Improving the activity and thermostability of GH2 ß-glucuronidases via domain reassembly.
Biotechnol Bioeng
; 118(5): 1962-1972, 2021 05.
Article
em En
| MEDLINE
| ID: mdl-33559890
ABSTRACT
Glycoside hydrolase family 2 (GH2) enzymes are generally composed of three domains TIM-barrel domain (TIM), immunoglobulin-like ß-sandwich domain (ISD), and sugar-binding domain (SBD). The combination of these three domains yields multiple structural combinations with different properties. Theoretically, the drawbacks of a given GH2 fold may be circumvented by efficiently reassembling the three domains. However, very few successful cases have been reported. In this study, we used six GH2 ß-glucuronidases (GUSs) from bacteria, fungi, or humans as model enzymes and constructed a series of mutants by reassembling the domains from different GUSs. The mutants PGUS-At, GUS-PAA, and GUS-PAP, with reassembled domains from fungal GUSs, showed improved expression levels, activity, and thermostability, respectively. Specifically, compared to the parental enzyme, the mutant PGUS-At displayed 3.8 times higher expression, the mutant GUS-PAA displayed 1.0 time higher catalytic efficiency (kcat /Km ), and the mutant GUS-PAP displayed 7.5 times higher thermostability at 65°C. Furthermore, two-hybrid mutants, GUS-AEA and GUS-PEP, were constructed with the ISD from a bacterial GUS and SBD and TIM domain from fungal GUSs. GUS-AEA and GUS-PEP showed 30.4% and 23.0% higher thermostability than GUS-PAP, respectively. Finally, molecular dynamics simulations were conducted to uncover the molecular reasons for the increased thermostability of the mutant.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Engenharia de Proteínas
/
Domínios Proteicos
/
Glucuronidase
Limite:
Humans
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article