Rothlisberger U

References (2)

Title : Keys to Lipid Selection in Fatty Acid Amide Hydrolase Catalysis: Structural Flexibility, Gating Residues and Multiple Binding Pockets - Palermo_2015_PLoS.Comput.Biol_11_e1004231
Author(s) : Palermo G , Bauer I , Campomanes P , Cavalli A , Armirotti A , Girotto S , Rothlisberger U , De Vivo M
Ref : PLoS Comput Biol , 11 :e1004231 , 2015
Abstract : The fatty acid amide hydrolase (FAAH) regulates the endocannabinoid system cleaving primarily the lipid messenger anandamide. FAAH has been well characterized over the years and, importantly, it represents a promising drug target to treat several diseases, including inflammatory-related diseases and cancer. But its enzymatic mechanism for lipid selection to specifically hydrolyze anandamide, rather than similar bioactive lipids, remains elusive. Here, we clarify this mechanism in FAAH, examining the role of the dynamic paddle, which is formed by the gating residues Phe432 and Trp531 at the boundary between two cavities that form the FAAH catalytic site (the "membrane-access" and the "acyl chain-binding" pockets). We integrate microsecond-long MD simulations of wild type and double mutant model systems (Phe432Ala and Trp531Ala) of FAAH, embedded in a realistic membrane/water environment, with mutagenesis and kinetic experiments. We comparatively analyze three fatty acid substrates with different hydrolysis rates (anandamide > oleamide > palmitoylethanolamide). Our findings identify FAAH's mechanism to selectively accommodate anandamide into a multi-pocket binding site, and to properly orient the substrate in pre-reactive conformations for efficient hydrolysis that is interceded by the dynamic paddle. Our findings therefore endorse a structural framework for a lipid selection mechanism mediated by structural flexibility and gating residues between multiple binding cavities, as found in FAAH. Based on the available structural data, this exquisite catalytic strategy for substrate specificity seems to be shared by other lipid-degrading enzymes with similar enzymatic architecture. The mechanistic insights for lipid selection might assist de-novo enzyme design or drug discovery efforts.
ESTHER : Palermo_2015_PLoS.Comput.Biol_11_e1004231
PubMedSearch : Palermo_2015_PLoS.Comput.Biol_11_e1004231
PubMedID: 26111155

Title : Computational, structural, and kinetic evidence that Vibrio vulnificus FrsA is not a cofactor-independent pyruvate decarboxylase - Kellett_2013_Biochemistry_52_1842
Author(s) : Kellett WF , Brunk E , Desai BJ , Fedorov AA , Almo SC , Gerlt JA , Rothlisberger U , Richards NG
Ref : Biochemistry , 52 :1842 , 2013
Abstract : The fermentation-respiration switch (FrsA) protein in Vibrio vulnificus was recently reported to catalyze the cofactor-independent decarboxylation of pyruvate. We now report quantum mechanical/molecular mechenical calculations that examine the energetics of C-C bond cleavage for a pyruvate molecule bound within the putative active site of FrsA. These calculations suggest that the barrier to C-C bond cleavage in the bound substrate is 28 kcal/mol, which is similar to that estimated for the uncatalyzed decarboxylation of pyruvate in water at 25 degrees C. In agreement with the theoretical predictions, no pyruvate decarboxylase activity was detected for recombinant FrsA protein that could be crystallized and structurally characterized. These results suggest that the functional annotation of FrsA as a cofactor-independent pyruvate decarboxylase is incorrect.
ESTHER : Kellett_2013_Biochemistry_52_1842
PubMedSearch : Kellett_2013_Biochemistry_52_1842
PubMedID: 23452154
Gene_locus related to this paper: vibvy-y856