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Bjørn Dalhus

Publications and source records attributed to Bjørn Dalhus.

11 recordsLinked to original sources
PubMed2006

Crystal structure and enzymatic properties of a bacterial family 19 chitinase reveal differences from plant enzymes.

We describe the cloning, overexpression, purification, characterization and crystal structure of chitinase G, a single-domain family 19 chitinase from the Gram-positive bacterium Streptomyces coelicolor A3(2). Although chitinase G was not capable of releasing 4-methylumbelliferyl from artificial chitooligosaccharide substrates, it was capable of degrading longer chitooligosaccharides at rates similar to those observed for other chitinases. The enzyme was also capable of degrading a colored colloidal chitin substrate (carboxymethyl-chitin-remazol-brilliant violet) and a small, presumably amorphous, subfraction of alpha-chitin and beta-chitin, but was not capable of degrading crystalline chitin completely. The crystal structures of chitinase G and a related Streptomyces chitinase, chitinase C [Kezuka Y, Ohishi M, Itoh Y, Watanabe J, Mitsutomi M, Watanabe T & Nonaka T (2006) J Mol Biol358, 472-484], showed that these bacterial family 19 chitinases lack several loops that extend the substrate-binding grooves in family 19 chitinases from plants. In accordance with these structural features, detailed analysis of the degradation of chitooligosaccharides by chitinase G showed that the enzyme has only four subsites (- 2 to + 2), as opposed to six (- 3 to + 3) for plant enzymes. The most prominent structural difference leading to reduced size of the substrate-binding groove is the deletion of a 13-residue loop between the two putatively catalytic glutamates. The importance of these two residues for catalysis was confirmed by a site-directed mutagenesis study.

Amino Acid Sequence↗
PubMed2005

1.6-Angstroms crystal structure of EntA-im. A bacterial immunity protein conferring immunity to the antimicrobial activity of the pediocin-like bacteriocin enterocin A.

Many Gram-positive bacteria produce ribosomally synthesized antimicrobial peptides, often termed bacteriocins. Genes encoding pediocin-like bacteriocins are generally cotranscribed with or in close vicinity to a gene encoding a cognate immunity protein that protects the bacteriocin-producer from their own bacteriocin. We present the first crystal structure of a pediocin-like immunity protein, EntA-im, conferring immunity to the bacteriocin enterocin A. Determination of the structure of this 103-amino acid protein revealed that it folds into an antiparallel four-helix bundle with a flexible C-terminal part. The fact that the immunity protein conferring immunity to carnobacteriocin B2 also consists of a four-helix bundle (Sprules, T., Kawulka, K. E., and Vederas, J. C. (2004) Biochemistry 43, 11740-11749) strongly indicates that this is a conserved structural motif in all pediocin-like immunity proteins. The C-terminal half of the immunity protein contains a region that recognizes the C-terminal half of the cognate bacteriocin, and the flexibility in the C-terminal end of the immunity protein might thus be an important characteristic that enables the immunity protein to interact with its cognate bacteriocin. By homology modeling of three other pediocin-like immunity proteins and calculation of the surface charge distribution for EntA-im and the three structure models, different charge distributions were observed. The differences in the latter part of helix 3, the beginning of helix 4, and the loop connecting these helices might also be of importance in determining the specificity.

Amino Acid Motifs↗
PubMed2005

Pediocin-like antimicrobial peptides (class IIa bacteriocins) and their immunity proteins: biosynthesis, structure, and mode of action.

Pediocin-like antimicrobial peptides (AMPs) form a group of lactic acid bacteria produced, cationic membrane-permeabilizing peptides with 37 to 48 residues. Upon exposure to membrane-mimicking entities, their hydrophilic, cationic, and highly conserved N-terminal region forms a three-stranded antiparallel beta-sheet supported by a conserved disulfide bridge. This N-terminal beta-sheet region is followed by a central amphiphilic alpha-helix and this in most (if not all) of these peptides is followed by a rather extended C-terminal tail that folds back onto the central alpha-helix, thereby creating a hairpin-like structure in the C-terminal half. There is a flexible hinge between the beta-sheet N-terminal region and the hairpin C-terminal region and one thus obtains two domains that may move relative to each other. The cationic N-terminal beta-sheet domain mediates binding of the pediocin-like AMPs to the target-cell surface through electrostatic interactions, while the more hydrophobic and amphiphilic C-terminal hairpin domain penetrates into the hydrophobic part of the target-cell membrane, thereby mediating leakage through the membrane. The hinge provides the structural flexibility that enables the C-terminal hairpin domain to dip into the hydrophobic part of the membrane. Despite extensive sequence similarities, these AMPs differ markedly in their target-cell specificity, and results obtained with hybrid AMPs indicate that the membrane-penetrating hairpin-like C-terminal domain is the major specificity determinant. Bacteria that produce pediocin-like AMPs also produce a 11-kDa cognate immunity protein that protects the producer. The immunity proteins are well-structured, 4-helix bundle cytosolic proteins. They show a high degree of specificity in that they largely recognize and confer immunity only to their cognate AMP and in some cases to a few AMPs that are closely related to their cognate AMP. The C-terminal half of the immunity proteins contains a domain that is involved in specific recognition of the C-terminal membrane-penetrating specificity-determining hairpin domain of the cognate AMP.

