which possesses the catalytic centre-defining motifs of the penicilloyl serine transferases superfamily. In in vitro assays with the lipid precursor and in the presence of penicillin at concentrations sufficient to derivatize the active-site serine 510 of the acyl transferase, the rate of glycan chain synthesis is unmodified, showing that the functioning of the glycosyl transferase is acyl transferase independent. In the absence of penicillin, the products of the Ser-510-assisted double-proton shuttle are glycan strands substituted by cross-linked tetrapeptide-pentapeptide and tetrapeptide-tetrapeptide dimers and uncross-linked pentapeptide and tetrapeptide monomers. The acyl transferase of the PBP also catalyses aminolysis and hydrolysis of properly structured thiolesters, but it lacks activity on D-alanyl-D-alanine-terminated peptides. This substrate specificity suggests that carbonyl donor activity requires the attachment of the pentapeptides to the glycan chains made by the glycosyl transferase, and it implies that one and the same PBP molecule catalyses transglycosylation and peptide cross-linking in a sequential manner. Attempts to produce truncated forms of the PBP lead to the conclusion that the multimodular polypeptide chain behaves as an integrated folding entity during PBP1b biogenesis.

Acyltransferases↗

PubMed1999

Crystallization and preliminary X-ray analysis of a new L-aminopeptidase-D-amidase/D-esterase activated by a Gly-Ser peptide bond hydrolysis.

Ochrobactrum anthropi possesses an L-aminopeptidase (DmpA) also able to act as a D-amidase/D-esterase. DmpA (40 kDa) is activated by auto-catalyzed protein splicing liberating an alpha-amino group presumably used as a general base in the catalytic mechanism. Two crystal forms were obtained at 294 K in 13-16% PEG 2000 mono-methylether at pH 9.0, adding either 0.2 M magnesium chloride or 1 M lithium chloride. Crystals of the first form belong to the space group C2221 and diffract to 3.0 A resolution, whereas crystals of the second form belong to the space group P21212 and diffract to 2.3 A resolution. Initial screening for heavy-atom derivatives on form II crystals, has led to a well substituted Hg derivative.

Aminopeptidases↗

PubMed1999

Purification and characterization of Aspergillus ficuum endoinulinase.

Endoinulinase from Aspergillus ficuum, which catalyzes the hydrolysis of inulin via an endo-cleavage mode, was purified by chromatography from Novozym 230 as a starting commercial enzyme mixture on CM-Sephadex and DEAE-Sepharose, and by preparative electrophoresis under native conditions. The enzyme was estimated to be pure on the basis of its I/S ratio, whose value was infinite in our assay conditions. Two forms separated by using this method. SDS gel electrophoresis showed the two purified forms to respectively exhibit molecular weights of 64,000 +/- 500 and 66,000 +/- 1,000. The results of deglycosylation indicated that the two forms were originally the same protein but with different sugar contents. A molecular weight of 54,800 +/- 1,500 was found by gel filtration of the native enzyme, indicating the native functional protein to be a monomer. The enzyme showed nearly absolute substrate specificity towards inulin and inulooligosaccharides, and acted via an endo-attack to produce mainly inulotriose during the late stage of the reaction. The apparent Km and Vmax values for inulin hydrolysis were 8.1 +/- 1.0 mM and 773 +/- 60 U/mg, respectively. The internal peptides of the enzyme showed sequence homology to the endoinulinase of Penicillium purpurogenum.

Amino Acid Sequence↗

PubMed1998

The surface-charge asymmetry and dimerisation of cytochrome c550 from Paracoccus denitrificans--implications for the interaction with cytochrome c peroxidase.

The implications of the dimeric state of cytochrome c550 for its binding to Paracoccus cytochrome c peroxidase and its delivery of the two electrons required to restore the active enzyme during catalysis have been investigated. The amino acid sequence of cytochrome c550 of Paracoccus denitrificans strain LMD 52.44 was determined and showed 21 differences from that of strain LMD 22.21. Based on the X-ray structure of the latter, a structure for the cytochrome c550 monomer from strain 52.44 is proposed and a dipole moment of 945 debye was calculated with an orientation close to the exposed haem edge. The behaviour of the cytochrome on molecular-exclusion chromatography is indicative of an ionic strength-dependent monomer (15 kDa)/dimer (30 kDa) equilibrium that can also be detected by 1H-NMR spectroscopy. The apparent mass of 50 kDa observed at very low ionic strength was consistent with the presence of a strongly asymmetric dimer. This was confirmed by cross-linking studies, which showed that a cross-linked species of mass 30 kDa on SDS behaved with an apparent mass of 50 kDa on molecular-exclusion chromatography. A programme which carried out and evaluated molecular docking of two monomers to give a dimer generated a most probable dimer in which the monomer dipoles lay almost antiparallel to each other. The resultant dipole moment of the dimer is therefore small. Although this finding calls into question the possibility of preorientation of a strongly asymmetrically charged cytochrome as it collides with a redox partner, the stoichiometry of complex formation with cytochrome c peroxidase as studied by 1H-NMR spectroscopy shows that it is the monomer that binds.

Amino Acid Sequence↗

PubMed1998

Characterization of cytochrome c-556 from the purple phototrophic bacterium Rhodobacter capsulatus as a cytochrome-c peroxidase.

