1,721,342 research outputs found
The causative role and therapeutic potential of the kynurenine pathway in neurodegenerative disease
No full textMetabolites of the kynurenine pathway (KP), which arise from the degradation of tryptophan, have been studied in detail for over a century and garnered the interest of the neuroscience community in the late 1970s and early 1980s with work uncovering the neuromodulatory potential of this pathway. Much research in the following decades has found that perturbations in the levels of KP metabolites likely contribute to the pathogenesis of several neurodegenerative diseases. More recently, it has become apparent that targeting KP enzymes, in particular kynurenine 3-monooxygenase (KMO), may hold substantial therapeutic potential for these disorders. Here we provide an overview of the KP, the neuroactive properties of KP metabolites and their role in neurodegeneration. We also discuss KMO as a therapeutic target for these disorders, and our recent resolution of the crystallographic structure of KMO, which will permit the development of new and improved KMO inhibitors which may ultimately expedite clinical application of these compounds. © 2013 Springer-Verlag Berlin Heidelberg
The dimeric form of flavocytochrome P450 BM3 is catalytically functional as a fatty acid hydroxylase
No full textIn the model P450 BM3 system, the P450 is fused to its diflavin reductase partner in a single polypeptide. BM3 dimerizes in solution, but the catalytic relevance of the phenomenon was hitherto unknown. We show that BM3 fatty acid hydroxylase specific activity decreases sharply at low enzyme concentrations, consistent with separation of active dimer into inactive monomer. Reductase-dependent specific activities are maintained or enhanced at low concentration, suggesting inter-flavin electron transfer is unaffected. Fatty acid oxidation is reconstituted by mixing inactive oxygenase (A264H) and FMN-depleted (G570D) mutants, demonstrating that inter-monomer (FMN1-to-heme2) electron transfer supports oxygenase activity in the BM3 dimer
Flavocytochrome P450 BM3 substrate selectivity and electron transfer in a model cytochrome P450
No full text
Beyond the Historical Perspective on Hydrogen and Electron Transfers
Get PDFA brief overview of proton and electron transfer history is given, and various
features influencing enzymatic catalysis are discussed. Examples of generic
behavior are considered, together with questions that can be addressed for both
experimental and computational results. Examples of high and low pre-exponential
factors A of the intrinsic rate constant k_H ranging from ~10^(17) s^(-1) to ~10^4 s^(-1) and normal (~10^(13)) are noted with significant error bars and
discussed
Production of the Fragrance Geraniol in Peroxisomes of a Product-Tolerant Baker’s Yeast
No full textMonoterpenoids, such as the plant metabolite geraniol, are of high industrial relevance since they are important fragrance materials for perfumes, cosmetics, and household products. Chemical synthesis or extraction from plant material for industry purposes are complex, environmentally harmful or expensive and depend on seasonal variations. Heterologous microbial production offers a cost-efficient and sustainable alternative but suffers from low metabolic flux of the precursors and toxicity of the monoterpenoid to the cells. In this study, we evaluated two approaches to counteract both issues by compartmentalizing the biosynthetic enzymes for geraniol to the peroxisomes of Saccharomyces cerevisiae as production sites and by improving the geraniol tolerance of the yeast cells. The combination of both approaches led to an 80% increase in the geraniol titers. In the future, the inclusion of product tolerance and peroxisomal compartmentalization into the general chassis engineering toolbox for monoterpenoids or other host-damaging, industrially relevant metabolites may lead to an efficient, low-cost, and eco-friendly microbial production for industrial purposes
Structure and mechanism of kynurenine 3-monooxygenase, a candidate huntington’s disease drug target
No full textTese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2013Huntington’s disease (HD) is a neurodegenerative disorder caused by a
polyglutamine expansion in the huntingtin protein. There are currently no effective
therapeutics available to treat this disorder despite intense research in the field. Recently,
however, the flavoenzyme kynurenine 3-monooxygenase (KMO) emerged as a promising
candidate therapeutic target for HD. KMO is an FAD-dependent outer mitochondrial
membrane protein which catalyses the conversion of L-kynurenine (L-KYN) to 3-
hydroxykynurenine (3-HK). It has been shown that inhibition of KMO activity is
protective in yeast, fruit fly, and mouse models of HD [1–5]. Additionally, it has been
also implicated in the pathophysiology of several other neurological conditions such as
Alzheimer’s and Parkinson’s Disease, AIDS-dementia complex, amyotrophic lateral
sclerosis, depression and schizophrenia [6, 7].Despite major interest in pharmacological targeting of KMO, only a few potent
inhibitors are currently available, and none are known to appreciably penetrate the bloodbrain
barrier in adult animals [3, 8]. Furthermore, the molecular basis of KMO inhibition
by available lead compounds has remained unknown and for that reason KMO crystal
