Behind substrate specificity switch in lactate dehydrogenase: Active-site loop dynamics and epistatic enhancement

Area of Science:

• Enzyme engineering
• Protein biochemistry
• Metabolic flux control

Background:

• Substrate specificity in lactate dehydrogenases (LDHs) is crucial for controlling metabolic flux but challenging to engineer, especially for substrates differing by a single carboxylate group.
• The role of glutamine (Q102) as a substrate-specificity determinant is known, but its interplay with steric effects, dynamics, and scaffold context in substrate recognition is unclear.

Purpose of the Study:

• To investigate specificity switching from pyruvate to oxaloacetate in Geobacillus stearothermophilus L-LDH (GsLDH) using combined experimental and computational approaches.
• To elucidate the cooperative mechanisms of electrostatic and steric factors in governing substrate recognition and catalytic efficiency.

Main Methods:

• Steady-state kinetics were employed to measure enzyme activity and efficiency.
• Molecular dynamics simulations were used to analyze substrate binding, conformational dynamics, and productive geometries.
• Principal component analysis was utilized to study the redistribution of conformational ensembles.

Main Results:

• The Q86R substitution significantly enhanced oxaloacetate efficiency via electrostatic stabilization and substrate retention, while reducing pyruvate activity.
• The I227V substitution primarily improved steric accommodation, broadening substrate acceptance for both pyruvate and oxaloacetate.
• The combined Q86R/I227V variant showed positive epistasis, achieving the highest oxaloacetate efficiency, and mutations were found to shift conformational ensembles towards productive states.

Conclusions:

• Specificity switching in LDH is driven by a combination of electrostatic stabilization and steric optimization, operating through an ensemble-selection mechanism.
• Catalysis requires the coordinated interplay of loop conformation, substrate retention, and precise geometric arrangements for hydride/proton transfer.
• Specificity switching is asymmetric and scaffold-dependent, as demonstrated by reciprocal mutations in malate dehydrogenase (MDH).