RESUMO
The recombinant dihydropyrimidinase from Sinorhizobium meliloti CECT4114 (SmelDhp) has been characterised and its crystal structure elucidated at 1.85A. The global architecture of the protein is reminiscent of that of the amidohydrolase superfamily, consisting of two domains; an (alpha/beta)(8) TIM-like barrel domain, where the catalytic centre is located, and a smaller beta-sheet sandwich domain of unknown function. The c-terminal tails of each subunit extend toward another monomer in a swapping-like manner, creating a hydrogen bond network which suggests its implication in protein oligomerisation. Mutational and structural evidence suggest the involvement of a conserved tyrosine in the reaction mechanism of the enzyme. SmelDhp presents both hydantoinase and dihydropyrimidinase activities, with higher affinity for the natural six-membered ring substrates. For the five-membered ring substrates, affinity was greater for those with aliphatic and apolar groups in the 5th carbon atom, with the highest rates of hydrolysis for d-5-methyl and d-5-ethyl hydantoin (k(cat)/K(m)=2736+/-380 and 944+/-52M(-1)s(-1), respectively). The optimal conditions for the enzyme activity were found to be 60 degrees C of temperature at pH 8.0. SmelDhp retains 95% of its activity after 6-hour preincubation at 60 degrees C. This is the first dihydropyrimidinase used for the hydrolytic opening of non-natural 6-monosubstituted dihydrouracils, which may be exploited for the production of beta-amino acids.
Assuntos
Amidoidrolases/química , Modelos Moleculares , Conformação Proteica , Sinorhizobium meliloti/enzimologia , Amidoidrolases/genética , Sequência de Aminoácidos , Sequência de Bases , Evolução Molecular , Dados de Sequência Molecular , Ligação Proteica , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade por SubstratoRESUMO
N-Carbamoyl-L-amino-acid amidohydrolases (L-N-carbamoylases; EC 3.5.1.87) hydrolyze the carbon-nitrogen bond of the ureido group in N-carbamoyl-L-alpha-amino acids. These enzymes are commonly used in the production of optically pure natural and non-natural L-amino acids using the ;hydantoinase process'. Recombinant L-N-carbamoylase from Geobacillus stearothermophilus CECT43 has been expressed, purified and crystallized by hanging-drop vapour diffusion. X-ray data were collected to a resolution of 2.75 A. The crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 103.2, b = 211.7, c = 43.1 A and two subunits in the asymmetric unit.
Assuntos
Amidoidrolases/química , Proteínas de Bactérias/química , Geobacillus stearothermophilus/enzimologia , Amidoidrolases/genética , Amidoidrolases/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Geobacillus stearothermophilus/metabolismo , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
A recombinant active-site mutant of hydantoin racemase (C76A) from Sinorhizobium meliloti CECT 4114 (SmeHyuA) has been crystallized in the presence and absence of the substrate D,L-5-isopropyl hydantoin. Crystals of the SmeHyuA mutant suitable for data collection and structure determination were grown using the counter-diffusion method. X-ray data were collected to resolutions of 2.17 and 1.85 A for the free and bound enzymes, respectively. Both crystals belong to space group R3 and contain two molecules of SmeHyuA per asymmetric unit. The crystals of the free and complexed SmeHyuA have unit-cell parameters a = b = 85.43, c = 152.37 A and a = b = 85.69, c = 154.38 A, crystal volumes per protein weight (V(M)) of 1.94 and 1.98 A3 Da(-1) and solvent contents of 36.7 and 37.9%, respectively.
Assuntos
Racemases e Epimerases/química , Sinorhizobium meliloti/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação , Indicadores e Reagentes , Racemases e Epimerases/genética , Racemases e Epimerases/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoAssuntos
Cristalização/métodos , Proteínas/química , Ação Capilar , Difusão , Géis , Proteínas/isolamento & purificaçãoRESUMO
Granada Crystallisation Box (GCB) is a new crystallisation device designed to perform counter-diffusion experiments. Here we describe the device and its use for protein crystallisation purposes. GCB allows one to explore and exploit the coupling between crystallisation and diffusion. The role of viscous fluids, gels and/or microgravity can be enhanced by using capillary volumes, creating a perfect diffusive mass transport scenario. The use of capillaries also reduces the consumption of macromolecules and avoids the handling of crystals for X-ray diffraction data collection.