Amino Acid Sequence↗
PubMed2004

Synthesis and characterization of half-sandwich N-heterocyclic carbene complexes of cobalt and rhodium.

A series of novel half-sandwich M(I) and M(III) complexes (M = Co, Rh) bearing the N-heterocyclic carbene ligand 1,3-dimesitylimidazol-2-ylidene (IMes) have been prepared and characterized. Thus, (eta5-C(5)R(5))M(IMes)(C(2)H(4))(M = Co, Rh; R = H, Me) were obtained from the corresponding bis(ethene) complexes (eta5-C(5)R(5))M(C(2)H(4))(2), except for CpRh(IMes)(C(2)H(4)) which was prepared via the novel 16-electron Rh(I) compound Rh(IMes)(C(2)H(4))(2)Cl. The carbonyl compounds (eta5-C(5)R(5))Co(IMes)(CO)(R = H, Me) were synthesized by thermal CO substitution of (eta5-C(5)R(5))Co(CO)(2). A diamagnetic, apparently 16-electron Co(III) compound [CpCo(IMes)I](+)[I(3)(-)] was obtained from CpCo(IMes)(CO) and I(2). Finally, Co(III) and Rh(III) complexes CpCo(IMes)Me(2) and Cp*Rh(IMes)Me(2) were prepared by methylation of [CpCo(IMes)I](+)[I(3)(-)], and ligand exchange at Cp*Rh(Me(2)SO)Me(2), respectively. The molecular structures of CpCo(IMes)(CO), CpRh(IMes)(C(2)H(4)), Cp*Rh(IMes)(C(2)H(4)), and Cp*Rh(IMes)Me(2) were determined by single crystal X-ray diffraction. Steric and electronic factors imposed by the strongly donating and sterically demanding IMes ligand are discussed on the basis of X-ray crystallographic, NMR, and IR spectroscopic analyses. Very poor correlations are found between values for (1)J(Rh-C(carbene)) and dRh-C(carbene) data for Rh(i) N,N-heterocyclic carbene complexes including literature data and this work.

Journal Article↗
PubMed2004

Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface.

The stability of tetrameric malate dehydrogenase from the green phototrophic bacterium Chloroflexus aurantiacus (CaMDH) is at least in part determined by electrostatic interactions at the dimer-dimer interface. Since previous studies had indicated that the thermal stability of CaMDH becomes lower with increasing pH, attempts were made to increase the stability by removal of (excess) negative charge at the dimer-dimer interface. Mutation of Glu165 to Gln or Lys yielded a dramatic increase in thermal stability at pH 7.5 (+23.6 -- + 23.9 degrees C increase in apparent t(m)) and a more moderate increase at pH 4.4 (+4.6 -- + 5.4 degrees C). The drastically increased stability at neutral pH was achieved without forfeiture of catalytic performance at low temperatures. The crystal structures of the two mutants showed only minor structural changes close to the mutated residues, and indicated that the observed stability effects are solely due to subtle changes in the complex network of electrostatic interactions in the dimer-dimer interface. Both mutations reduced the concentration dependency of thermal stability, suggesting that the oligomeric structure had been reinforced. Interestingly, the two mutations had similar effects on stability, despite the charge difference between the introduced side-chains. Together with the loss of concentration dependency, this may indicate that both E165Q and E165K stabilize CaMDH to such an extent that disruption of the inter-dimer electrostatic network around residue 165 no longer limits kinetic thermal stability.

