The Structural Biology Research On Cab2(phosphopantothenoylcysteine Synthetase)from Saccharomyces Cerevisiae

CoA,an essential cofactor,plays an important role in metabolism in almost all living organisms.CoA is biosynthesized from pantothenate,ATP and L-cysteine using five steps enzymatic reaction catalyzed by pantothenate kinase,phosphopantothenoylcysteine synthetase(PPCS),phosphopantothenoylcysteine decarboxylase,phosphopantetheine adenylyl transferase and dephosphocoenzyme A kinase.PPCS catalyzes the second step of CoA biosynthesis,the nucleoside triphosphate-dependent condensation reaction between 4'-phosphopantothenate(PPA)and L-cysteine to form phosphopantothenoylcysteine(PPC).Coenzyme A biosynthesis protein 2(Cab2)possesses activity of PPCS in Saccharomyces cerevisiae.To address the catalytic mechanism of Cab2,relevant researches were carried out in present study.The crystal structures of Cab2 and its complex with PPA,the reaction intermediate 4'-phosphopantothenoyl-CMP(PMT)and the reaction product PPC were determined in our study.These complex structures provide structural information on the ligands binding and catalytic mechanism of Cab2.Firstly,the y-amino of Asn97,an extremely conserved residue,interact with the carboxyl group of PPA,a-phosphate of CMP-moiety of PMT and the carbonyl of PPA-moiety of PPC through water-mediated hydrogen bonds.Secondly,the carboxyl of cysteine-moiety of PPC binds with the ?-amino groups of Lys281 and Lys226(from adjacent monomer)of Cab2.The thiol of cysteine-moiety is immobilized by the hydrophobic core of the protein,which might be important for the amino acid selectivity of Cab2.Thirdly,the helix ?7 and the preceding loop of Cab2 likely plays an important role in the ATP/CTP selectivity of PPCS.Lastly,the carboxyl of PPA-moiety of the ligands and the y-amino group of Asn97 possess different conformations in these complex structures.Furthermore,ITC and enzymatic assays were performed to study the biochemical properties of Cab2 in this paper.The results of ITC indicate that PPA,CTP and ATP could bind to Cab2 individually and the foremost has the highest binding affinity to Cab2.The enzymatic assays demonstrate that Mn2+/Mg2+ are indispensable to the enzymatic reaction of Cab2 in yeast;similar to human PPCS,Cab2 also could utilize both ATP and CTP to activate PPA;Cab2 could utlize both cysteine and cystine in vitro,which indicates that the amino group rather than the thiol group of L-cysteine attacks the carbonyl of PMT to form PPC;highly conserved residues Asn97,Asp214,Lys226 and Lys281 are important for the activity of PPCS.Based on structural and biochemical data,the catalytic mechanism of Cab2 was proposed in current study,which would further our understanding of PPCS.