RESUMO
Avidins represent an interesting group of proteins showing high structural similarity and ligand-binding properties but low similarity in primary structure. In this study, we show that it is possible to create functional chimeric proteins from the avidin protein family when applying DNA family shuffling to the genes of the avidin protein family: avidin, avidin related gene 2 and biotin-binding protein A. The novel chimeric proteins were selected by phage display biopanning against biotin, and the selected enriched proteins were characterized, displaying diverse features distinct from the parental genes, including binding to cysteine.
Assuntos
Avidina/genética , Embaralhamento de DNA/métodos , Proteínas Recombinantes de Fusão/genética , Sequência de Aminoácidos , Animais , Avidina/química , Avidina/metabolismo , Biotina/química , Biotina/genética , Biotina/metabolismo , Varredura Diferencial de Calorimetria , Galinhas , Cisteína , Evolução Molecular Direcionada , Dados de Sequência Molecular , Mutação , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de SequênciaRESUMO
BACKGROUND: The beta-carbonic anhydrase (CA, EC 4.2.1.1) enzymes have been reported in a variety of organisms, but their existence in animals has been unclear. The purpose of the present study was to perform extensive sequence analysis to show that the beta-CAs are present in invertebrates and to clone and characterize a member of this enzyme family from a representative model organism of the animal kingdom, e.g., Drosophila melanogaster. RESULTS: The novel beta-CA gene, here named DmBCA, was identified from FlyBase, and its orthologs were searched and reconstructed from sequence databases, confirming the presence of beta-CA sequences in 55 metazoan species. The corresponding recombinant enzyme was produced in Sf9 insect cells, purified, kinetically characterized, and its inhibition was investigated with a series of simple, inorganic anions. Holoenzyme molecular mass was defined by dynamic light scattering analysis and gel filtration, and the results suggested that the holoenzyme is a dimer. Double immunostaining confirmed predictions based on sequence analysis and localized DmBCA protein to mitochondria. The enzyme showed high CO2 hydratase activity, with a kcat of 9.5 x 105 s-1 and a kcat/KM of 1.1 x 108 M-1s-1. DmBCA was appreciably inhibited by the clinically-used sulfonamide acetazolamide, with an inhibition constant of 49 nM. It was moderately inhibited by halides, pseudohalides, hydrogen sulfide, bisulfite and sulfate (KI values of 0.67 - 1.36 mM) and more potently by sulfamide (KI of 0.15 mM). Bicarbonate, nitrate, nitrite and phenylarsonic/boronic acids were much weaker inhibitors (KIs of 26.9 - 43.7 mM). CONCLUSIONS: The Drosophila beta-CA represents a highly active mitochondrial enzyme that is a potential model enzyme for anti-parasitic drug development.
Assuntos
Anidrases Carbônicas/química , Anidrases Carbônicas/classificação , Proteínas de Drosophila/química , Proteínas de Drosophila/classificação , Drosophila melanogaster/enzimologia , Sequência de Aminoácidos , Animais , Anidrases Carbônicas/genética , Bases de Dados de Proteínas , Dimerização , Proteínas de Drosophila/genética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Cinésica , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
The hexameric membrane-spanning ATP-dependent metalloprotease FtsH is universally conserved in eubacteria, mitochondria, and chloroplasts, where it fulfills key functions in quality control and signaling. As a member of the self-compartmentalizing ATPases associated with various cellular activities (AAA+ proteases), FtsH converts the chemical energy stored in ATP via conformational rearrangements into a mechanical force that is used for substrate unfolding and translocation into the proteolytic chamber. The crystal structure of the ADP state of Thermotoga maritima FtsH showed a hexameric assembly consisting of a 6-fold symmetric protease disk and a 2-fold symmetric AAA ring. The 2.6 A resolution structure of the cytosolic region of apo-FtsH presented here reveals a new arrangement where the ATPase ring shows perfect 6-fold symmetry with the crucial pore residues lining an open circular entrance. Triggered by this conformational change, a substrate-binding edge beta strand appears within the proteolytic domain. Comparison of the apo- and ADP-bound structure visualizes an inward movement of the aromatic pore residues and generates a model of substrate translocation by AAA+ proteases. Furthermore, we demonstrate that mutation of a conserved glycine in the linker region inactivates FtsH.
