Designs for a broad substrate specificity keto acid dehydrogenase

Area of Science:

• Biochemistry
• Enzyme Engineering
• Structural Biology

Background:

• Nicotinamide adenine dinucleotide (NAD)-dependent L-lactate dehydrogenase (LDH) from Bacillus stearothermophilus exhibits specific substrate preferences.
• The enzyme's active site regions are crucial for determining specificity towards alpha-hydroxy acids.

Purpose of the Study:

• To engineer Bacillus stearothermophilus LDH to accommodate larger hydrophobic substrate side chains.
• To broaden the substrate specificity of LDH beyond its natural preference for pyruvate.

Main Methods:

• Rational protein engineering was employed to introduce specific mutations in two regions of the LDH enzyme.
• Mutations 102-105GlnLysPro to MetValSer and 236-237AlaAla to GlyGly were introduced.
• The catalytic efficiency (kcat) of wild-type and mutant enzymes was assessed using various alpha-hydroxy acid substrates.

Main Results:

• Single and double mutants showed limited improvement in accommodating branched-chain-substituted pyruvates.
• A combined five-residue change resulted in a significant broadening of substrate specificity.
• The engineered enzyme exhibited a 55-fold increased kcat for alpha-ketoisocaproate compared to the wild-type enzyme's activity with pyruvate.

Conclusions:

• Rational protein engineering can effectively alter enzyme substrate specificity.
• The modified LDH demonstrates enhanced tolerance for larger hydrophobic side chains, creating a versatile alpha-hydroxy acid dehydrogenase.
• This approach offers a higher probability of success for targeted enzyme modifications compared to random mutagenesis.