Role of a remote leucine residue in the catalytic function of polyol dehydrogenase

Abstract

Studies on the protein-metal binding sites have mainly focused on the residues immediately surrounding the reacting substrate, cofactors, and metal ions. The contribution of residues in remote layers to the highly optimized microenvironments of catalytic active sites is not well understood. To improve our understanding, the present study examined the role of remote residues on the structure and function of zinc-dependent polyol dehydrogenases. We used an integrated computational and biochemical approach to determine the role of L136 in the third shell of the l-arabinitol 4-dehydrogenase (LAD) from Neurospora crassa. Substitution of L136 with charged (Asp, Lys, or His) and bulky (Trp) side chain amino acids abolished enzyme activity. Whereas the L136A mutant exhibited a 95% decrease in catalytic efficiency (kcat/Km), the L136C mutant exhibited a 39% decrease in kcat/Km. Additionally, molecular docking and dynamic simulations on the mutant (L136A, L136C, L136H, and L136P) complexes showed the loss of crucial H-bonds between G77 and the corresponding mutated residue. It is evident from theoretical and biochemical studies that the L136 is part of the extensive hydrogen bonding network coupled to the reaction catalyzed at the active site. We propose that L136, critically positioned behind the active site residues H78 and E79 in the third shell of LAD, plays a crucial role in modulating catalysis or substrate binding by stabilizing the GHE motif in the LAD active site.

A Single Second Shell Amino Acid Determines Affinity and Kinetics of Linagliptin Binding to Type 4 Dipeptidyl Peptidase and Fibroblast Activation Protein

“…The effect of second shell residues on binding affinity and kinetics in protein‐ligand interactions is poorly documented. Several groups have reported observations similar to ours [25–28] . One study described a role for the remote residue Phe46 in myoglobin on ligand binding [25] .…”

“…One study described a role for the remote residue Phe46 in myoglobin on ligand binding [25] . Another highlighted the role of the second shell in protein‐metal recognition [27] . Specifically, a study of zinc finger structures demonstrated the significance of second layer packing in shielding the negatively charged zinc finger cores.…”

Binding and Catalytic Mechanisms of Veratryl Alcohol Oxidation by Lignin Peroxidase: A Theoretical and Experimental Study

“…The loss of VA affinity, in the case of some mutants, was correctly predicted by the ΔE calculations. Similar studies supplied evidence that substrate binding differences affect the enzyme catalysis [45]. Therefore, the SQM methodology enabled us to explain the experimental results obtained with the different LiP variants.…”

Asp271 is critical for substrate interaction with the surface binding site in β‐agarase a from Zobellia galactanivorans

“…This destabilizes the interaction between residues in AgaA‐E147S/D271A and agaro‐octaose hence binding more loosely in agreement with the decreased affinity determined by SPR (Table ). In previous studies, focusing on H‐bond forming ability, it was suggested that perturbed H‐bonds may affect substrate binding affinities, turnover, and the kinetically preferred reaction pathway . Therefore, it is not surprising that loss of H‐bond network interactions by the single point mutation D271A impairs binding affinity.…”