Research in the Meilleur lab focuses on mapping intra- and intermolecular interactions that govern enzyme function within the microenvironment of the catalytic site. Knowledge of local electrostatics, hydrogen-bonding networks and protonation states of titratable catalytic residues is essential for proper thermodynamic modeling and for predicting binding interactions in molecular recognition and catalysis.

Biological catalysts, or enzymes, fluctuate to function. While the prevailing structure based view of enzymes is of a scaffold that precisely positions a few key catalytic residues, the process is intrinsically dynamic and relies upon a series of coordinated chemical and structural changes throughout the reaction cycle. As a result, the microenvironment at and around the active site of the enzyme can fluctuate significantly, altering and perturbing local electrostatics, H-bonding interactions and the pKa of titratable catalytic residues. While small structural changes that result in minor conformations can dominate reactivity in some systems, many enzymes require substantial conformational changes in order to bind, position, process and release their reactants. When these active sites are buried within extended tunnels or clefts, additional questions arise on how local conformational flexibility and chemical environment couple to control binding and release of substrate and product.

The enzymatic systems currently targeted in our lab to investigate the role and significance of conformational flexibility in enzymatic mechanism include the cellulose degrading enzymes, Hypocrea jecorina Cel7a and Neurospora Crassa PMO-2.The active site of HjCel7a and NcPMO-2 are very extended and contain a number of flexible loops and numerous aromatic residues.

HjCel7a, a member of the Glycosyl Hydrolase family 7 (GH7), catalyzes the cleavage of glycosidic bonds in cellulose to produce cellobiose. Full length Cel7a consists of a small Carbohydrate Binding Module (CBM), a flexible linker and a Catalytic Core Domain (CCD). The CCD fold consists of a β-sandwich formed by two anti-parallel β-sheets. The catalytic site of Cel7a sits towards the end of a long substrate binding tunnel (~50 Å) formed along the concave side of the β-sandwich and structurally maintained, in part, by disulfide bridges. The bottom of the tunnel is composed of six flexible loops. The potential role of these loops in binding and catalysis has been the subject of recent investigations, but their functional implication remains unclear. The glycosidic bond is hydrolyzed at the active site of Cel7a by the concerted action of the catalytic residues Glu 212 and Glu 217. A third carboxylic residue, Asp 214 is thought to play a role in fine tuning the pKa of Glu 212.

Polysaccharide monooxyganses (PMOs), previously characterized as Glycosyl Hydrolase family 61(GH61) and now classified as Auxiliary Activity family 9 (AA9), have recently been characterized as cellulase enhancers. PMOs catalyze the oxidative degradation of cellulose. While GH7 family members act on the reducing ends of isolated chains of cellodextrin as a cellobiohydrolase, PMOs oxidize crystalline cellulose randomly and create new reducing ends for GH7 members to act on. PMOs are metal containing oxygenase enzymes. The active site is composed a copper ion liganded to the N-terminal His residue and another His residue. The overall fold is an immunoglobulin like β-sandwich accompanied by numerous loops. Recent structural studies have given insight into the C1’/C4 specificity of these enzymes; however, much remains to be learned about both the oxidation mechanism and the redox partners involved in the cellulose oxidative cycle.

We primarily combine atomic structure determination using X-ray and neutron crystallography and molecular dynamics approaches to map relationships between the electrostatic micro-environment of amino-acids, local dynamics and protonation states.