André Fernandes

A robust metallo-oxidase from the hyperthermophilic bacterium Aquifex aeolicus.

The gene, Aquifex aeolicus AAC07157.1, encoding a multicopper oxidase (McoA) and localized in the genome as part of a putative copper‐resistance determinant, has been cloned, over‐expressed in Escherichia coli and the recombinant enzyme purified to homogeneity. The purified enzyme shows spectroscopic and biochemical characteristics typical of the well‐characterized multicopper oxidase family of enzymes. McoA presents higher specificity (kcat/Km) for cuprous and ferrous ions than for aromatic substrates and is therefore designated as a metallo‐oxidase. Addition of copper is required for maximal catalytic efficiency. A comparative model structure of McoA has been constructed and a striking structural feature is the presence of a methionine‐rich region (residues 321–363), reminiscent of those found in copper homeostasis proteins. The kinetic properties of a mutant enzyme, McoAΔP321‐V363, deleted in the methionine‐rich region, provide evidence for the key role of this region in the modulation of the catalytic mechanism. McoA has an optimal temperature of 75 °C and presents remarkable heat stability at 80 and 90 °C, with activity lasting for up to 9 and 5 h, respectively. McoA probably contributes to copper and iron homeostasis in A. aeolicus.

The multicopper oxidase from the archaeon Pyrobaculum aerophilum shows nitrous oxide reductase activity

The multicopper oxidase from the hyperthermophilic archaeon Pyrobaculum aerophilum (McoP) was overproduced in Escherichia coli and purified to homogeneity. The enzyme consists of a single 49.6 kDa subunit, and the combined results of UV–visible, CD, EPR and resonance Raman spectroscopies showed the characteristic features of the multicopper oxidases. Analysis of the McoP sequence allowed its structure to be derived by comparative modeling methods. This model provided a criterion for designing meaningful site‐directed mutants of the enzyme. McoP is a hyperthermoactive and thermostable enzyme with an optimum reaction temperature of 85 °C, a half‐life of inactivation of ∼ 6 h at 80 °C, and temperature values at the midpoint from 97 to 112 °C. McoP is an efficient metallo‐oxidase that catalyzes the oxidation of cuprous and ferrous ions with turnover rate constants of 356 and 128 min−1, respectively, at 40 °C. It is noteworthy that McoP follows a ping‐pong mechanism, with three‐fold higher catalytic efficiency when using nitrous oxide as electron acceptor than when using dioxygen, the typical oxidizing substrate of multicopper oxidases. This finding led us to propose that McoP represents a novel archaeal nitrous oxide reductase that is most probably involved in the final step of the denitrification pathway of P. aerophilum.

The hyperthermophilic nature of the metallo-oxidase from Aquifex aeolicus.

The stability of the Aquifex aeolicus multicopper oxidase (McoA) was studied by spectroscopy, calorimetry and chromatography to understand its thermophilic nature. The enzyme is hyperthermostable as deconvolution of the differential scanning calorimetry trace shows that thermal unfolding is characterized by temperature values at the mid-point of 105, 110 and 114 degrees C. Chemical denaturation revealed however a very low stability at room temperature (2.8 kcal/mol) because copper bleaching/depletion occur before the unfolding of the tertiary structure and McoA is highly prone to aggregate. Indeed, unfolding kinetics measured with the stopped-flow technique quantified the stabilizing effect of copper on McoA (1.5 kcal/mol) and revealed quite an uncommon observation further confirmed by light scattering and gel filtration chromatography: McoA aggregates in the presence of guanidinium hydrochloride, i.e., under unfolding conditions. The aggregation process results from the accumulation of a quasi-native state of McoA that binds to ANS and is the main determinant of the stability curve of McoA. Kinetic partitioning between aggregation and unfolding leads to a very low heat capacity change and determines a flat dependence of stability on temperature.

Insight into stability of CotA laccase from the spore coat of Bacillus subtilis.

The axial ligand of the catalytic mononuclear T1 copper site (Met(502)) of the CotA laccase was replaced by a leucine or phenylalanine residue to increase the redox potential of the enzyme. These mutations led to an increase in the redox potential by approx. 100 mV relative to the wild-type enzyme but the catalytic constant k(cat) in the mutant enzymes was severely compromised. This decrease in the catalytic efficiency was unexpected as the X-ray analysis of mutants has shown that replacement of methionine ligand did not lead to major structural changes in the geometry of the T1 centre or in the overall fold of the enzyme. However, the mutations have a profound impact on the thermodynamic stability of the enzyme. The fold of the enzyme has become unstable especially with the introduction of the larger phenylalanine residue and this instability should be related to the decrease in the catalytic efficiency. The instability of the fold for the mutant proteins resulted in the accumulation of an intermediate state, partly unfolded, in-between native and unfolded states. Quenching of tryptophan fluorescence by acrylamide has further revealed that the intermediate state is partly unfolded.