structures in complex with tight binding inhibitors would be of undeniable interest for the
future design of new small molecule inhibitors that can penetrate the blood-brain barrier
and could ultimately have major therapeutic value.The aim of this thesis was to produce high levels of KMO protein for structural,
functional, and mechanistic studies, with the final goal of developing novel inhibitors that
possess the selectivity and affinity to open up new opportunities for therapeutic
intervention and inform the development of brain-penetrant KMO inhibitors.Several constructs, including both full length and truncated forms of human KMO
(HsKMO), were efficiently overexpressed and purified and kinetic analysis of pure
recombinant KMO showed a Km value for L-kynurenine of 22.62± 4 M which is very
similar to that observed for the rat liver mitochondria preparations (16 M) [9] and
human liver enzyme (13.0 ± 3.3 M) [10]. The tight-binding substrate-like inhibitor UPF
648 was found to bind recombinant KMO tightly (Ki 56.7 nM). The poor stability and low
expression yield of human KMO however prevented crystallisation. We thus turned our
attention to Saccharomyces cerevisiae KMO (ScKMO), which is highly related to human
KMO (38 % identity and 51 % similarity). The biochemical characterisation of ScKMO was carried out by using a combination of UV/Visible absorbance spectroscopy,
fluorescence spectroscopy, HPLC-based assays and stopped-flow analyses and revealed
that Sc enzyme was active as a flavin-dependent monooxygenases, generated
authentic 3-HK in HPLC-based assays and was inhibited by UPF 648 (Ki 74 nM) with
potency similar to that with HsKMO.The structure of ScKMO was determined using selenomethionine single anomalous
diffraction and subsequent crystal structures were solved to 1.85 Å resolution. We were
unable to obtain a complex with the kynurenine substrate but succeeded in cocrystallising
the enzyme with UPF 648, a tight-binding substrate-like inhibitor. UPF 648
binds close to the FAD cofactor and perturbs the local active-site structure, preventing
productive binding of the substrate kynurenine.Functional assays and targeted mutagenesis revealed that the active-site architecture
and UPF 648 binding are essentially identical in human KMO, validating the ScKMO–
UPF 648 structure as a template for structure-based drug design. This will inform the
search for new KMO inhibitors that are able to cross the blood–brain barrier in targeted
therapies against HD and other neurological diseases.A doença de Huntington é uma doença neurodegenerativa causada por uma
mutação no gene que codifica a proteína huntingtina. Apesar do grande desenvolvimento
cientifico no campo das neurociências não existe actualmente nenhum tratamento capaz
de tratar ou retardar o progresso desta doença. A enzima quinurenina mono oxigenase
(KMO) surgiu recentemente como importante alvo terapêutico para a doença de
Huntington. KMO é uma flavoproteína mitocondrial que catalisa a conversão do substrato
quinorinina em 3-hidroxiquinorinina. Vários estudos mostraram que a inibição da
actividade da KMO é neuroprotectora em modelos animais de Huntington, incluindo
modelos de levedura, de Drosophila e de rato [1–5]. Esta enzima está igualmente
relacionada com outras doenças neurológicas como por exemplo Alzheimer, Parkinson,
complexo AIDS demência, esclerose lateral amiotrófica, depressão e esquizofrenia [6, 7].
Apesar do grande interesse terapêutico da KMO, até à data foram desenvolvidos
poucos inibidores especificos para esta enzima, e nenhum deles comprovou ser eficiente
a atravessar a barreira hemoto-encefálica em modelos animais.O conhecimento da estrutura molecular da KMO em complexo com substratos ou
potenciais inibidores é desta forma fundamental para o desenvolvimento de novas
moléculas capazes de penetrar a barreira hemoto-encefálica. O objectivo principal da
presente tese é produzir quantidades significativas da proteína KMO para o
desenvolvimentos de estudos estruturais, funcionais e mecanisticos com o objectivo
futuro de desenvolver novos fármacos que possuam a selectividade e afinidade
necessárias para intervenções terapêuticas.A proteina humana KMO foi eficientemente expressa e purificada, e estudos
cinéticos da respectiva proteina revelaram um valor de Km para quinorinina (22.62± 4
M) bastante semelhante ao valor calculado para preparações mitocondriais de fígado de
rato (16 M) [9] e humano (13.0 ± 3.3 M) [10]. Estudos de inibição enzimática com o
inibidor forte da KMO (substrato análogo) UPF 648, revelaram também que este se liga
com uma afinidade nanomolar (Ki 56.7 nM). Os baixos niveis de expressão proteica em
conjunto com a instabilidade da proteina impossibilitaram o processo de crsitalização e
futuros estudos estruturais. Consequentemente, o projecto foi direccionado para a
proteína homologa KMO de Saccharomyces cerevisiae (ScKMO), que apresenta 38 % de
identidade e 51 % de similaridade com a proteina humana.A caracterização bioquimica da proteina Sc foi realizada utilizando uma
combinação de várias técnicas biofisicas, como espectroscopia de UV-visivel e de
fluorescência, HPLC e ensaios de stopped-flow, mostrando que o mecanismo enzimático
da ScKMO se assemelha a maioria das flavoproteinas, e mostrando que esta enzima é
inibida pela molécula UPF 648 de modo semelhante a homóloga humana (Ki 74 nM).
