RESUMO
Lately it has been proposed that interaction between two positively charged side chains can stabilize the folded state of proteins. To further explore this point, we studied the effect of histidine-histidine interactions on thermostability of methylglyoxal synthase from Thermus sp. GH5 (TMGS). The crystal structure of TMGS revealed that His23, Arg22, and Phe19 are in close distance and form a surface loop. Here, two modified enzymes were produced by site-directed mutagenesis (SDM); one of them, one histidine (TMGS-HH(O)), and another two histidines (TMGS-HHH(O)) were inserted between Arg22 and His23 (H(O)). In comparison with the wild type, TMGS-HH(O) thermostability increased remarkably, whereas TMGS-HHH(O) was very unstable. To explore the role of His23 in the observed phenomenon, the original His23 in TMGS-HHH(O) was replaced with Ala (TMGS-HHA). Our data showed that the half-life of TMGS-HHA decreased in relation to the wild type. However, its half-life increased in comparison with TMGS-HHH(O). These results demonstrated that histidine-histidine interactions at position 23 in TMGS-HH(O) probably have the main role in TMGS thermostability.
Assuntos
Carbono-Oxigênio Liases/química , Carbono-Oxigênio Liases/genética , Dipeptídeos , Mutagênese Sítio-Dirigida , Sequência de Aminoácidos , Carbono-Oxigênio Liases/metabolismo , Ativação Enzimática , Estabilidade Enzimática , Meia-Vida , Histidina , Cinética , Modelos Moleculares , Mutação , Conformação Proteica , Temperatura , Thermus/enzimologiaRESUMO
Asparagine deamidation is one of the important determinants of protein thermostability. Here, structure based mutagenesis has been done in order to probe the role of Asn residues in thermostability of a Ca independent Bacillus sp. KR-8104 α-amylase (BKA). Residues involved in potential deamidation processes have been selected and replaced using a site directed mutagenesis. Fourteen different variants were tested for thermostability by measuring residual activities after incubation at high temperature. In comparison to the wild-type enzyme, four mutated variants are able to increase the half life of the protein at high temperatures. The highest stabilization resulted from the substitution of asparatate in place of asparagine at position 112, leading to a nearly fivefold increase of the enzyme's half-life at 70°C. Also replacement of Asn129 to aspartic acid and Asn312 to serine markedly increased the half-life of the enzyme at 70°C indicating that the deamination of these residues may have a deleterious effect on BKA.
