A Study On The Enzymatic Characteristics And Crystal Structure Of Malassezia Globosa Lipases

Mono-and diacylglycerol lipase which considerably differs from typical lipase insubstrate selectivity, is strictly specific for mono-and diacylglycerol but not triacylglycerol.SMG1is a lipase secreted from Malassezia globosa and closely involved in common skindisorders such as the Dandruff and seborrheic dermatitis （D/SD）, suggesting that SMG1would be a novel target for treatment of D/SD. Additionally, SMG1is a mono-anddiacylglycerol lipase, which has been successfully applied to the synthesis of high-purediacylglycerol that is proved to be beneficial in lowering the risk factors for obesity-relateddiseases. Important physiological function and high practical value of mono-anddiacylglycerol lipase SMG1underscore the needs for a comprehensive understanding of itsstructure and function. In this study, a series of experiments were carried out to investigate theenzymatic characteristics of lipase SMG1and its crystal structures was solved. Specificcontents and results are as follows:（1） The cloning and expression of the mono-and diacylglycerol lipase from Malasseziaglobosa in the Pichia pastorisPrediction of signal peptide in the target gene of Malassezia globosa Lip1（Genbank ID:XP₀01732204.1.） obtained from NCBI data bank suggested that the gene of mono-anddiacylglycerol lipase SMG1existed in57bp signal peptide in the N-terminal of SMG1. Inorder to be successfully expressed in the Pichia pastoris, the coding sequence correspondingto mature peptide of SMG1was carried out codon optimization by DNAworks software andthen it was divided into18oligonucleotide primers. The coding sequence of SMG1synthesizeby two-step overlapping PCR was integrated in to plasmid pGAPZαA and then theconstructed pGAPZαA-smg1plasmid was transformed into Pichia pastoris X-33. The stain inwhich SMG1can be overexpressed was screened and identified.（2） The purification and enzymatic characteristics of mono-and diacylglycerol lipaseSMG1Pichia pastoris only secreted a small amount of background proteins, so the primarypurification of mono-and diacylglycerol lipase SMG1can be achieved by one step usinganion exchange chromatography （Q-Sepharose XL）. A series of experiments were carried out to investigate the enzymatic characteristics of SMG1in detail. The optimum temperature forthe reaction catalyzed by lipase SMG1was25℃, once reaction temperature over40℃would result in the denaturalization and deactivation of lipase SMG1quickly. The optimumpH of lipase SMG1catalyzing reaction was6.0, but the alkaline environment was beneficialto maintaining the enzymatic activity of lipase SMG1. In this study, the effect of differentlength of acyl chain on the enzymatic activity of lipase SMG1was investigated by usingp-nitrophenol as substrate. Lipase SMG1displayed little difference in activity for substrateswith short or medium chain and the maximum activity was observed in substrate with C8. Theinfluence of different metal ions at the concentration of5mM on the enzymatic activity oflipase SMG1was detected, and these results suggested that no activation in enzymatic activityof lipase SMG1was observed and the activity was decreased significantly by Hg2+, forexample. Methanol, ethanol, isopropanol and acetone were used to treat lipase SMG1sampleand that resulted in the improvement to varying degrees of enzymatic activity of lipase SMG1.A striking characteristic of lipases was the interfacial activation: the catalytic activity of lipaseon monomeric substrate was very low, but it increases tremendously when the substratereached concentrations above its supersaturation level. Compared with the activity of SMG1that displayed on the monomeric substrate, a dramatic improvement of enzymatic activity forthe substrate in the aggregated state was not observed. In this study, the effect of four types ofsurfactant on enzymatic activity of lipase SMG1was studied and these experiments showedthat lipase SMG1was very sensitive to all surfactants tested: they had no significantactivation effect on its activity, and relative low concentrations of these detergents couldimpair greatly the catalytic efficiency of lipase SMG1.（3） the crystallographic study on the mono-and diacylglycerol lipase SMG1In order to fulfill the requirement of purity for crystallization, lipase SMG1sample wasfurther purified by molecular exclusion chromatography S75. The pre-crystallization test wascarried out to determine the appropriate concentration for crystallization screening and in thecase of lipase SMG1,10mg/mL was finally subject to the crystallization screening. Afteroptimization, two crystals with high quality were obtained under the conditions0.1MBis-Tris pH6.5,20%PEGMME5000and10%PEG1000,10%PEG8000, respectively.Two crystal structures （PDB ID:3UUE and3UUF） were resolved respectively at1.45and 2.60by molecular replacement using the Gibberella zeae lipase （PDB ID:3NGM） as initialmodel.The structure of mono-and diacylglycerol lipase SMG1consisted of a core domain（residue20-100and120-304）, showing the typical features of the α/β hydrolase fold topology,and a lid domain （residue101-119）. The central β sheet was formed by8β-strands, two ofwhich were antiparallel （β2and β8）, and was surrounded by4long helices, several shorthelices, and connective loops on both sides. The residues forming the catalytic triad in lipaseSMG1were Ser171, Asp228and His281. The nucleophilic residue Ser171was part of theconsensus sequence G-X-Ser-X-G and located at the conserved tight turn between theC-terminus of β4strand and α3helix. The catalytic Asp228and His281of lipase SMG1alsolocalized in a position well conserved within the α/β hydrolase fold. The “oxyanion hole” wasconstituted by backbone NH from Thr101and Leu172and would stabilize the tetrahedralintermediate during hydrolysis.The most striking difference in structure between mono-and diacylglycerol lipase SMG1and its homologous lipases occurred in the lid region: typical lipases always contained one ormore amphiphilic helices, in which one face is exclusively composed of hydrophobic residuesand the other of hydrophilic residues; but the lid of lipase SMG1adopted mostly a loopconformation with a small supplementary310helical turn containing three residues at theC-terminus.The interfacial activation of lipase generally resulted from the lid movement. In the caseof mono-and diacylglycerol lipase SMG1, the lid was a loose and flexible loop, suggestingthat it was easier to displace the lid in lipase SMG1. Above all, there were only twohydrophobic residues （Leu106and Phe115） embedded into the interior of lipase SMG1, theconformational changes from the closed state （catalytically inactive） to open （catalyticallyactive） state could take place easily by breaking these hydrophobic interactions between lidand core domain of SMG1even in the absence of interface.（4） The study on the substrate selectivity of mono-and diacylglycerol lipase SMG1Two crystal structures of lipase SMG1were solved in the closed conformation, wherethe active site was shielded from the reaction medium by lid domain, considered catalyticallyinactive. In order to investigate the molecular basis responsible for this unique selectivity, we constructed a structural model in the open, active conformation and modeled a substrateanalogue to the active site to mimic the interactions between enzyme and substrate. Moleculardynamics simulations （MD） were performed to relax steric clashes in the SMG1-analoguecomplex and to identify the binding sites.The model of the SMG1-analogue complex revealed that Leu103and Phe278divided thewhole catalytic pocket into two separated moieties, an exposed groove and a narrow tunnel,which were restricted to accommodating the two alkyl chains of diacylglycerol only.