Insight into the multicopper oxidases stability

Dissertation presented to obtain the PhD degree in BiochemistryThis dissertation portrays recent development on the knowledge of the stability determinants and of functional characteristics of multicopper oxidases (MCO). Multicopper oxidases are a family of enzymes that includes laccases (benzenediol oxygen oxidoreductase; EC 1.10.3.2), ascorbate oxidase (L-ascorbate oxygen oxidoreductase, EC 1.10.3.3) and ceruloplasmin (Fe2+ oxygen oxidoreductase, EC 1.16.3.1). MCO are characterized by having four copper ions that are classified into three distinct types of copper sites, namely type 1 (T1), type 2 (T2) and type 3 (T3). The classical T1 copper site comprises two histidine residues and a cysteine residue arranged in a distorted trigonal geometry around the copper ion with bonding distances approx. 2.0 Å (1 Å=0.1 nm); a weaker fourth methionine ligand completes the tetrahedral geometry. The copper–cysteine linkage is characterized by an intense S(π)→Cu(dx2−y2) CT (charge transfer) absorption band at approximately 600 nm, and a narrow parallel hyperfine splitting A\\ = (43–90)×10−4 cm−1 in the electron paramagnetic resonance (EPR) spectrum. The function of the T1 copper site is to shuttle electrons from substrates to the trinuclear copper centre where molecular oxygen is reduced to two molecules of water during the complete four-electron catalytic cycle. The trinuclear center contains a T2 copper coordinated by two histidine residues and one water molecule, lacks strong absorption bands and exhibits a large parallel hyperfine splitting in the EPR spectrum (A\\ = (150–201)×10−4 cm−1). The T2 copper site is in close proximity to two T3 copper ions, which are each coordinated by three histidine residues and typically coupled, for example, through a dioxygen molecule. The T3 or coupled binuclear copper site is characterized by an intense absorption band at 330 nm originating from the bridging ligand and by the absence of an EPR signal due to the antiferromagnetically coupling of the copper ions.(...)Apoio financeiro da FCT e do FSE no âmbito do Quadro Comunitário de Apoio, BD nº SFRH/BD/31444/200

Protein Engineering Applications On Candida Methylica Formate Dehydrogenase To Elucidate Folding Mechanisms And To Increase The Thermostability

Tez (Doktora) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2010Thesis (PhD) -- İstanbul Technical University, Institute of Science and Technology, 2010NAD+-bağımlı format dehidrogenaz (EC 1.2.1.2, FDH) metilotrofik mikroorganizmalarda metanol metabolizmasındaki son enzimdir ve formatın karbondioksite oksidasyonunu katalizlerken NAD+’yi NADH’e indirger. Endüstriyel açıdan en önemli rolü NAD+ ve format kullanarak NADH’i rejenere etmesidir. Birçok avantajına rağmen düşük termostabilitesi FDH enzimin en önemli dezavantajıdır. Bu tez çalışması başlıca üç kısma ayrılmaktadır. Öncelikle protein mühendisliği çalışmalarında oluşturulacak mutant enzimlerin kolay ve verimli saflaştırılabilmesi amacıyla Candida methylica mayasından klonlanmış olan NAD+-bağımlı format dehidrogenaz (cmFDH) enzimi için basit ve etkili bir protein saflaştırma metodu geliştirilmiştir. İkinci bölümde orjinal cmFDH enziminin katlanma mekanizması ve stabilitesi incelenmiştir. Protein dissosiayon sabitinin 10-13 M olarak hesaplandığı denge denaturasyon deneyleri cmFDH’in iki yapılı tek adım geçiş modeline göre katlanmamış hale geçtiğini göstermiştir. Kinetik deneyler sonucunda katlanma işleminin iki adımda gerçekleştiği gözlenmiştir. Fizyolojik koşullarda dimerik yapının dissosiasyon sabiti oldukça yavaştır (3x10-7 s-1). Son olarak, termostabiliteyi arttırmak amacıyla Pseudomonas sp. 101 ve Candida boidinii FDH’lerinin kristal yapıları temel alınarak hazırlanan cmFDH homolji modeli kullanılarak orjinal enzim üzerine protein mühendisliği stratejileri uygulanmıştır. Termodinamik ve kinetik sonuçlar dört mutasyonun, N187E, Q105R and N147R, M1C, orjinal cmFDH enziminin katlanma biçimini değiştirmeden stabiliteyi arttırdığını ortaya koymaktadır.NAD+-dependent formate dehydrogenase (EC 1.2.1.2, FDH) is the last enzyme in the metabolism of methanol in methylotrophs and catalyzes the oxidation of formate anion into carbondioxide concomitant with the reduction of NAD+ to NADH. One crucial role of this enzyme in industrial redox chemistry is to regenerate NADH from NAD+ and formate. Although there are many advantages using FDH for this purpose, one problem is a lack of thermostability. The studies described in this thesis are divided into three parts. Initially, a simple and efficient molecular biological method was described to improve the purification of NAD+-dependent FDH from yeast Candida methylica (cmFDH). It allowed us to easily purify the constructed thermostabile mutants. The second section describes the folding mechanism and stability of dimeric native cmFDH. Equilibrium denaturation data yielded a dissociation constant of about 10-13 M. Findings showed that native cmFDH unfolds by two state single transition model in equilibrium. The kinetics of refolding and unfolding reactions revealed that the overall process comprises 2 steps. The rate of dissociation of the dimeric state in physiological conditions is extremely slow (k-2 ~ 3x10-7 s-1). Finally, protein engineering strategies were applied to increase the thermostability by using a homology model of cmFDH based on Pseudomonas sp. 101 and Candida boidinii. The thermodynamic and kinetic results suggested that four mutations, N187E, Q105R and N147R, M1C increased the stability while the folding and unfolding patterns of native cmFDH was not altered.DoktoraPh

Kinetic stability and temperature adaptation. Observations from a cold adapted subtilisin-like serine protease.