A estrutura da ScKMO foi inicialmente determinada usando o método da dispersão
anómala a um unico comprimento de onda e estruturas subsequentes foram determinadas
com uma resolução de 1.85 Å. Foi igualmente determinada a estrutura da ScKMO em
complexo com a molécula UPF 648. Este inibidor liga-se na proximidade do cofactor
FAD e perturba a estrutura do sitio activo, impossibilitando a ligação do substrato
quinorinina.Estudos de mutagenese sitio-dirigida em aminoácidos do sitio activo e respectivos
ensaios enzimáticos revelaram que a arquitectura do sitio activo é identico à proteina
humana, validando a estrutura ScKMO–UPF 648 como base para o futuro
desenvolvimento de moléculas baseadas na estrutura da proteina, e consequentemente a
pesquisa de novos farmacos capazes de atravessar a barreira hemato-encefalica e com
potencial terapêutico para a doença de Huntington e outras doenças neurológicas.Fundação para a Ciência e a Tecnologia (FCT
Structure and mechanism of kynurenine 3-monooxygenase, a candidate huntington’s disease drug target
No full textTese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2013Huntington’s disease (HD) is a neurodegenerative disorder caused by a
polyglutamine expansion in the huntingtin protein. There are currently no effective
therapeutics available to treat this disorder despite intense research in the field. Recently,
however, the flavoenzyme kynurenine 3-monooxygenase (KMO) emerged as a promising
candidate therapeutic target for HD. KMO is an FAD-dependent outer mitochondrial
membrane protein which catalyses the conversion of L-kynurenine (L-KYN) to 3-
hydroxykynurenine (3-HK). It has been shown that inhibition of KMO activity is
protective in yeast, fruit fly, and mouse models of HD [1–5]. Additionally, it has been
also implicated in the pathophysiology of several other neurological conditions such as
Alzheimer’s and Parkinson’s Disease, AIDS-dementia complex, amyotrophic lateral
sclerosis, depression and schizophrenia [6, 7].Despite major interest in pharmacological targeting of KMO, only a few potent
inhibitors are currently available, and none are known to appreciably penetrate the bloodbrain
barrier in adult animals [3, 8]. Furthermore, the molecular basis of KMO inhibition
by available lead compounds has remained unknown and for that reason KMO crystal
structures in complex with tight binding inhibitors would be of undeniable interest for the
future design of new small molecule inhibitors that can penetrate the blood-brain barrier
and could ultimately have major therapeutic value.The aim of this thesis was to produce high levels of KMO protein for structural,
functional, and mechanistic studies, with the final goal of developing novel inhibitors that
possess the selectivity and affinity to open up new opportunities for therapeutic
intervention and inform the development of brain-penetrant KMO inhibitors.Several constructs, including both full length and truncated forms of human KMO
(HsKMO), were efficiently overexpressed and purified and kinetic analysis of pure
recombinant KMO showed a Km value for L-kynurenine of 22.62± 4 M which is very
similar to that observed for the rat liver mitochondria preparations (16 M) [9] and
human liver enzyme (13.0 ± 3.3 M) [10]. The tight-binding substrate-like inhibitor UPF
648 was found to bind recombinant KMO tightly (Ki 56.7 nM). The poor stability and low
expression yield of human KMO however prevented crystallisation. We thus turned our
attention to Saccharomyces cerevisiae KMO (ScKMO), which is highly related to human
KMO (38 % identity and 51 % similarity). The biochemical characterisation of ScKMO was carried out by using a combination of UV/Visible absorbance spectroscopy,
fluorescence spectroscopy, HPLC-based assays and stopped-flow analyses and revealed
that Sc enzyme was active as a flavin-dependent monooxygenases, generated
authentic 3-HK in HPLC-based assays and was inhibited by UPF 648 (Ki 74 nM) with
potency similar to that with HsKMO.The structure of ScKMO was determined using selenomethionine single anomalous
diffraction and subsequent crystal structures were solved to 1.85 Å resolution. We were
unable to obtain a complex with the kynurenine substrate but succeeded in cocrystallising
the enzyme with UPF 648, a tight-binding substrate-like inhibitor. UPF 648
