Acceleration of an aldo-keto reductase by minimal loop engineering

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering

Background:

• Aldo-keto reductases (AKRs) are crucial enzymes in metabolic pathways.
• Coenzyme binding, specifically nicotinamide adenine dinucleotide (phosphate) (NAD(P)(H)), is essential for AKR activity.
• A conserved hydrogen bond interaction involving Lysine 24 (Lys24) is proposed to stabilize coenzyme binding in AKRs.

Purpose of the Study:

• To investigate the role of the Lys24 hydrogen bond in coenzyme binding and catalytic activity of AKRs.
• To determine the impact of disrupting this hydrogen bond on the kinetic parameters of xylose reductase from Candida tenuis.

Main Methods:

• Site-directed mutagenesis was used to alter the Lys24 residue in Candida tenuis xylose reductase.
• Enzyme kinetics assays were performed to measure substrate and coenzyme affinities (K(m)) and turnover numbers (k(cat)).
• Kinetic parameters were analyzed for wild-type and mutant enzymes using various ketone substrates.

Main Results:

• Mutation of Lys24 disrupted the hydrogen bond anchoring the nicotinamide adenine dinucleotide (NADH) pyrophosphate group.
• The Lys24 mutation resulted in loosened NAD(H) binding.
• Increased turnover numbers (k(cat)) were observed for the reduction of bulky-bulky ketones, while substrate and coenzyme affinities remained constant.

Conclusions:

• The Lys24 residue and its associated hydrogen bond are critical for optimal coenzyme binding and catalytic efficiency in aldo-keto reductases.
• Disruption of this "safety belt" mechanism enhances the enzyme's ability to process sterically hindered substrates.
• This finding provides insights into enzyme engineering strategies for modifying substrate specificity and catalytic activity.