Biomedical subjects

Peter F Lindley

Publications and source records attributed to Peter F Lindley.

9 recordsLinked to original sources

PubMed2006

Purification, crystallization and preliminary X-ray study of the fungal laccase from Cerrena maxima.

Laccases are members of the blue multi-copper oxidase family that oxidize substrate molecules by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear centre. Dioxygen binds to the trinuclear centre and, following the transfer of four electrons, is reduced to two molecules of water. Crystals of the laccase from Cerrena maxima have been obtained and X-ray data were collected to 1.9 A resolution using synchrotron radiation. A preliminary analysis shows that the enzyme has the typical laccase structure and several carbohydrate sites have been identified. The carbohydrate chains appear to be involved in stabilization of the intermolecular contacts in the crystal structure, thus promoting the formation of well ordered crystals of the enzyme. Here, the results of an X-ray crystallographic study on the laccase from the fungus Cerrena maxima are reported. Crystals that diffract well to a resolution of at least 1.9 A (R factor = 18.953%; R(free) = 23.835; r.m.s.d. bond lengths, 0.06 A; r.m.s.d. bond angles, 1.07 degrees) have been obtained despite the presence of glycan moieties. The overall spatial organization of C. maxima laccase and the structure of its copper-containing active centre have been determined by the molecular-replacement method using the laccase from Trametes versicolor (Piontek et al., 2002) as a structural template. In addition, four glycan-binding sites were identified and the 1.9 A X-ray data were used to determine the previously unknown primary structure of this protein. The identity (calculated from sequence alignment) between the C. maxima laccase and the T. versicolor laccase is about 87%. Tyr196 and Tyr372 show significant extra density at the ortho positions and this has been interpreted in terms of NO(2) substituents.

Basidiomycota↗

PubMed2006

X-ray structural studies of the fungal laccase from Cerrena maxima.

Laccases are members of the blue multi-copper oxidase family. These enzymes oxidize substrate molecules by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear centre. Dioxygen binds to the trinuclear centre and following the transfer of four electrons is reduced to two molecules of water. The X-ray structure of a laccase from Cerrena maxima has been elucidated at 1.9 A resolution using synchrotron data and the molecular replacement technique. The final refinement coefficients are Rcryst = 16.8% and Rfree = 23.0%, with root mean square deviations on bond lengths and bond angles of 0.015 A and 1.51 degrees , respectively. The type 1 copper centre has an isoleucine residue at the axial position and the "resting" state of the trinuclear centre comprises a single oxygen (OH) moiety asymmetrically disposed between the two type 3 copper ions and a water molecule attached to the type 2 ion. Several carbohydrate binding sites have been identified and the glycan chains appear to promote the formation of well-ordered crystals. Two tyrosine residues near the protein surface have been found in a nitrated state.

Binding Sites↗

PubMed2006

The role of the hybrid cluster protein in oxidative stress defense.

Hybrid cluster proteins (HCP) contain two types of Fe/S clusters, namely a [4Fe-4S](2+/1+) or [2Fe-2S](2+/1+) cluster and a novel type of hybrid cluster, [4Fe-2S-2O], in the as-isolated state. Although first isolated from anaerobic sulfate-reducing bacteria, the analysis of the genomic sequences reveals that genes encoding putative hybrid cluster proteins are present in a wide range of organisms, aerobic, anaerobic, or facultative, from the Bacteria, Archaea, and Eukarya domains. Despite a detailed spectroscopic and structural characterization, the precise physiological function of these proteins remained unknown. The present work shows that the transcription of the Escherichia coli hcp gene is induced by hydrogen peroxide, and this induction is regulated by the redox-sensitive transcriptional activator, OxyR. The E. coli hcp mutant strain exhibits higher sensitivity to hydrogen peroxide, a behavior that reverts to the wild type phenotype once a plasmid carrying the hcp gene is reintroduced. Furthermore, the purified HCPs from E. coli and Desulfovibrio desulfuricans ATCC 27774 show an alternative enzymatic activity, which under physiological conditions exhibited K(m) values for hydrogen peroxide (approximately 0.3 mM) within the range of other peroxidases. Altogether, the results reveal that HCP is involved in oxidative stress protection.

Desulfovibrio desulfuricans↗

PubMed2006

Reduction of dioxygen by enzymes containing copper.

The reduction of dioxygen is a key step in many important biological processes including respiration and ligand oxidation. Enzymes containing either iron or copper or, indeed, both elements are often involved in this process, yet the catalytic mechanisms employed are not fully understood at the current time despite intensive biochemical, spectroscopic and structural studies. The aim of this article is to highlight the current structural knowledge regarding the process of dioxygen reduction using examples of copper-containing enzymes.

Copper↗

PubMed2006

Perturbations of the T1 copper site in the CotA laccase from Bacillus subtilis: structural, biochemical, enzymatic and stability studies.

