Staphylococcus aureuslipase: purification, kinetic characterization, crystallization and crystallographic study

Abstract

Staphylococcus aureus lipase (SAL), a triacylglycerol esterase, is an important virulence factor in S. aureus and may be a therapeutic target for infectious diseases caused by S. aureus. For the purposes of anti-SAL drug development using structure-based drug design, X-ray crystallographic analysis of SAL overexpressed in Escherichia coli was performed. The recombinant protein was purified using a three-step protocol involving immobilized metal-affinity chromatography, cation-exchange chromatography and anion-exchange chromatography flowthrough methods, yielding 40 mg of protein per litre of bacterial culture. Crystals were obtained using the sitting-drop vapor-diffusion technique. Diffraction data to 3.0 Å resolution were collected on the BL44XU beamline at SPring-8 at the zinc peak of 1.2842 Å for SAD phasing. The crystals belonged to space group P422 or P422, with unit-cell parameters a = 131.0, b = 131.0, c = 250.6 Å, and are likely to contain four SAL molecules (408 residues) per asymmetric unit.

Crystal structure of pathogenic Staphylococcus aureus lipase complex with the anti-obesity drug orlistat

“…cloning and expression of SAL. The SAL gene was cloned into a pCold II vector (Takara Bio) and amplified by PCR with the KOD FX DNA polymerase (Toyobo) using chromosomal DNA as previously reported 32 . The plasmid for the expression of the Ser116Ala mutant was constructed using site-directed mutagenesis with 5′-CTTGTAGGGCATgcTATGGGTGG-3′ as the mutagenesis primer.…”

“…Recombinant SAL and S116A-SAL were expressed in E. coli and purified as described previously 32 with three-step chromatography. The eluted protein containing SAL was further purified using size-exclusion chromatography to analyze its molecular size in solution ( Supplementary Fig.…”

“…Determination of specific activity. Enzymatic activities were determined using p-nitrophenylbutyrate (pNPB) ester substrate as previously reported 32,33 . Enzyme concentration was adjusted to 0.002 mM and pNPB concentration to 0.08 mM.…”

Crystal structure of Staphylococcus aureus lipase complex with unsaturated petroselinic acid

“…Diffraction data sets of SAL/PSA complex crystals were measured and analyzed at the BL44XU beamline at SPring-8 at 2. 19 A resolution (Table 1). The space group was P4 1 22, cell constants a = b = 132.3, c = 248.…”

“…SAL and PSA inhibitors were cocrystallized, and diffraction data sets were collected to 2. 19 A resolution at SPring-8 to determine the crystal structure and elucidate the detailed structural interactions. The results show that the fatty acid moiety of PSA is tightly bound to a hydrophobic pocket extending in two directions around the catalytic residue Ser116.…”