binds close to the FAD cofactor and perturbs the local active-site structure, preventing
productive binding of the substrate kynurenine.Functional assays and targeted mutagenesis revealed that the active-site architecture
and UPF 648 binding are essentially identical in human KMO, validating the ScKMO–
UPF 648 structure as a template for structure-based drug design. This will inform the
search for new KMO inhibitors that are able to cross the blood–brain barrier in targeted
therapies against HD and other neurological diseases.A doença de Huntington é uma doença neurodegenerativa causada por uma
mutação no gene que codifica a proteína huntingtina. Apesar do grande desenvolvimento
cientifico no campo das neurociências não existe actualmente nenhum tratamento capaz
de tratar ou retardar o progresso desta doença. A enzima quinurenina mono oxigenase
(KMO) surgiu recentemente como importante alvo terapêutico para a doença de
Huntington. KMO é uma flavoproteína mitocondrial que catalisa a conversão do substrato
quinorinina em 3-hidroxiquinorinina. Vários estudos mostraram que a inibição da
actividade da KMO é neuroprotectora em modelos animais de Huntington, incluindo
modelos de levedura, de Drosophila e de rato [1–5]. Esta enzima está igualmente
relacionada com outras doenças neurológicas como por exemplo Alzheimer, Parkinson,
complexo AIDS demência, esclerose lateral amiotrófica, depressão e esquizofrenia [6, 7].
Apesar do grande interesse terapêutico da KMO, até à data foram desenvolvidos
poucos inibidores especificos para esta enzima, e nenhum deles comprovou ser eficiente
a atravessar a barreira hemoto-encefálica em modelos animais.O conhecimento da estrutura molecular da KMO em complexo com substratos ou
potenciais inibidores é desta forma fundamental para o desenvolvimento de novas
moléculas capazes de penetrar a barreira hemoto-encefálica. O objectivo principal da
presente tese é produzir quantidades significativas da proteína KMO para o
desenvolvimentos de estudos estruturais, funcionais e mecanisticos com o objectivo
futuro de desenvolver novos fármacos que possuam a selectividade e afinidade
necessárias para intervenções terapêuticas.A proteina humana KMO foi eficientemente expressa e purificada, e estudos
cinéticos da respectiva proteina revelaram um valor de Km para quinorinina (22.62± 4
M) bastante semelhante ao valor calculado para preparações mitocondriais de fígado de
rato (16 M) [9] e humano (13.0 ± 3.3 M) [10]. Estudos de inibição enzimática com o
inibidor forte da KMO (substrato análogo) UPF 648, revelaram também que este se liga
com uma afinidade nanomolar (Ki 56.7 nM). Os baixos niveis de expressão proteica em
conjunto com a instabilidade da proteina impossibilitaram o processo de crsitalização e
futuros estudos estruturais. Consequentemente, o projecto foi direccionado para a
proteína homologa KMO de Saccharomyces cerevisiae (ScKMO), que apresenta 38 % de
identidade e 51 % de similaridade com a proteina humana.A caracterização bioquimica da proteina Sc foi realizada utilizando uma
combinação de várias técnicas biofisicas, como espectroscopia de UV-visivel e de
fluorescência, HPLC e ensaios de stopped-flow, mostrando que o mecanismo enzimático
da ScKMO se assemelha a maioria das flavoproteinas, e mostrando que esta enzima é
inibida pela molécula UPF 648 de modo semelhante a homóloga humana (Ki 74 nM).
A estrutura da ScKMO foi inicialmente determinada usando o método da dispersão
anómala a um unico comprimento de onda e estruturas subsequentes foram determinadas
com uma resolução de 1.85 Å. Foi igualmente determinada a estrutura da ScKMO em
complexo com a molécula UPF 648. Este inibidor liga-se na proximidade do cofactor
FAD e perturba a estrutura do sitio activo, impossibilitando a ligação do substrato
quinorinina.Estudos de mutagenese sitio-dirigida em aminoácidos do sitio activo e respectivos
ensaios enzimáticos revelaram que a arquitectura do sitio activo é identico à proteina
humana, validando a estrutura ScKMO–UPF 648 como base para o futuro
desenvolvimento de moléculas baseadas na estrutura da proteina, e consequentemente a
pesquisa de novos farmacos capazes de atravessar a barreira hemato-encefalica e com
potencial terapêutico para a doença de Huntington e outras doenças neurológicas.Fundação para a Ciência e a Tecnologia (FCT
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