The removal of a disulfide bridge in CotA-laccase changes the slower motion dynamics involved in copper binding but has no effect on the thermodynamic stability

The contribution of the disulfide bridge in CotA-laccase from Bacillus subtilis is assessed with respect to the enzyme’s functional and structural properties. The removal of the disulfide bond by site-directed mutagenesis, creating the C322A mutant, does not affect the spectroscopic or catalytic properties and, surprisingly, neither the long-term nor the thermodynamic stability parameters of the enzyme. Furthermore, the crystal structure of the C322A mutant indicates that the overall structure is essentially the same as that of the wild type, with only slight alterations evident in the immediate proximity of the mutation. In the mutant enzyme, the loop containing the C322 residue becomes less ordered, suggesting perturbations to the substrate binding pocket. Despite the wild type and the C322A mutant showing similar thermodynamic stability in equilibrium, the holo or apo forms of the mutant unfold at faster rates than the wild-type enzyme. The picosecond to nanosecond time range dynamics of the mutant enzyme was not affected as shown by acrylamide collisional fluorescence quenching analysis. Interestingly, copper uptake or copper release as measured by the stopped-flow technique also occurs more rapidly in the C322A mutant than in the wild-type enzyme. Overall the structural and kinetic data presented here suggest that the disulfide bridge in CotA-laccase contributes to the conformational dynamics of the protein on the microsecond to millisecond timescale, with implications for the rates of copper incorporation into and release from the catalytic centres.

Unfolding pathway of CotA-laccase and the role of copper on the prevention of refolding through aggregation of the unfolded state.

Copper is a redox-active metal and the main player in electron transfer reactions occurring in multicopper oxidases. The role of copper in the unfolding pathway and refolding of the multicopper oxidase CotA laccase in vitro was solved using double-jump stopped-flow experiments. Unfolding of apo- and holo-CotA was described as a three-state process with accumulation of an intermediate in between the native and unfolded state. Copper stabilizes the native holo-CotA but also the intermediate state showing that copper is still bound to this state. Also, copper binds to unfolded holo-CotA in a non-native coordination promoting CotA aggregation and preventing refolding to the native structure. These results gather information on unfolding/folding pathways of multicopper oxidases and show that copper incorporation in vivo should be a tight controlled process as copper binding to the unfolded state under native conditions promotes protein aggregation.

The Cultural Heritage in the Postindustrial Waterfront: A Case Study of the South Bank of the Tagus Estuary, Portugal

In examining the process of waterfront revitalization of the South bank of the Tagus Estuary during the postindustrial era, it is possible to discern a commitment to appropriation and valorization of heritage and cultural identity inherited from preceding economic cycles. Using a qualitative approach, backed up by a detailed analysis of territorial planning instruments, strategic documents, and intervention projects, we identify three main ways of appropriation, aimed to add value to the heritage and cultural identity, considering them as follows: (a) resources for the promotion of various activities, (b) drivers in strengthening territorial identity, and (c) elements of territorial differentiation. Analyzing these aspects, the article presents a proposal for the classification of the actions proposed for their implementation, namely: conservation of cultural heritage, adaptation of heritage to new uses and functions, appropriation of symbolic elements, promotion of cultural events, and raising awareness of cultural heritage among local communities.

Metamorfoses da cidade portuária: transformações da relação entre o porto e a cidade de Lisboa

O processo de evolucao dos portos no periodo moderno tem sido determinado pela mutabilidade das funcoes portuarias, induzida por factores como as evolucoes tecnologicas nos navios, nos equipamentos de apoio a movimentacao de mercadorias ou nas tecnicas de armazenagem, transformacoes nos padroes do comercio internacional, modificacoes na organizacao dos transportes maritimos ou alargamento e complexificacao das cadeias logisticas globais. Como resultado destas mutacoes, assiste-se a uma concomitante evolucao da estrutura, morfologia e extensao dos espacos ocupados por actividades portuarias e actividades complementares. Neste contexto, a relacao entre o porto e a cidade de Lisboa (Portugal) e marcada por uma grande complexidade desde logo imprimida pela tradicao historica que a mesma encerra e pela magnitude e intensidade de um processo que envolve a principal infra-estrutura do sistema portuario nacional e a maior cidade do pais. Note-se, porem, que de uma relacao simbiotica cidade-porto, em que o porto exercia um papel estrutural na organizacao da cidade e das suas funcoes, evoluiu-se para uma relacao em que a cidade, polarizadora do sistema urbano regional e principal elemento de integracao nacional na rede urbana peninsular e europeia, se autonomiza e assume um caracter eminentemente generalista, economicamente diversificado e funcionalmente complexo. Nao obstante esta evolucao, o porto mantem-se como ponto nodal de integracao da cidade-regiao em sistemas globais de fluxos materiais e imateriais. Tratando-se de um elemento progressivamente menos integrado na estrutura urbana e funcional da cidade, o porto continua assim a marcar a cadencia de importantes mutacoes e transformacoes. Com efeito, a luz de conceptualizacoes e sistematizacoes teoricas e da analise das evidencias empiricas, analisa-se a evolucao da relacao entre o porto e a cidade de Lisboa (incidindo nas dimensoes funcional, urbanistica e economica) e apresenta-se um esquema de base para a definicao de um modelo original que identifique, explique e caracterize as grandes fases desta dinâmica relacional.