Life on earth is found everywhere where water is found, meaning that life has adapted to extremely varied environments. Thus, protein structures must adapt to a myriad of environmental stressors while maintaining their functional forms. In the case of enzymes, temperature is one of the main evolutionary pressures, affecting both the stability of the structure and the rate of catalysis. One of the solutions Nature has come up with to maintain activity and stability in harsh environments over biological relevant timescales, are kinetically stable proteins. This thesis will outline work carried out on the kinetically stable VPR, a cold active subtilisin-like serine protease and discuss our current understanding of protein kinetic stability, temperature adaptation and our current hypothesis of the molecular interactions contributing to the stability of VPR. The research model that we have used to study these attributes consists of the cold active VPR and its thermostable structural homolog AQUI. The results discussed in this thesis will be on the importance of calcium, the role of prolines in loops, the role of a conserved N-terminal tryptophan residue and lastly primary observations on differences in active site dynamics between VPR and AQUI. A model is proposed of a native structure that unfolds in a highly cooperative manner. This cooperativity can be disrupted, however, by modifying calcium binding of the protein or via mutations that affect how the N-terminus interacts with the rest of the protein. The N-terminus likely acts as a kinetic lock that infers stability to the rest of the structure through many different interactions. Some of these interactions may be strengthened via proline residues, that seemingly act as anchor points that tend to maintain correct orientation between these parts of the protein as thermal energy is increased in the system. Our results give a deeper insight into the nature of the kinetic stability, the importance of cooperativity during unfolding of kinetically stable proteases, synergy between distant parts of the protein through proline mutations and how different calcium binding sites have vastly differing roles. The results provide a solid ground for continuing work in designing enzyme variants with desired stabilities and activities and improve our understanding of kinetically stable systems.The Icelandic Research Fund [grant number 162977-051

Metal ions and protein folding: conformational and functional interplay

Dissertation presented to obtain a PhD degree in Biochemistry at Instituto de Tecnologia Química e Biológica, Universidade Nova de LisboaMetal ions are cofactors in about 30% of all proteins, where they fulfill catalytical and structural roles. Due to their unique chemistry and coordination properties they effectively expand the intrinsic polypeptide properties (by participating in catalysis or electron transfer reactions), stabilize protein conformations (like in zinc fingers) and mediate signal transduction (by promoting functionally relevant protein conformational changes). However, metal ions can also exert have deleterious effects in living systems by incorporating in non-native binding sites, promoting aberrant protein aggregation or mediating redox cycling with generation of reactive oxygen and nitrogen species. For this reason, the characterization of the roles of metal ions as modulators of protein conformation and stability provides fundamental knowledge on protein folding properties and is instrumental in establishing the molecular basis of disease. In this thesis we have analyzed protein folding processes using model protein systems incorporating covalently bound metal cofactors – iron-sulfur (FeS) proteins – or where metal ion binding is reversible and associated conformational readjustments – the S100 proteins.(...

Diseño e ingeniería de lacasas quiméricas por evolución dirigida para el tratamiento de biomasa vegetal

Las lacasas son oxidasas multicobre que se encuentran ampliamente distribuidas en la naturaleza aunque los principales productores de estas enzimas son los hongos basidiomicetos de la podredumbre blanca de la madera, los únicos organismos capaces de degradar completamente la lignina. Las lacasas catalizan la oxidación de una gran variedad de compuestos aromáticos acoplada a la reducción de oxígeno a agua. Además, su capacidad catalítica puede ampliarse en presencia de mediadores redox, compuestos de bajo peso molecular que, una vez oxidados por la lacasa, pueden oxidar otros compuestos que en principio no son sustrato de la enzima. Debido a su amplio rango de sustratos y a sus bajos requerimientos catalíticos, las lacasas y los sistemas lacasa-mediador tienen un gran potencial biotecnológico, particularmente en procesos de tratamiento de la biomasa vegetal como son la producción de pasta de papel o de biocombustibles de segunda generación. Además, en estos procesos se generan una gran cantidad de residuos ricos en fenoles derivados de la lignina que pueden actuar como mediadores de lacasa, y cuyo uso podría reducir los costes y el impacto medioambiental del proceso. Sin embargo, para la aplicación industrial de las lacasas puede ser necesario recurrir a la ingeniería de proteínas para ajustar sus propiedades catalíticas a las condiciones de operación. En trabajos previos se ha descrito la evolución dirigida de dos lacasas de alto potencial redox procedentes de los basidiomicetos PM1 (PM1L) y Pycnoporus cinnabarinus (PcL). Las variantes evolucionadas de estas lacasas, OB1 y 3PO, respectivamente, son el punto de partida de esta tesis doctoral..