Structural And Functional Study Of Aminotransferase CrmG And Human SNX7

(?)CrmG is a pyridoxal-5-phosphate dependent enzyme that belongs to aspartate Aminotransferase fold-type I family.It is involved in the biosynthesis of Caerulomycin A.Caerulomycin A was found to have strong antifungal,antitumor activity and moderate antibacterial activity.The CrmG gene is located in the gene cluster from a marine actinomycete Actinoalloteichus cyanogriseus WH1-2216-6.The aminotransferase CrmG consists of 523 amino acid residues,folding into two domains:a large domain that binds with the cofactor PLP and a small domain consisting of the C-terminus and the beginning part of N-terminus.In order to understand the catalytic mechanism of the enzyme,we carried out structural studies on CrmG.We cloned the target gene.It was then inserted into the vector pET-28a,tansformed into E.coli BL21.We successfully obtained overexpression protein.After affinity and gel filtration purification,the purity of CrmG reached over 95%.We determined the structures of wild-type CrmG and the complex of CrmG with Glutamate to 2.3 A and 1.8 A resolution.PISA analysis shows that a tetramer is formed through weak interactions by homodimer.The rod-shaped crystals belonged to space group 1222 and contained one molecule in the asymmetric unit.The overall structure of the enzyme is similar to those of other aminotransferase subgroup ?enzymes.There is no large conformational change between the two structures.Both structures lack electron density in the active site.The complex structure also has missing electron density corresponding to Val202-His208 and Ser231-Val249.Based on structural analysis,the flexible region may contain one ?-sheet and two a-helices.Whether the flexible region is involved in the interaction with the substrate or the formation of dimers is unknown.In addition,we found a unique feature in this family proteins,an outwardly extending tail shaped conformation formed by two a-helices(residue:121-196)and two ?-sheet(residue:256-266).Multiple sequence alignment indicated that this region is not conserved in the family.This area is away from the active pocket.Site-directed mutagenesis show that mutation Phe55Gln did not affect the activity of the enzyme.The aldehyde group of PLP is covalently linked to the Lys344 at the active site of the enzyme,which can form a Schiff base as the key intermediate for the enzyme activity.When the Lys344 is mutated into Ala,the enzyme losses its activity.Additionally,when Val317 is mutated to Ala,the protein abolishes the enzymatic activity as well.Presumably,the ?-branched side chain of V317 may facilitate formation of external aldimine intermediates with primary amine substrates.(?)Sorting nexin7(SNX7)is a family member of SNXs,which are a large group of proteins involved in regulating endocytosis and endosome sorting.SNX7 belongs to the subclass of SNX-PX-BAR that contains a PX domain and a BAR domain.In zebrafish,SNX7 is a liver-enriched anti-apoptotic protein and is indispensible for the liver development.In order to study the structure and function of SNX7 at the protein level,we subcloned a fragment of SNX7 cDNA(SNX7PX-BAR),encoding the PX domain and the BAR domain.It was inserted into the expressing vector p28a,transformed into E.coli Rosseta2(DE3)after amplification,and then induced by IPTG.The induced fusion protein(43 kDa)was expressed successfully in soluble form.After nickel column affinity,ion exchange and gel filtration purification,the purity of the protein SNX7PX-BAR reached over 95%,confirmed by Western-Blotting experiment.Dynamic light scattering(DLS)experiment indicated SNX7PX-BAR was homogeneous in solution.Lipid overlay assay showed that SNX7PX-BAR can strongly bind to PtdIns(5)P,PtdIns(4,5)P2 and PtdIns(3,4,5)P3,while weakly bind to PtdIns(4)P.So far,we have not yet obtained the crystal of SNX7PX-BAR protein,and further work is ongoing.