RESUMEN
Maltooligosyltrehalose synthase (MTSase) is a key enzyme in the synthesis of trehalose. Computer simulations using AutoDock and NAMD were employed to assess the substrate-binding and selectivity-related residues of MTSase. We introduced mutations at residues D411, D610, and R614 to determine the substrate-binding residues of Sulfolobus solfataricus ATCC 35092 MTSase, and introduced mutations at residues P402, A406, and V426 to investigate the enzyme's selectivity-related residues. Kinetic studies of D411A, D610A, and R614A MTSases reveal significant reductions in catalytic efficiency and cause increase in the transition-state energy of mutant MTSases, indicating that residues D411, D610, and R614 form hydrogen bonds to the substrate. Compared with wild-type MTSase, the hydrolysis: transglycosylation selectivity ratio was significantly decreased for P402Q and significantly increased for A406S MTSases, while the ratio for V426T MTSase showed little change. The results suggest that P402 and A406 residues are selectivity-related.
Asunto(s)
Proteínas Arqueales/metabolismo , Glucosiltransferasas/metabolismo , Sulfolobus solfataricus/enzimología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/aislamiento & purificación , Catálisis , Dominio Catalítico , Glucosiltransferasas/química , Glucosiltransferasas/genética , Glucosiltransferasas/aislamiento & purificación , Glicosilación , Enlace de Hidrógeno , Hidrólisis , Cinética , Modelos Moleculares , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Especificidad por Sustrato , Sulfolobus solfataricus/genéticaRESUMEN
In this study, we report a novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method modified from the QuikChange site-directed mutagenesis (QCM). One mutagenic oligonucleotide and one universal flanking primer were used to produce the complementary megaprimers that were then used to amplify the whole plasmid template. This method yields a mutagenesis efficiency ( approximately 90%) similar to that of QCM but uses only one mutagenic oligonucleotide instead of two of them, and the length of the oligonucleotide could be shorter. This method can be further extended to double mutations that are located at distant sites by using two mutagenic oligonucleotides and even to site saturation mutagenesis by introducing randomized codons.
