Preliminary investigation of crystals of the neutral lipase from Pseudomonas fluorescens

Abstract

The neutral lipase from the bacteria Pseudomonas jluorescens, marketed under the trade name LpL-BOOS, has been crystallized in a form suitable for X-ray diffraction analysis from 35% n-propanol at pH 8.5. The crystals are monoclinic prisms and are of space group C2 with a = 9160 A, b = 47.17 A, c = 35.21 A and /Y? = 121.43". There is one molecule of the protein as the asymmetric unit of the crystals. The diffraction pattern extends to at least 1.6 A resolution and the crystals are extremely robust in terms of X-ray exposure.

The open conformation of a Pseudomonas lipase

“…The GBF crystals were grown at 12°C, those at BRI at 18°C and those at P&G at room temperature (~20°C). Despite these differences, the crystals obtained were all isomorphous with those of Pseudomonas fluorescens lipase [19]. They belong to space group C2 and the unit cell dimensions varied only slightly from one group to another.…”

“…The dialyzed protein was crystallized from 50% n-propanol in 50 mM Tris buffer, pH 8.5 by either hangingdrop or sitting-drop vapor diffusion at 18°C. These conditions were adapted from the crystallization conditions reported for P. fluorescens lipase by Larson et al [19]. The crystals had unit cell dimensions of a = 91.5, b = 47.3, c = 85.2 Å and ␤ = 121.25° and belonged to space group C2.…”

“…Prior to crystallization, the enzyme was lyophilized and redissolved in double-distilled water to a protein concentration of 16 mg ml -1 . Diffraction quality crystals were obtained by hanging-drop vapor diffusion in Linbro plates at room temperature using the crystallization conditions reported for P. fluorescens lipase by Larson et al [19] as a guide. The droplets were made by mixing 5 l of the enzyme stock with 5 l of the reservoir solution which contained 21-24% npropanol, 0.2 M sodium citrate, and 0.1 M MES, pH 6.5 and were suspended over a 1 ml reservoir.…”

“…Consistent with the other lipases crystallized from PEG, the closed conformation was observed. Pseudomonas lipases have also been crystallized from n-propanol [19,20]. Based on the behavior of other lipases, Candida rugosa lipase in particular, one might expect that these conditions would produce the open conformation of the enzyme.…”

Quantitative structure–activity relationship correlation between molecular structure and the Rayleigh enantiomeric enrichment factor

“…Pleiss et al 52 analyzed and compared the shape of the binding site of six lipases and subdivided them into three sub groups: (1) lipases with a crevice-like binding site (lipases from Rhizomucor and Rhizopus); (2) lipases with a funnel-like binding site (lipases from Candida antarctica, Pseudomonas and mammalian pancreas and cutinase); and (3) lipases with a tunnel-like binding site (lipase from Candida rugosa). 46,54 Fig. 5A and B show that the binding pocket of Pseudomonas cepacia lipase (PCL) is an elliptical funnel (the length is 17Å and the width at the base is 4.5Å that increases to 10.5Å at the entrance to the binding site) and the catalytic active site lies in the base of the funnel.…”