Bacterial Proteins↗
PubMed2003

Stabilization of a tetrameric malate dehydrogenase by introduction of a disulfide bridge at the dimer-dimer interface.

Malate dehydrogenase (MDH) from the moderately thermophilic bacterium Chloroflexus aurantiacus (CaMDH) is a tetrameric enzyme, while MDHs from mesophilic organisms usually are dimers. To investigate the potential contribution of the extra dimer-dimer interface in CaMDH with respect to thermal stability, we have engineered an intersubunit disulfide bridge designed to strengthen dimer-dimer interactions. The resulting mutant (T187C, containing two 187-187 disulfide bridges in the tetramer) showed a 200-fold increase in half-life at 75 degrees C and an increase of 15 deg. C in apparent melting temperature compared to the wild-type. The crystal structure of the mutant (solved at 1.75 A resolution) was essentially identical with that of the wild-type, with the exception of the added inter-dimer disulfide bridge and the loss of an aromatic intra-dimer contact. Remarkably, the mutant and the wild-type had similar temperature optima and activities at their temperature optima, thus providing a clear case of uncoupling of thermal stability and thermoactivity. The results show that tetramerization may contribute to MDH stability to an extent that depends strongly on the number of stabilizing interactions in the dimer-dimer interface.

Bacterial Proteins↗
PubMed2003

Crystallization and preliminary X-ray data investigation of the bacterial enterocin A immunity protein at 1.65 A resolution.

Crystals of the bacterial enterocin A immunity protein have been prepared by the hanging-drop vapour-diffusion technique at 293 K. The crystals diffract to better than 1.7 A resolution and X-ray diffraction data to 1.65 A have been collected at 110 K using synchrotron radiation. The enterocin A immunity protein crystals belong to the monoclinic crystal system, with unit-cell parameters a = 116.32, b = 42.35, c = 66.17 A, beta = 111.3 degrees. The symmetry and systematic absences in the diffraction pattern are consistent with space group C2. The presence of two molecules in the asymmetric unit with a molecular weight of approximately 12.2 kDa gives a crystal volume per protein mass (V(M)) of approximately 3.1 A(3) Da(-1) and a solvent content of approximately 60% by volume.

Bacteriocins↗
PubMed2002

An organometallic RhIII complex with a distorted octahedral structure: (acetonitrile-kappaN)dimethyl(1,4,7-trimethyl-1,4,7-triazacyclononane-kappa3N,N',N")rhodium(III) tetraphenylborate.

In the title compound, [Rh(CH(3))(2)(C(2)H(3)N)(C(9)H(21)N(3))](C(24)H(20)B), the geometry around the Rh(III) centre is distorted octahedral, with elongated Rh-N bonds trans to the metal-bonded methyl groups. The metal-containing cations are located in channels formed by an anionic supramolecular mesh, in which aromatic pi-pi interactions between anionic [B(Ph)(4)](-) units play a major role.

Journal Article↗
PubMed2002

Structural basis for thermophilic protein stability: structures of thermophilic and mesophilic malate dehydrogenases.

The three-dimensional structure of four malate dehydrogenases (MDH) from thermophilic and mesophilic phototropic bacteria have been determined by X-ray crystallography and the corresponding structures compared. In contrast to the dimeric quaternary structure of most MDHs, these MDHs are tetramers and are structurally related to tetrameric malate dehydrogenases from Archaea and to lactate dehydrogenases. The tetramers are dimers of dimers, where the structures of each subunit and the dimers are similar to the dimeric malate dehydrogenases. The difference in optimal growth temperature of the corresponding organisms is relatively small, ranging from 32 to 55 degrees C. Nevertheless, on the basis of the four crystal structures, a number of factors that are likely to contribute to the relative thermostability in the present series have been identified. It appears from the results obtained, that the difference in thermostability between MDH from the mesophilic Chlorobium vibrioforme on one hand and from the moderate thermophile Chlorobium tepidum on the other hand is mainly due to the presence of polar residues that form additional hydrogen bonds within each subunit. Furthermore, for the even more thermostable Chloroflexus aurantiacus MDH, the use of charged residues to form additional ionic interactions across the dimer-dimer interface is favored. This enzyme has a favorable intercalation of His-Trp as well as additional aromatic contacts at the monomer-monomer interface in each dimer. A structural alignment of tetrameric and dimeric prokaryotic MDHs reveal that structural elements that differ among dimeric and tetrameric MDHs are located in a few loop regions.

Amino Acid Sequence↗