RESUMO
Drawbacks of industrially-used fructosyltransferases (FTs) such as low optimum temperature and low fructooligosaccharides (FOS) yield necessitates the search for engineered FTs that are highly thermostable and active. With the availability of the first plant FT crystal structure from Pachysandra terminalis (PDB ID: 3UGH), computer-aided protein engineering of plant FT is now feasible. To obtain insights on the effect of specific mutations i.e. disulfide bridge introduction, wild-type and mutant FTs were subjected to a 15 µs Martini Coarse-grained Molecular Dynamics (CGMD) simulations at 303â¯K and 334â¯K. We report here the five mutants, M31C-Q49C, L144C-S193C, P34C-W300C, S219C-L226C and V470C-S498C with enhanced thermostabilities and/or activities relative to the wild type. Interestingly, M31C-Q49C, which is located within the catalytic-carrying blade of the catalytic domain, has an activity enhancement at both temperatures. At 334â¯K, three mutations, L144C-S193C, P34C-W300C and V470C-S498C, achieved thermostability relative to the wild type. Intriguingly, both activity and stability enhancement exhibited only at 334â¯K can be achieved provided that the mutation is located either on the catalytic-carrying residue blade of the catalytic domain or on the non-catalytic domain. Our results suggest that V470C-S498C and L144C-S193C are promising mutants and that domain-specific approach may be exploited to customize enzyme properties.
Assuntos
Dissulfetos/química , Hexosiltransferases/química , Modelos Moleculares , Pachysandra/enzimologia , Termodinâmica , Sítios de Ligação , Estabilidade Enzimática , Hexosiltransferases/genética , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , TemperaturaRESUMO
Half-lives of α-tocopherol in plasma have been reported as 2-3 d, whereas the Elgin Study required >2 y to deplete α-tocopherol, so gaps exist in our quantitative understanding of human α-tocopherol metabolism. Therefore, 6 men and 6 women aged 27 ± 6 y (mean ± SD) ingested 1.81 nmol, 3.70 kBq of [5-(14)CH(3)]-(2R, 4'R, 8'R)-α-tocopherol. The levels of (14)C in blood plasma and washed RBC were monitored frequently from 0 to 460 d while the levels of (14)C in urine and feces were monitored from 0 to 21 d. Total fecal elimination (fecal + metabolic fecal) was 23.24 ± 5.81% of the (14)C dose, so feces over urine was the major route of elimination of the ingested [5-(14)CH(3)]-(2R, 4'R, 8'R)-α-tocopherol, consistent with prior estimates. The half-life of α-tocopherol varied in plasma and RBC according to the duration of study. The minute dose coupled with frequent monitoring over 460 d and 21 d for blood, urine, and feces ensured the [5-(14)CH(3)]-(2R, 4'R, 8'R)-α-tocopherol (the tracer) had the chance to fully mix with the endogenous [5-(14)CH(3)]-(2R, 4'R, 8'R)-α-tocopherol (the tracee). The (14)C levels in neither plasma nor RBC had returned to baseline by d 460, indicating that the t(1/2) of [5-CH(3)]-(2R, 4'R, 8'R)-α-tocopherol in human blood was longer than prior estimates.