The Characterization Of Quinoprotein Glucose Dehydrogenase From Serratia Marcescens FS14

Pyrroloquinoline quinone(PQQ)is a water soluble,heat-stable,tricyclic ortho-quinone,identified as the third class of redox cofactors in bacteria in addition to the well-known cofactors,nicotinamide(NAD(P)+)and flavin(FAD,FMN).Apart from NAD/FAD-dependent glucose dehydrogenases(GDH),quino protein glucose dehydrogenase,regarding PQQ as cofactor,is a new GDH and existed in Gram negative bacteria.To identify quino-dependent glucose dehydrogenases in S.marcescens FS14,three genes of hypothetic quino-dependent glucose dehydrogenase(12765,13040 and 20445)were identified via genome analyses of FS14,deletion mutant of 12765,13040,20445,including single mutant,double mutant and triple mutant were then constructed respectively.The pH of culture supernatant of these mutants were then measured in glycerol peptone broth and glycerol peptone glucose broth respectively.Intersetingly,the pH of the culture supernatant and prodigiosin production of single mutants and double mutants decreased in glycerol peptone glucose broth while only the triple mutant did not.As FS14 PQQ deletion mutant could not produce gluconate to decrease the pH of the cuture supernatant,therefore,these results implied that the three proteins are PQQ-dependent glucose dehydrogenase.However,unfortunately,the complementation assaies of these three genes were not successful except the 13040.To identify these three proteins are PQQ-dependent glucose dehydrogenase and elucidate the catalytic mechanism of quinoprotein glucose dehydrogenases,the gene of 12765,13040 and 20445 were amplified by PCR using the genomic DNA of FS14 as template and expressed in E.coli C43.20445 protein was then further purified and used for crystallization.The best crystal was obtained in 0.1 M sodium citrate pH6.0,27%PEG4000 and0.1 M Calcium Chloride condition at 4?.A complete 1.33 A diffraction dataset was collected by X-ray and subsequently crystal structure of 20445 was resolved by molecular replacement.The crystal of 20445 belongs to space group P121 and the asymmetric unit of the crystal contains two 20445 molecules.Each monomer of the structure comprised of six four-stranded,antiparallel ?-sheets aligned around a 6-fold pseudo-symmetry axis which showes a ?-propeller fold characteristic of PQQ-dependent glucose dehydrogenase.Structural comparison showed that the overall fold of the enzyme from FS14 exhibits significant similarity to that of the PQQ-dependent glucose dehydrogenase from E.coil and Acinetobacter calcoaceticus,and most of the residues related with the active site are conserved.These results confirmed that 20445 is a PQQ-dependent glucose dehydrogenase.