4 structures(e.g. : 5W95, 5V3Z, 4BE9... more)(less)5W95: Mtb Rv3802c with PEG bound, 5V3Z: Crystal Structure of the D1607N mutant form of Thioesterase domain of Mtb Pks13, 4BE9: Open conformation of O. piceae sterol esterase, 3GUU: X-ray structure of Candida Antarctica lipase A
1 moreTitle: Structural basis for lipid binding and mechanism of the Mycobacterium tuberculosis Rv3802 phospholipase Goins CM, Schreidah CM, Dajnowicz S, Ronning DR Ref: Journal of Biological Chemistry, 293:1363, 2018 : PubMed
The Mycobacterium tuberculosis rv3802c gene encodes an essential enzyme with thioesterase and phospholipase A activity. Overexpression of Rv3802 orthologs in Mycobacterium smegmatis and Corynebacterium glutamicum increases mycolate content and decreases glycerophospholipids. Although a role in modulating the lipid composition of the unique mycomembrane has been proposed, the true biological function of Rv3802 remains uncertain. In this study, we present the first M. tuberculosis Rv3802 X-ray crystal structure, solved to 1.7 A resolution. On the basis of the binding of PEG molecules to Rv3802, we identified its lipid-binding site and the structural basis for phosphatidyl-based substrate binding and phospholipase A activity. We found that movement of the alpha8-helix affords lipid binding and is required for catalytic turnover through covalent tethering. We gained insights into the mechanism of acyl hydrolysis by observing differing arrangements of PEG and water molecules within the active site. This study provides structural insights into biological function and facilitates future structure-based drug design toward Rv3802.
Sterol esterases are able to efficiently hydrolyze both sterol esters and triglycerides and to carry out synthesis reactions in the presence of organic solvents. Their high versatility makes them excellent candidates for biotechnological purposes. Sterol esterase from fungus Ophiostoma piceae (OPE) belongs to the family abH03.01 of the Candida rugosa lipase-like proteins. Crystal structures of OPE were solved in this study for the closed and open conformations. Enzyme activation involves a large displacement of the conserved lid, structural rearrangements of loop alpha16-alpha17, and formation of a dimer with a large opening. Three PEG molecules are placed in the active site, mimicking chains of the triglyceride substrate, demonstrating the position of the oxyanion hole and the three pockets that accommodate the sn-1, sn-2 and sn-3 fatty acids chains. One of them is an internal tunnel, connecting the active center with the outer surface of the enzyme 30A far from the catalytic Ser220. Based on our structural and biochemical results we propose a mechanism by which a great variety of different substrates can be hydrolyzed in OPE paving the way for the construction of new variants to improve the catalytic properties of these enzymes and their biotechnological applications.
Lipases are widely used as catalysts in industrial applications due to their thermostability and enantioselectivity. Candida antarctica lipase A (CAL-A) is highly thermostable in organic solvents and has therefore become a frequently used catalyst in chemical and pharmaceutical industry. CAL-A shows some unusual properties, which makes it a highly attractive enzyme. CAL-A is the only known lipase to have Sn2-preference towards triglycerides. It is able to hydrolyze sterically hindered alcohols, both secondary and tertiary alcohols. In addition, it shows a high chemoselectivity for the N-acylation of beta-amio esters, which makes CAL-A an important catalyst in the production of enantiopure amino acids. We have determined the crystal structure of CAL-A at 2.1 A resolution. CAL-A exhibits a typical alpha/beta hydrolase fold, consisting of a central beta-sheet and surrounding alpha-helices. The active site pocket is formed like a deep L-shaped tunnel covered by a lid that regulates the interfacial activation. Residues Ser184, Asp334 and His366 form the catalytic triad at the bottom of the pocket.
