Structural basis of the chiral selectivity of Pseudomonas cepacia lipase

Abstract

To investigate the enantioselectivity of Pseudomonas cepacia lipase, inhibition studies were performed with S C -and R C -(R P ,S P )-1,2-dialkylcarbamoylglycero-3-O-p-nitrophenyl alkylphosphonates of different alkyl chain lengths. P. cepacia lipase was most rapidly inactivated by R C -(R P ,S P )-1,2-dioctylcarbamoylglycero-3-O-p-nitrophenyl octylphosphonate (R C-trioctyl) with an inactivation half-time of 75 min, while that for the S C -(R P ,S P )-1,2-dioctylcarbamoylglycero-3-O-p-nitrophenyl octyl-phosphonate (S C -trioctyl) compound was 530 min. X-ray structures were obtained of P. cepacia lipase after reaction with R Ctrioctyl to 0.29-nm resolution at pH 4 and covalently modified with R C-(RP ,SP)-1,2-dibutylcarbamoylglycero-3-O-p-nitrophenyl butyl-phosphonate (R C -tributyl) to 0.175-nm resolution at pH 8.5. The three-dimensional structures reveal that both triacylglycerol analogues had reacted with the active-site Ser87, forming a covalent complex. The bound phosphorus atom shows the same chirality (S P) in both complexes despite the use of a racemic (R P ,S P ) mixture at the phosphorus atom of the triacylglycerol analogues. In the structure of R C -tributyl-complexed P. cepacia lipase, the diacylglycerol moiety has been lost due to an aging reaction, and only the butyl phosphonate remains visible in the electron density. In the R C-trioctyl complex the complete inhibitor is clearly defined; it adopts a bent tuning fork conformation. Unambiguously, four binding pockets for the triacylglycerol could be detected : an oxyanion hole and three pockets which accommodate the sn-1, sn-2, and sn-3 fatty acid chains. Van der Waals' interactions are the main forces that keep the radyl groups of the triacylglycerol analogue in position and, in addition, a hydrogen bond to the carbonyl oxygen of the sn-2 chain contributes to fixing the position of the inhibitor.Keywords : crystal structure; transition-state analog; enantioselectivity; lipase; stereospecificity.Lipases are lipolytic enzymes, which hydrolyze ester bonds fold, a structural motif common to a wide variety of hydrolases of triacylglycerols. However, their substrate specificity is not [3]. Their active sites contain a catalytic triad, Ser-His-Asp/Glu, limited to triacylglycerols. They may also hydrolyze ester bonds similar to those of serine proteases [4Ϫ6]. Studies with lipases of other compounds such as acetyl-arylpropionic acid esters, covalently complexed with organosulfates [7], organophosphates which are precursors for the nonsteroidal anti-inflammatory [8, 9], or organophosphonates [10Ϫ12] demonstrated that, in the agents naproxen and ibuprofen [1]. Because of this broad sub-presence of lipid-like compounds or organic solvents, their strate specificity, and because of their distinct stereopreferences, active-site regions may undergo drastic conformational changes, lipases have found widespread application in the enantioselec-exposing the catalytic residues and the surrounding hydrophobic tive synthesis of organic compounds, and in the resolution of ...

The crystal structure of Bacillus subtili lipase: a minimal α/β hydrolase fold enzyme

“…40 around the C-2 glycerol backbone atom could be explained by a speci®c hydrogen bonding interaction of Thr18 with the carbonyl oxygen atom of the sn-2 chain. 27 In agreement with the low stereoselectivity of B. subtilis lipase, there is no such hydrophilic amino acid residue present at a position equivalent to Thr18 in B. cepacia lipase. Instead, Asn18 of B. subtilis lipase (which occupies a position different from Thr18 in B. cepacia lipase) is at interacting distance from the scissile bond carbonyl oxygen atom of both substrate enantiomers.…”

Crystal Structure and Biochemical Properties of a Novel Thermostable Esterase Containing an Immunoglobulin-Like Domain

“…To visualize this, the structure of EstA in complex with the tributyrin transition state was manually modeled based on the structure of Pseudomonas cepacia lipase (PDB 4LIP) with the covalently bound tributyrin transition state analogue R C -(R P ,S P )-1,2-dibutylcarbamoylglycero-3-O-p-nitrophenyl butylphosphonate (data not shown). 21 According to the model, tributyrin could bind in the active site, resembling the way paraoxon binds. However, the size of the binding pocket is limited, thus making it impossible to bind longer acyl-chain triacylglycerols as in P. cepacia lipase.…”

Cultivation conditions and properties of extracellular crude lipase from the psychrotrophic fungus Penicillium chrysogenum 9′

“…Knowledge about molecular mechanisms of protein-lipid interaction is essential for our understanding of lipase stereoselectivity. X-ray crystallography studies on lipase-inhibitor complexes provide detailed information about lipasesubstrate interactions at submolecular levels [17,18].…”