Engineering of the Conformational Dynamics of an Enzyme for Relieving the Product Inhibition

Abstract

Enzymes with a product inhibition are generally considered a limiting step of the metabolic pathway in producing valuable compounds. Most attempts to relieve the product inhibition have relied on mutations at the productbinding site of enzymes. However, such an approach has resulted in a severe decrease in the catalytic activity, mainly owing to the shared binding site of product with substrate. Herein, we present the modulation of the conformational dynamics of chorismate-pyruvate lyase (CPL) for relieving the product inhibition without a decrease in the catalytic efficiency. CPL is a key enzyme in the biosynthesis of diverse aromatics but incurs a severe product inhibition owing to a strong product binding. On the basis of a structural analysis and molecular dynamics simulations, two key residues were identified for increasing the conformational dynamics of the flaps and thereby facilitating the product release. The designed mutants exhibited almost an 8-fold reduction in product inhibition and a 3-fold higher catalytic rate in comparison to the wild type. We demonstrate that mutation at two key residues leads to a significant increase in the conformational dynamics of the flaps, enhancing the product release through an opening of the flaps and thereby relieving the product inhibition.

Biocatalytic Potential of Enzymes Involved in the Biosynthesis of Isoprenoid Quinones

“…[45] Recently Han et al performed as tructurala nalysisa nd molecular dynamics simulations on E. coli UbiC protein and identified key mutations that were predicted to increase conformational dynamics of flaps to enhance product release. [46] Using this approach, they attempted to solve the problem of product inhibition encountered with the wild-type UbiC enzyme. The rationally designed mutants displayed an up to 8-fold decrease in product inhibition and an up to threefold increaseincatalytic rate, if compared to the wild-type enzyme.…”

“…The rationally designed mutants displayed an up to 8-fold decrease in product inhibition and an up to threefold increaseincatalytic rate, if compared to the wild-type enzyme. [46] Pohl and co-workers have engineered (S)-selective variants of MenD. They identified two residues (I474 and F475) that were hypothesised to be crucial for the stereoselectivity of E. coli MenD enzyme, using structural analysis and docking studies.…”

“…Recently Han et al. performed a structural analysis and molecular dynamics simulations on E. coli UbiC protein and identified key mutations that were predicted to increase conformational dynamics of flaps to enhance product release . Using this approach, they attempted to solve the problem of product inhibition encountered with the wild‐type UbiC enzyme.…”

“…Using this approach, they attempted to solve the problem of product inhibition encountered with the wild‐type UbiC enzyme. The rationally designed mutants displayed an up to 8‐fold decrease in product inhibition and an up to threefold increase in catalytic rate, if compared to the wild‐type enzyme …”

Conformational Dynamics‐Guided Loop Engineering of an Alcohol Dehydrogenase: Capture, Turnover and Enantioselective Transformation of Difficult‐to‐Reduce Ketones

“…In recent years, there has been growing interest in the conformational dynamics of proteins, which play an important role in enzyme catalysis, and engineering of enzymes guided by conformational dynamics has become an effective strategy for protein evolution, achieving successes in expanding substrate scope, increasing enantioselectivity, relieving product inhibition, and improving thermostability . The motion of amino acid residues in loops involved in the pocket has been recognized to influence the catalytic properties of enzymes .…”