A cytochrome c-556 was purified from Rhodobacter capsulatus and the complete amino acid sequence was determined. It contains 328 amino acid residues and two typical heme-binding sites at cysteine residues 54 and 57 and at residues 200 and 203. It is homologous to the family of bacterial cytochrome c peroxidases (BCCP) with 69% identity to Paracoccus denitrificans BCCP and 60% identity to Pseudomonas aeruginosa BCCP for which there is a three-dimensional structure. There is lesser similarity to the mauG gene products from methylotrophic bacteria which are thought to be involved in biosynthesis of the quinone cofactor of methylamine dehydrogenase. Translated genes from Escherichia coli and Helicobacter pylori are also related to the bacterial cytochrome c peroxidases. The divergence of this family of proteins is reflected in the fact that the reported sixth heme ligands are not conserved, except in Pseudomonas, Rhodobacter and Paracoccus. This suggests that homologs of BCCP may fold differently and/or may not have the same enzymatic activity as the prototypic protein from Ps. aeruginosa. We found that the Rb. capsulatus BCCP is active with both Rb. capsulatus cytochrome c2 and with horse cytochrome c as substrates (Km values 60 microm and 6 microm, respectively). The turnover number was 40 s(-1) and the Km for peroxide was 33 microm. We have thus confirmed that the Rb. capsulatus protein is a cytochrome c peroxidase.

Amino Acid Sequence↗

PubMed1998

The primary structures of the low-redox potential diheme cytochromes c from the phototrophic bacteria Rhodobacter sphaeroides and Rhodobacter adriaticus reveal a new structural family of c-type cytochromes.

The complete amino acid sequence of the low-redox potential cytochrome c-551.5 from Rhodobacter sphaeroides was determined by automated Edman degradation combined with mass spectroscopy. There are 139 residues and two typical Cys-X-X-Cys-His heme-binding sites. A homologous low-redox potential cytochrome was also sequenced from Rhodobacter adriaticus and was found to contain 126 residues. It is 53% identical to that of Rb. sphaeroides and has two internal deletions of one and five residues. The Rhodobacter diheme cytochromes are 21-24% identical to the translated open reading frame SLL1886 from Synechocystis sp. PCC6801. There are at least two deletions of five and eight residues in the 188-residue cyanobacterial protein. Each of the three cytochromes has more histidines than it needs to bind the two hemes, but conserved histidines located 23 residues after the first heme and 14-19 residues before the second heme are likely to be the sixth heme ligands. There is no evidence for gene doubling and no similarity to any other known cytochromes. The measured helix content of 24% is much less than normal for c-type cytochromes. These proteins thus appear to be representative of an entirely new class of c-type cytochromes.

Amino Acid Sequence↗

PubMed1998

X-ray structure of a blue-copper nitrite reductase in two crystal forms. The nature of the copper sites, mode of substrate binding and recognition by redox partner.

Denitrification is one of the main steps of the global nitrogen cycle that is sustained by prokaryotic organisms. Denitrifying bacteria use two entirely different enzymes in this process, one based on haem cd1 prosthetic groups and the other on type 1-type 2 Cu centres. Copper-containing nitrite reductases (NiRs) are sub-divided into blue and green NiRs, which are respectively thought to be redox partners of azurins and pseudo-azurins. Crystallographic structures of the blue nitrite reductase from Alcaligenes xylosoxidans (AxNiR) are presented in the oxidised hexagonal form and the substrate-bound orthorhombic form to 2.1 A and 2.8 A resolution, respectively. The complete amino acid sequence of AxNiR has been determined by conventional chemical analysis. A 3 A structure of AxNiR has been published where the modelling was based on the sequence of another blue NiR. The higher resolution of the hexagonal form together with the correct sequence allows a detailed comparison with the crystallographic structures of the green NiRs. There is a striking difference in the overall surface charge distribution between the two sub-groups, providing a neat structural explanation for their different reactivities to pseudoazurin or azurin and supporting the view that electron transfer proceeds via complex formation. A detailed examination of the type-1 Cu site, the site responsible for the colour, reveals several subtle differences, including a lateral displacement of 0.7 A for Smet. The structure of the type-2 Cu site, and changes that occur upon substrate binding are discussed in terms of the catalytic mechanism. The similarity of the type 2 Cu site to the catalytic Zn site in carbonic anhydrase and the catalytic Cu site of superoxide dismutase is re-examined in view of the high-resolution (2.1 A) structure.

Alcaligenes↗

PubMed1998

The primary structure of soluble cytochrome c-551 from the phototrophic green sulfur bacterium Chlorobium limicola, strain Tassajara, reveals a novel c-type cytochrome.

Chlorobium limicola, strain Tassajara, cytochrome c-551 is a soluble dimeric protein containing identical subunits of about 30 kDa. The amino acid sequence was determined by a combination of automated Edman degradation and mass analysis. There are 258 residues with a single heme binding site located at cysteine positions 172 and 175. In addition, there is a disulfide bridge between Cys78 and Cys109, and a free cysteine at position 219 which was found to occur as cysteic acid. The only homologue of soluble cytochrome c-551 is the soxA protein which is part of the thiosulfate utilization operon of Paracoccus denitrificans. They are 32% identical with three small gaps. This is consistent with the observation that cytochrome c-551 is the electron acceptor for a thiosulfate-oxidizing enzyme. On the basis of the redox potential of 135 mV, the sixth heme ligand should be a methionine. Among the seven methionine residues that are present in c-551, only one is conserved, two residues ahead of the heme-binding site. The far-UV circular dichroism spectrum indicates 40% alpha helix and 25% beta secondary structure. No other known cytochrome c has such a mixed structure; they are either all helical or all beta. Thus, Chlorobium soluble cytochrome c-551 and soxA are likely to be representative of a new class of c-type cytochromes.

Amino Acid Sequence↗