Structural characterization of monoglyceride lipases

Monoglyceride lipase (MGL) catalyzes the hydrolysis of monoacylglycerol (MG) into free fatty acid and glycerol. Members of this ubiquitous enzyme family were found in different kingdoms of life including bacteria, yeasts, and animals. The known biologic role appears to be greatly species dependent. In mammals, MGLs have been shown to catalyze the last reaction step in intracellular and extracellular lipid catabolism, to be responsible for the degradation of the endocannabinoid 2-arachidonoyl glycerol, and to play a role in cancer progression. Interestingly, MGs and especially short chain MGs are toxic for bacteria, thus assigning MGL a crucial role in the survival of the organism. Although first reports on MGLs were published decades ago, strikingly little is known on the molecular basis accounting for their selective hydrolytic specificity towards MGs of different chain lengths. Currently, the only published crystal structures of MGLs are that of human MGL, all published only within the last two years. The structures revealed an a/ß hydrolase fold with a cap region in open and closed conformation. In our previous work, we determined the crystal structure of MGL from Bacillus sp. H257 which shares only 17% overall sequence identity, whereas the sequence identity drops even to 13% within the cap region. Nevertheless and quite surprisingly, the overall architecture of the cap region is identical in both, human MGL and MGL from Bacillus sp. H257. In the research proposed here, we will explore the hypothesis that the cap architecture of MGLs is conserved within this enzyme family. To achieve this, we will determine the structure of YJU3p, the MGL ortholog from yeast, and test the substrate specificity of biochemically uncharacterized proteins with similar cap architecture. Determinants for substrate specificity will be identified after analysis of crystal structure of MGL with substrate analogs and inhibitors mimicking the MGL substrate. Based on these structures, specific mutants will further delineate important residues conferring selectivity to the enzyme. We will also determine the role of the cap region with respect to substrate specificity by generating chimeric proteins with cap regions from MGLs with different preferences for their acyl chain lengths. Conformational flexibility of the cap region from MGL will be determined using X-ray crystallographic methods. This will be accomplished by identifying new crystallization conditions for the uncomplexed protein or crystallizing the protein in presence of ligands. Additionally, NMR dynamics experiments will be employed to provide insights into the dynamics and specific structural changes of the protein on a per-residue basis. The outcome of this work will significantly enhance our understanding of MGLs, their dynamics, factors determining substrate specificities, and evolutionary restraints beyond sequence conservation. This knowledge then can also help to understand structure-function relationships of proteins from different enzyme classes.

Monoglyceride lipases are important enzymes that catalyze the cleavage of the lipid molecules termed monoglycerides into their components of a corresponding fatty acid and a glycerol molecule. By virtue of this function, monoglyceride lipases are of great importance in many physiological processes in health and disease, e.g. intracellular and extracellular energy metabolism, assembly and re-organization of biomembranes, signaling and inflammatory processes, as well as in the development and proliferation of some types of cancer. Lipases have recently also attracted increasing interest as new therapeutic options for bacterial pathogens, since well-orchestrated energy and fat metabolism is essential for the survival and infection mechanism of pathogens. In addition, lipases and monoglycerides are important components in biotechnological processes as well as in the cosmetic- and food industries. In order to understand the catalyzed reaction, the specific selection of the natural substrates, and to develop possible inhibitory molecules for therapeutic purposes on a rational basis, a detailed molecular understanding is needed. This project provided insights into the structural relationship between monoglyceride lipases from different organisms (Bacillus sp. H-257, Mycobacterium tuberculosis, baker's yeast). The previously existing knowledge of three-dimensional structures of monoglyceride lipases of three crystal structures was expanded by ten high-resolution structures of monoglyceride lipases and thus the knowledge base more than tripled. We were able to study substrate binding in detail, identify flexible regions and experimentally verify different conformations of the protein. The fold of the protein revealed many similarities to other hydrolases. Additionally, an unexpected conservation of the general architecture of the so-called lid or cap region, which is involved in forming the substrate binding pocket, has been discovered in monoglyceride lipases of bacteria, yeast and humans. This structural feature has already been successfully used now in the identification of monoglyceride lipase from previously poorly characterized proteins. Small differences in the binding pockets of different species may be exploited in future studies for the development of specific inhibitors of monoglyceride lipases from pathogenic bacteria.