Crystallization And Structure Studies On ?-galactosidase Aga2 And Phytoene Desaturase CrtI_Ra

This thesis consists of two parts:crystallization condition and crystal structure analysis of a-galactosidase Aga2,and crystallization study on the phytoene desaturase CrtI from Rhodobacter azotoformans Y6.a-Galactosidase is an important class of glycosidase,and can catalyze the hydrolysis of a-galactoside bond.Certain types of a-galactosidase can also synthesize a-galactooligosaccharide and a-galactoside compounds through transglycosylation reaction.It has been reported that this class of enzymes mainly synthesize Gal?1-6 and Galal-3 linkages.Only three ?-galactosidases from Bifidobacterium breve 203(reported in our previous work),Stachys affinis and Lactobacillus could synthsize the Galal-4 linkages.The Gal?l-4 linkages naturally exist in glycoconjugates recptors on the cell surface,such as oligosaccharides Gb3(globotriose,Gal?l-4Ga1?1-4Glc)of Shiga toxin receptor,and tumor-associate glycan Globo-H.Synthesis of Gal?l-4 sugar chain in vitro may provide a basis for the development of pharmaceutical products such as antibiotics and cancer vaccines.In our previous work,in combination with random and site-directed mutation,we selected an Aga2 V564N mutant,with highly increased transglycosylation activity.We found that Aga2 and V564N showed different regional selectivity for oligosaccharide receptors during transglycosylation.Since the published a-galactosidase crystal structure studies are mainly focused on the explaination of the hydrolysis properties,and the crystal structure of the a-galactosidase of bifidobacteria has not been reported yet.Morever,the similarity between Aga2 and a-galactosidase with known crystal structures is relatively low.It's difficult to comprehensively explain the transglycosylation molecular mechanisms and regional selectivity using simulated structure model.In this thesis,we hetrogeneously expressed and purified Aga2,and screened fifteen crystallization kits including more than six hundred conditions.We selected and optimized an appropriate condition in kit PEGRx-2 33(4%2-propanol,0.1 M Bis-Tris pH 9.0 and 20%PEG5000),and finally obtained a 3.0 A crystal.The crystal structure was determined by X-ray and molecular replacement(MR).By analyzing the crystal structure,we found that the Aga2 crystal consisted of four identical monomers which packed into a close symmetrical tetramer,with vertical symmetry axises.Each monomer had a large twisted "?-sandwich" N-terminal domain,connected by a long a-helix to the catalytic(?/a)8-barrel domain,and a C-terminal domain including five?-sheets.Since there was no substrate co-crystallized in the active region of the A catalytic domain,electron density of some regions' were invisible.Structure comparison of Aga2 with LaMel36A(PDB 2xn0,structure contains a substate galactose)revealed that Asp537,Cys515,Gly518,Lys469,Asp355,Trp403,Asp471,Arg436 and Asp356 were identical to those corresponding residues of LaMel36A,suggesting that these residues might play great roles in the substrate recognition and stabilization.Aga2 showed a wide catalytic pocket,which might be related to transglycosylation activity modulation and substrate regional selectivity.We also attempted to co-crystallize substrate galactose,lactose and methyl ?-lactoside with Aga2 to obtain high quality single crystal for further explalining the molecular mechanism of the increased transglycosylation efficiency and enzymatic regional selectivity of mutant V564N.The second part of this thesis is crystallization study of the phytoene desaturase CrtlRa.Carotenoids are tetraterpenoid organic pigments that containing nine to thirteen conjugated double bonds,and reaveal very important biological functions and high medicinal values.Phytoene can be dehydrogenated to generate three types of carotenoids,which are neurosporene in the three-step,lycopene in four-step,and 3,4-didehydrolycopene in five-step desaturations.Such reactions are catalyzed by phytoene desaturase CrtI Carotenoids are tetraterpenoid organic pigments that containing nine to thirteen conjugated double bonds,and reaveal very important biological functions and high medicinal values.The carotenoids production through chemical synthesis or extraction from plants is currently limited,so.that the development of microbial fermentation method has become very attractive.Under this background,studies on carotene synthesis pathway have considerable theoretical significance and application values.Phytoene desaturase(CrtI)is a key enzyme in carotenoids biosynthetic pathway in bacteria and fungi.However,the studies on CrtIs are mainly limited to their enzymatic properties,and the key amino acid residues of CrtI and specific catalytic mechanism in carotenoids synthesis pathway remain unclear.We reported carotenoids biosynthetic pathway and CrtIRa dehydrogenated phytoene in three,four and five-steps in Rba.Azotoformans.The CrtIRa have two functions:dehydrogenation and isomerism,and was predicted to be an integral membrane protein with three transmembrane regions,which caused the difficulty in CrtIRa crystallization.To date,there is only one published crystal structure of CrtI from Pantoea ananatis with amino acid similarity of 40%to CrtIRa from Rba.azotoformans.The published crystal structure of CrtI was not completely solved,neither with substrate co-crystallization.Therefore,it is worthwhile to screen crystallization conditions of CrtIRa to obtain the crystal structure of full-length protein with the substrate co-crystallization.Further study of CrtIRa structure will elucidate catalytic mechanism of the enzyme and directed molecular modification.Using the pET28b-crtIRa(crtIRa gene was derived from Rba.azotoformans Y6)recombinant,we selected BL21(DE3)as expression host,and then expressed and purificated the recombinant CrtIRa.For the purification strategies,we employed ultracentrifugation 150,000g for one hour,and dissolved precipitated membrane protein in a buffer with 1.5%(w/v)DDM(n-Dodecyl-p-D-Maltopyranoside).We purified CrtI through nickel affinity chromatography with 0.05%DDM,and then used low concentrations of DDM,C8E4(n-Octyl-Tetraoxyethylene),LDAO(N,N-Dimethyldodecylamine-N-Oxide),OG(n-Octyl-?-D-Glucopyranoside),MNG(Maltose-Neopentyl Glycol)respectively to replace DDM by molecular exclusion chromatography.Proteins from each elution peac were harvested and enzymic activity of each portion was analyzed.The crystallization conditions were screend using about 10 mg/mL proteins and five membrane protein crystal screening kits:MemGold Box 1,MemGold Box 2,Memsys,Memstart,and Memplus with up to 1200 typies of variations in total.After preliminary screening,we obtained some irregular crystals of membrane protein CrtIRa using MemGold Boxl and Memsys kit with 0.02%DDM.Further studies on screening and optimizing crystallization conditions of CrtIRa are in progress.