Enhancing the Thermal Stability and Enzyme Activity of Ketopantoate Hydroxymethyltransferase through Interface Modification Engineering.
J Agric Food Chem
; 72(23): 13186-13195, 2024 Jun 12.
Article
em En
| MEDLINE
| ID: mdl-38814711
ABSTRACT
Ketopantoate hydroxymethyltransferase (KPHMT) plays a pivotal role in d-pantothenic acid biosynthesis. Most KPHMTs are homodecamers with low thermal stability, posing challenges for protein engineering and limiting output enhancement. Previously, a high-enzyme activity KPHMT mutant (K25A/E189S) from Corynebacterium glutamicum was screened as mother strain (M0). Building upon this strain, our study focused on interface engineering modifications, employing a multifaceted approach including integrating folding-free energy calculation, B-factor analysis, and conserved site analysis. Preliminary screening led to the selection of five mutants in the interfaceâE106S, E98T, E98N, S247I, and S247Dâshowing improved thermal stability, culminating in the double-site mutant M8 (M0-E98N/S247D). M8 exhibited a T1/2 value of 288.79 min at 50 °C, showing a 3.29-fold increase compared to M0. Meanwhile, the Tm value of M8 was elevated from 53.2 to 59.6 °C. Investigations of structural and molecular dynamics simulations revealed alterations in surface electrostatic charge distribution and the formation of increased hydrogen bonds between subunits, contributing to enhanced thermal stability. This investigation corroborates the efficacy of interface engineering modifications in bolstering KPHMT stability while showing its potential for positively impacting industrial d-pantothenic acid synthesis.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
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Estabilidade Enzimática
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Engenharia de Proteínas
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Corynebacterium glutamicum
Idioma:
En
Revista:
J Agric Food Chem
Ano de publicação:
2024
Tipo de documento:
Article
País de publicação:
EEUU
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ESTADOS UNIDOS
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ESTADOS UNIDOS DA AMERICA
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EUA
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UNITED STATES
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UNITED STATES OF AMERICA
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US
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USA