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The crystal structure of Pseudomonas cepacia lipase with bound transition state analog


Leščić, Ivana; Luić, Marija; Kojić-Prodić, Biserka
The crystal structure of Pseudomonas cepacia lipase with bound transition state analog // Seventh International Summer School on Biophysics: Supramolecular Structure and Function
Rovinj, Hrvatska, 2000. str. 103-103 (poster, međunarodna recenzija, sažetak, znanstveni)


Naslov
The crystal structure of Pseudomonas cepacia lipase with bound transition state analog

Autori
Leščić, Ivana ; Luić, Marija ; Kojić-Prodić, Biserka

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Seventh International Summer School on Biophysics: Supramolecular Structure and Function / - , 2000, 103-103

Skup
Seventh International Summer School on Biophysics: Supramolecular Structure and Function

Mjesto i datum
Rovinj, Hrvatska, 14-26.09.2000

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
Pseudomonas cepacia; lipase; inhibitor; structure

Sažetak
Lipases (triacylglycerol acylhydrolases) are enzymes that initiate the catabolism of fats and oils in biological systems. Hydrolysis and other reactions catalyzed by lipases (for instance, transesterification) are usually highly regio- and enantioselective, which makes these enzymes very important for stereoselective synthesis of organic compounds. For studying the mechanism of action and factors that influence stereoselectivity of lipases, knowledge of three-dimensional structures of native lipases and of lipases with bound transition state analogs (or inhibitors) is essential. The aim of this study was to determine the three-dimensional structure of Pseudomonas cepacia (recently renamed as Burkholderia cepacia) lipase (PCL) in complex with (R_P, S_P)-methyl-O-((2'R)-1-phenoxybut-2'-yl)phosphonic acid chloride, which mimicked the transition state (TS). Complex was prepared by adding freshly prepared TS analog dissolved in acetonitrile to PCL solution (12mg/ml in 6.3 mM Na-phosphate, 5 mM piperazine, pH=6.0) in a molar excess of 60:1. Hanging drop vapour-diffusion technique was used, and crystals grew within a few days at 5^o C. The crystal grown from 42% 2-methyl-2,4-pentanediol, 0.1 M sodium citrate, 0.1 M Hepes, pH=7.4 was used for data collection. X-ray diffraction data to 2.3 A were collected using MAR research 345 mm diameter imaging plate detector system mounted on a Rigaku rotating-anode X-ray generator operated at 50 kV and 100 mA at 100 K. The crystal structure of this complex turned out to be isomorphous with the structure of the native PCL. The space group was determined as C2, with cell dimensions a=89.06 A, b=46.63 A, c=84.30 A, beta=120.86^o and one molecule in the asymmetric unit. After refinement, the R-factor was 17% and R_free was 22.3% (10% of the data). The three-dimensional structure showed that phosphorus atom is covalently bound to Ser87 oxygen atom. The inhibitor is clearly visible in the electron density maps. Bound TS analog exhibits S comfiguration on phosphorus atom. Catalytic residues Ser87, His286 and Asp264 are arranged similarly as in native PCL structure. One of the phosphonyl oxygen atoms is hydrogen bonded to main-chain nitrogen atoms of Leu17 and Gln88 that form the oxyanion hole.

Izvorni jezik
Engleski

Znanstvena područja
Kemija



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Projekt / tema
00980608

Ustanove
Institut "Ruđer Bošković", Zagreb