Towards resolving the complex paramagnetic NMR spectrum of small laccase: Assignments of resonances to residue specific nuclei

Laccases efficiently reduce dioxygen to water in an active site containing a tri-nuclear copper centre (TNC). One reason for its efficiency in catalysis of this complex reaction can be the presence of mobility of active site residues. To probe mobility, NMR spectroscopy is highly suitable. However, several factors complicate the assignment of resonances to active site nuclei in laccases. The paramagnetic nature causes large shifts and line broadening. Furthermore, the presence of slow chemical exchange processes of the imidazole rings of copper ligands result in peak doubling. A third complicating factor is that the enzyme occurs in two states, the native intermediate (NI) and resting oxidized (RO) states, with different paramagnetic properties. The present study aims at resolving the complex paramagnetic NMR spectra of the TNC of Streptomyces coelicolor small laccase (SLAC). With a combination of paramagnetically tailored NMR experiments, all eight His Nδ1 and Hδ1 resonances for the NI state are identified, as well as His Hβ protons for the RO state. With the help of second shell mutagenesis, selective resonances are tentatively assigned to the T2 histidines. This study demonstrates approaches that can be used for sequence specific assignment of the paramagnetic NMR spectra of ligands in the TNC that ultimately may lead to a description of the underlying motions.

Computational Design of Copper Ligands with Controlled Metal Chelating, Pharmacokinetics, and Redox Properties for Alzheimer’s Disease

Background: Redox active metal cations, such as Cu2 +, have been related to induce amyloid plaques formation and oxidative stress, which are two of the key events in the development of Alzheimer’s disease (AD) and others metal promoted neurodegenerative diseases. In these oxidative events, standard reduction potential (SRP) is an important property especially relevant in the reactive oxygen species formation. Objective: The SRP is not usually considered for the selection of drug candidates in anti-AD treatments. In this work, we present a computational protocol for the selection of multifunctional ligands with suitable metal chelating, pharmacokinetics, and redox properties. Methods: The filtering process is based on quantum chemical calculations and the use of in silico tools. Calculations of SRP were performed by using the M06-2X density functional and the isodesmic approach. Then, a virtual screening technique (VS) was used for similar structure search. Results: Protocol application allowed the assessment of chelating, drug likeness, and redox properties of copper ligands. Those molecules showing the best features were selected as molecular scaffolds for a VS procedure in order to obtain related compounds. After applying this process, we present a list of candidates with suitable properties to prevent the redox reactions mediated by copper(II) ion. Conclusion: The protocol incorporates SRP in the filtering stage and can be effectively used to obtain a set of potential drug candidates for AD treatments.

Effect of the Ligand in the Synthesis of 1-Sulfonyl-1,2,3-triazoles Through Copper-Catalyzed Alkyne-Azide Cycloaddition

Copper-catalyzed alkyne-azide cycloaddition of <em>p</em>-toluenesulfonylazide and phenylacetylene was investigated, using catalytic amounts of copper (I) chloride and diverse additives which were tested as copper ligands. The best results were obtained using a catalytic amount of diphenyl disulfide

Mapping of a copper-binding site on the small CP12 chloroplastic protein of Chlamydomonas reinhardtii using top-down mass spectrometry and site-directed mutagenesis

CP12 is a small chloroplastic protein involved in the Calvin cycle that was shown to bind copper, a metal ion that is involved in the transition of CP12 from a reduced to an oxidized state. In order to describe CP12's copper-binding properties, copper-IMAC experiments and site-directed mutagenesis based on computational modelling, were coupled with top-down MS [electrospray-ionization MS and MS/MS (tandem MS)]. Immobilized-copper-ion-affinity-chromatographic experiments allowed the primary characterization of the effects of mutation on copper binding. Top-down MS/MS experiments carried out under non-denaturing conditions on wild-type and mutant CP12–Cu2+ complexes then allowed fragment ions specifically binding the copper ion to be determined. Comparison of MS/MS datasets defined three regions involved in metal ion binding: residues Asp16–Asp23, Asp38–Lys50 and Asp70–Glu76, with the two first regions containing selected residues for mutation. These data confirmed that copper ligands involved glutamic acid and aspartic residues, a situation that contrasts with that obtaining for typical protein copper chelators. We propose that copper might play a role in the regulation of the biological activity of CP12.

Identification of copper ligands in Aspergillus oryzae tyrosinase by site-directed mutagenesis

Copper ligands of the recombinant tyrosinase from the fungus Aspergillus oryzae expressed in Saccharomyces cerevisiae or Escherichia coli were identified by site-directed mutagenesis. The recombinant protyrosinases expressed in S. cerevisiae were assayed for catalytic activities of mono-oxygenase and L-dopa oxidase at pH 5.5 after acid shock at pH 3.0. Replacements of His-63, His-84, His-93, His-290, His-294, His-332 or His-333 with asparagine resulted in mutant enzymes exhibiting no activities. The site-directed mutant Cys82Ala showed that Cys-82 was also an essential residue for the activity. We obtained homogeneous preparations of activated tyrosinases from mutated thioredoxin fusion gene products expressed in E. coli by acid shock. The copper contents of engineered mutants and wild-type enzyme expressed in E. coli were determined by atomic absorption spectrophotometry. The wild-type enzyme contained 2 g-atoms of copper/mol of the subunit. The His63Asn, His84Asn, His93Asn, His290Asn, His294Asn, His332Asn, His333Asn or Cys82Ala substitution decreased copper binding by approx. 50%, indicating that the mutants contain only approx. 1 g-atom of copper/mol of the subunit. The five mutants His63Asn, His93Asn, His290Asn, His294Asn and Cys82Ala contain only one copper ion, which is fully detectable by EPR. From the correlation of g‖ and CuA‖, we deduced that the nitrogen or sulphur donors in the copper ligands should be in a square or a distorted tetrahedral geometric environment. In further atomic absorption spectrophotometry experiments, no copper atom was observed in the seven double mutants His63Asn/His290Asn, His63Asn/His294Asn, His63Asn/His332Asn, His63Asn/His333Asn, Cys82Ala/His290Asn, His84Asn/His333Asn and His93Asn/His290Asn. We propose a new structure of active sites of tyrosinase from A. oryzae: the most likely binding sites of tyrosinase for Cu(A) are His-63, His-84 and His-93, with the remaining conserved Cys-82 providing the fourth ligand. Cu(B) liganded by four histidine residues, His-290, His-294, His-332 and His-333, is identified as new binding motif of Cu(B).