Multivariate sequence analysis reveals additional function impacting residues in the SDR superfamily.

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• The short chain dehydrogenases/reductases (SDR) superfamily exhibits conserved tertiary structures despite diverse functions and sequences.
• Understanding the structural basis for functional divergence within SDRs is crucial for enzyme engineering and drug discovery.

Purpose of the Study:

• To identify residue positions critical for functional specificity within the SDR fold.
• To elucidate the mechanism by which specific residues influence enzyme activity and cofactor interactions.

Main Methods:

• Principal Component Analysis (PCA) applied to structurally aligned SDR families.
• Utilized information-theoretic measures: Jensen-Shannon divergence and average Shannon entropy.
• Site-directed mutagenesis of key residues in UDP-Galactose 4-epimerase.
• Molecular dynamics simulations to investigate reaction mechanisms.

Main Results:

• PCA classified SDR residue positions into six groups based on structural and informational properties.
• A specific group of residues, characterized by low Shannon entropy and high Jensen-Shannon divergence, was identified as crucial for functional identity.
• Mutagenesis of these residues in UDP-Galactose 4-epimerase led to the formation of abortive NADH complexes.
• Molecular dynamics simulations suggested a mechanism involving impaired NADH re-oxidation.

Conclusions:

• Specific residue positions within the SDR fold are key determinants of enzyme function.
• These identified residues modulate the enzyme's catalytic cycle, specifically affecting NADH re-oxidation and leading to non-productive complex formation.
• The study provides insights into structure-function relationships within the SDR superfamily.