Modified substrate specificity of L-hydroxyisocaproate dehydrogenase derived from structure-based protein engineering

Area of Science:

• Biochemistry
• Protein Engineering
• Enzyme Kinetics

Background:

• L-2-Hydroxyisocaproate dehydrogenase (L-HicDH) exhibits broad substrate specificity for branched 2-oxo acids.
• Understanding L-HicDH substrate specificity is crucial for designing enzymes with tailored keto acid preferences.

Purpose of the Study:

• To define and alter the substrate specificity region of NAD(H)-dependent L-HicDH from Lactobacillus confusus.
• To engineer novel dehydrogenases with improved specificity towards C3 or C4 branched keto acids.

Main Methods:

• Homology modeling of the L-HicDH active site using L-lactate dehydrogenase (L-LDH) X-ray coordinates.
• Site-directed mutagenesis of key active site residues (Gly234, Gly235, Phe236, Leu239, Thr245).
• Kinetic characterization of wild-type and mutant enzymes with various 2-oxocarbonic acids.

Main Results:

• Mutant enzymes, particularly T245A, showed significantly altered substrate specificities, with shifts exceeding 17,000-fold for keto-tert-leucine.
• Amino acid substitutions at Leu239, Phe236, and Thr245 resulted in enzymes with distinct substrate profiles.
• Experimental data validated the homology model of the L-HicDH active site.

Conclusions:

• Specific active site residues (Leu239, Phe236, Thr245) are critical for determining L-HicDH substrate recognition and specificity.
• Protein engineering of L-HicDH can successfully create variants with novel substrate specificities for branched keto acids.
• This study offers valuable insights into enzyme-substrate interactions and the structural differences between L-LDHs and L-HicDHs.