Site-directed mutagenesis has been used to replace Met502 in CotA laccase by the residues leucine and phenylalanine. X-ray structural comparison of M502L and M502F mutants with the wild-type CotA shows that the geometry of the T1 copper site is maintained as well as the overall fold of the proteins. The replacement of the weak so-called axial ligand of the T1 site leads to an increase in the redox potential by approximately 100 mV relative to that of the wild-type enzyme (E0 =455 mV). However the M502L mutant exhibits a twofold to fourfold decrease in the kcat values for the all substrates tested and the catalytic activity in M502F is even more severely compromised; 10% activity and 0.15-0.05% for the non-phenolic substrates and for the phenolic substrates tested when compared with the wild-type enzyme. T1 copper depletion is a key event in the inactivation and thus it is a determinant of the thermodynamic stability of wild-type and mutant proteins. Whilst the unfolding of the tertiary structure in the wild-type enzyme is a two-state process displaying a midpoint at a guanidinium hydrochloride concentration of 4.6 M and a free-energy exchange in water of 10 kcal/mol, the unfolding for both mutant enzymes is clearly not a two-state process. At 1.9 M guanidinium hydrochloride, half of the molecules are in an intermediate conformation, only slightly less stable than the native state (approximately 1.4 kcal/mol). The T1 copper centre clearly plays a key role, from the structural, catalytic and stability viewpoints, in the regulation of CotA laccase activity.

Amino Acid Substitution↗

PubMed2005

Dioxygen reduction by multi-copper oxidases; a structural perspective.

The multi-copper oxidases oxidise substrate molecules by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear centre. Dioxygen binds to the trinuclear centre and, following the transfer of four electrons, is reduced to two molecules of water. The precise mechanism of this reduction has been unclear, but recent X-ray structural studies using the CotA endospore coat protein from Bacillus subtilis have given further insights into the principal stages. It is proposed that the mechanism involves binding of the dioxygen into the trinuclear centre so that it is sited approximately symmetrically between the two type 3 copper ions with one oxygen atom close to the type 2 copper ion. Further stages involve the formation of a peroxide intermediate and following the splitting of this intermediate, the migration of the hydroxide moieties towards the solvent exit channel. The migration steps are likely to involve a movement of the type 2 copper ion and its environment. Details of a putative mechanism are described herein based both on structures already reported in the literature and on structures of the CotA protein in the oxidised and reduced states and with the addition of peroxide and the inhibitor, azide.

Azides↗

PubMed2004

Ligand-binding and metal-exchange crystallographic studies on shrimp alkaline phosphatase.

Alkaline phosphatases (APs) are homodimeric metalloenzymes that catalyze the hydrolysis and transphosphorylation of phosphate monoesters. Each monomer contains a metal-binding triad that for optimal activity is usually occupied by two zinc ions and one magnesium ion. The recently determined crystal structure of cold-active shrimp alkaline phosphatase (SAP) was, however, fully occupied by zinc ions. This paper describes a metal-exchange experiment in which the zinc ion in one binding site (referred to as the M3 site) is replaced by magnesium. Crystal structures revealed a concomitant structural change: the metal exchange causes movement of a ligating histidine into a conformation in which it does not coordinate to the metal ion. The M3 site is relevant to catalysis: its occupation by magnesium is postulated to favour catalysis and it has been suggested to be a regulatory site for other APs. Further crystallographic studies show that ligand binding can induce a conformational change of an active-site arginine from a 'non-docked' (non-interacting) to a 'docked' conformation (interacting with the ligand). The first conformation has only been observed in SAP, while the latter is common in available AP structures. The observation that the arginine does not always bind the substrate may explain the increased catalytic efficiency that is generally observed for cold-active enzymes.

Alkaline Phosphatase↗

PubMed2004

Substrate and dioxygen binding to the endospore coat laccase from Bacillus subtilis.

The CotA laccase from the endospore coat of Bacillus subtilis has been crystallized in the presence of the non-catalytic co-oxidant 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and the structure was determined using synchrotron radiation. The binding site for this adduct is well defined and indicates how ABTS, in conjunction with laccases, could act as an oxidative mediator toward non-phenolic moieties. In addition, a dioxygen moiety is clearly defined within the solvent channel oriented toward one of the T3 copper atoms in the trinuclear center.

Bacillus subtilis↗

PubMed2002

The mechanism of iron uptake by transferrins: the X-ray structures of the 18 kDa NII domain fragment of duck ovotransferrin and its nitrilotriacetate complex.

In a previous paper [Lindley et al. (1993), Acta Cryst. D49, 292-304], the X-ray structure analysis of the 18 kDa fragment of duck ovotransferrin, corresponding to the NII domain of the intact protein, was reported at a resolution of 2.3 A. In this structure, the Fe(III) cation binds to two tyrosine residues and the synergistic carbonate anion in an identical manner to that found in the intact protein. However, the aspartate and histidine residues, normally involved in iron binding in transferrins, are absent in the fragment and it was not possible to unequivocally define what had replaced them. The electron density was tentatively assigned to be a mixture of peptides, presumably resulting from the proteolytic preparation of the fragment, binding to the iron through their amino and carboxylate termini. A more recent X-ray analysis of the fragment, from a different preparation, has resulted in a structure at 1.95 A, in which glycine appears to be the predominant residue bound to the cation. In an alternative attempt to clarify the binding of iron to the 18 kDa fragment, the metal was removed by dialysis and replaced in the form of ferric nitrilotriacetate. Crystallization of this complex has resulted in an X-ray structure at 1.90 A in which the Fe(III) is bound to the synergistic carbonate anion and only one tyrosine residue in a manner almost identical to the intact protein. The carboxylate groups and the tertiary amino group of the nitrilotriacetate occupy the remaining coordination sites. The second tyrosine residue, Tyr95, is not bound directly to the iron. The implication of these structures with respect to the mechanism of iron binding by the transferrins is addressed.

Animals↗