        
1 lessTitle: Structural basis for lipid binding and mechanism of the Mycobacterium tuberculosis Rv3802 phospholipase Goins CM, Schreidah CM, Dajnowicz S, Ronning DR Ref: Journal of Biological Chemistry, 293:1363, 2018 : PubMed
The Mycobacterium tuberculosis rv3802c gene encodes an essential enzyme with thioesterase and phospholipase A activity. Overexpression of Rv3802 orthologs in Mycobacterium smegmatis and Corynebacterium glutamicum increases mycolate content and decreases glycerophospholipids. Although a role in modulating the lipid composition of the unique mycomembrane has been proposed, the true biological function of Rv3802 remains uncertain. In this study, we present the first M. tuberculosis Rv3802 X-ray crystal structure, solved to 1.7 A resolution. On the basis of the binding of PEG molecules to Rv3802, we identified its lipid-binding site and the structural basis for phosphatidyl-based substrate binding and phospholipase A activity. We found that movement of the alpha8-helix affords lipid binding and is required for catalytic turnover through covalent tethering. We gained insights into the mechanism of acyl hydrolysis by observing differing arrangements of PEG and water molecules within the active site. This study provides structural insights into biological function and facilitates future structure-based drug design toward Rv3802.
Lipases and esterases constitute a group of enzymes that catalyze the hydrolysis or synthesis of ester bonds. A major biotechnological interest corresponds to thermophilic esterases, due to their intrinsic stability at high temperatures. The Pf2001 esterase from Pyrococcus furiosus reaches its optimal activity between 70 degrees C and 80 degrees C. The crystal structure of the Pf2001 esterase shows two different conformations: monomer and dimer. The structures reveal important rearrangements in the "cap" subdomain between monomer and dimer, by the formation of an extensive intertwined helical interface. Moreover, the dimer interface is essential for the formation of the hydrophobic channel for substrate selectivity, as confirmed by mutagenesis and kinetic analysis. We also provide evidence for dimer formation at high temperatures, a process that correlates with its enzymatic activation. Thus, we propose a temperature-dependent activation mechanism of the Pf2001 esterase via dimerization that is necessary for the substrate channel formation in the active-site cleft.
Sterol esterases are able to efficiently hydrolyze both sterol esters and triglycerides and to carry out synthesis reactions in the presence of organic solvents. Their high versatility makes them excellent candidates for biotechnological purposes. Sterol esterase from fungus Ophiostoma piceae (OPE) belongs to the family abH03.01 of the Candida rugosa lipase-like proteins. Crystal structures of OPE were solved in this study for the closed and open conformations. Enzyme activation involves a large displacement of the conserved lid, structural rearrangements of loop alpha16-alpha17, and formation of a dimer with a large opening. Three PEG molecules are placed in the active site, mimicking chains of the triglyceride substrate, demonstrating the position of the oxyanion hole and the three pockets that accommodate the sn-1, sn-2 and sn-3 fatty acids chains. One of them is an internal tunnel, connecting the active center with the outer surface of the enzyme 30A far from the catalytic Ser220. Based on our structural and biochemical results we propose a mechanism by which a great variety of different substrates can be hydrolyzed in OPE paving the way for the construction of new variants to improve the catalytic properties of these enzymes and their biotechnological applications.
Lipases are widely used as catalysts in industrial applications due to their thermostability and enantioselectivity. Candida antarctica lipase A (CAL-A) is highly thermostable in organic solvents and has therefore become a frequently used catalyst in chemical and pharmaceutical industry. CAL-A shows some unusual properties, which makes it a highly attractive enzyme. CAL-A is the only known lipase to have Sn2-preference towards triglycerides. It is able to hydrolyze sterically hindered alcohols, both secondary and tertiary alcohols. In addition, it shows a high chemoselectivity for the N-acylation of beta-amio esters, which makes CAL-A an important catalyst in the production of enantiopure amino acids. We have determined the crystal structure of CAL-A at 2.1 A resolution. CAL-A exhibits a typical alpha/beta hydrolase fold, consisting of a central beta-sheet and surrounding alpha-helices. The active site pocket is formed like a deep L-shaped tunnel covered by a lid that regulates the interfacial activation. Residues Ser184, Asp334 and His366 form the catalytic triad at the bottom of the pocket.