The Structural Studies Of Legionella Pneumophila Histidine Acid Phosphatase(MapA) And Sachomyces Cerevisae Deoxyhypusine Synthase(Dys1)

As is well-known,biological macromolecules mainly made of proteins participate in a variety of functions for life,such as enzymes,immune globulins and so on.Their functions fundamentally depend on three-dimensional structures of proteins.Therefore,resolving the three-dimensional structures of proteins is crucial to understanding how proteins perform their functions.This paper analyzes the three-dimensional crystal structure of the two enzymes,as well as the three-dimensional crystal structures that binds to the substrates and ligands during the catalytic processes.These results explain the mechanisms of action of two enzymes at the atomic level,which provides the theoretical basis for the development of targeted drugs for related diseases.（Ⅰ）:Legionnaires’disease（LD）is a severe human pneumonia caused by Legionella pneumophila which is an intracellular parasitic pathogen.Legionella pneumophila infects human alveolar macrophages through the Dot/Icm type II secretion system widespread in nature and parasitizes a host of hosts.This process involves 330 effector proteins and histidine phosphatase（HAP）is one of them.HAP derives from the histidine phosphatase superfamily which is characterized by a conserved histidine residue and catalyzes the transformation of phosphoryl groups from substrates to water via an active-site histidine during the catalytic cycle.There are at least two HAPs in Legionella pneumophila.The major HAP is the tartrate-sensitive HAP,which called Map A.Map A plays an important role in the progress of extracellular survival and in vivo spread in Legionella pneumophila.Recently,the substrate recognition mechanism of Map A has been still unclear.For understanding the mechanism of substrate recognition and the optimum substrate of Map A,the crystal structures of apo-Map A and the Map A^D281A mutant in complex with5′-AMP(Map A^D281A/5’-AMP)were solved at 2.2 and 2.6?resolution,respectively,by methods of X-ray crystallography.The overall structures of the two crystal structures are similar through comparison analysis of these two structures,which are two dimers.Each dimer comprises two subunits formed from two distinct domains,the N-terminal cap domain and the C-terminal core domain.In the binding site of the Map A^D281A/5′-AMP complex,the phosphate group ofsubstrate 5’-AMP interacts with conserved amino acid residues Arg33,His 34,Arg37 and Arg101 via hydrogen bonds.The 2′-hydroxyl group of the ribose moiety of 5’-AMP is stabilized by Glu201 and the adenine moiety of 5’-AMP is sandwiched between His205and Phe237 forming a stableπ-πinteraction.Our results reveal a novel substrate bonding pattern,which is suggests that 5’-AMP might be the optimal substrate for Map A.It could provide the development of inhibitors or drugs in the treatment of LD with theoretical basis.（Ⅱ）:Hypusine is a unique amino acid in eukaryotes and archaea,which synthetized by post-translational modification.This modification closely related to the function of eukaryotic translation initiation factor 5A（e IF-5A）is only exists in e IF-5A.Hypusine modification was carried out in two steps.The first reaction is catalyzed by deoxyhypusine synthases（DHS）.DHS transfers the 4-aminobutyl moieties of substrates spermidines to the conserved lysine residues of e IF-5A precursors forming intermediate product deoxyhypusines.The second reaction is catalyzed by deoxyhypusine hydroxylases（DOHH）.The intermediate products are hydroxylated forming hypusine.Taking DHS（Dys1）in Saccharomyces cerevisiae as the research object,we solved the three-dimensional structure of Dys1 complexing with NAD⁺in 2.8?resolution by Experimental method of X-ray crystallography.The overall structure was similar to structures which have been reported.All of these structures are homologous tetramers composed of two tightly bound dimers.There are two catalytic sites and two NAD⁺binding sites in each dimer.Each subunit contains a N-terminal ball-chain motif and Rossman folds composed of 9β-strands and 16α-helices.In the NAD⁺binding site of Dys1,NAD⁺interacts with Ser108,Asn109,Ser134,Ala135,Gly136,Glu139,Glu140,Asp257,Gly304,Thr329,Asp337,Ala338,Ala362residues via hydrogen bonds.Compared to the NAD⁺binding sites of Dys1,h DHS,Ph DHS and Tb DHS,we found that the amino acid residues that stabilize the NAD⁺cofactor are highly conserved.In addition,we predicted the interaction interface between Dys1 and Saccharomyces cerevisiae DOHH（Hyp2）and the three-dimensional model of a complex by molecular docking method.Glu139,Glu140,Arg162,Tyr179,Ser259,Met317 of Dys1 and Lys48,Thr49,Lys51,His52,His54 and Lys56 of Hyp2 interact through hydrogen bonds in the complex.Through structural analysis,the results show that the interaction between Dys1and Hyp2 is a transient effect.Our research results could preliminarily clarify the mechanism of the first reaction of hypusine modification.