Biological Function And Crystal Structure Analysis Of A Prolyl 4-hydroxylase

Prolyl 4-hydroxylases?P4Hs?belong to the ferrous iron and 2-oxoglutarate-dependent oxygenases superfamily and exist in all eukaryotes and many prokaryotes.P4Hs emply Fe²⁺as a cofactor,and catalyze the hydroxylation of the substrate that is coupled to the decarboxylation of 2-oxoglutarate to yield succinate and CO₂.The hydroxylation reactions play central roles in many life processes,including collagen biosynthesis,nucleic acid repair,and the regulation of gene transcription and translation in vertebrates.In addition,in plants and bacteria,P4Hs are also involved in the biosynthesis of secondary metabolites and signaling molecules.Phytophthora capsici is a notorious phytopathogenic oomycete causing devastating diseases on many economically important vegetables and has a huge impact on agriculture.Hydroxyproline are important component of Phytophthora cell wall and these phytotoxic elicitor proteins containing hydroxyproline take part in pathogen-plant interaction.Studying the biological functions of P4Hs,which take part in the processes of Phytophthora growth,development and pathogenesis might provide insights into chemical fungicide screening.Using the bioinformatics analyses,we found five new PcP4Hs gene,named PcP4H1-5 here.Then,we investigated biological functions and structure of the PcP4Hs.The detailed data was summaried below:1.Five PcP4Hs genes were obtained from P.capsici standard strain LT1534 and named PcP4H1-5 according to the bioinformatics analysis.The expression levels of PcP4Hs were down-regulated in response to hypoxia,espectically PcP4H1 which expression level was significant decreased.The expression level of PcP4H1 was sharply up-regulated in the early stages during infection period,while that of PcP4H2-5 were stable in different development and infection stages.2.PcP4H1 structure was resolved at 2.35?resolution using prokaryotic expression system.The space group of PcP4H1 is P1.Two molucules are present in each asymmetric unit,and each molecule consists of the DSBH core fold.The ferrous iron and 2-oxoglutarate locate at the active pocket enclosed by DSBH core fold.The ferrous iron binds to the highly conserved H155-X-D157…H214 motif and 2-oxoglutarate might directly or indirectly bind to Arg223,Thr227 and Trp229 resideus.The peptide substrate binding shallow groove is shaped by??-strand,?9-helix and??-??thumb loop,and the active pocket is located at the center of the shallow groove.The disordered longer?2-?3 loop,?9-?10 loop,and??-??thumb loop might be involved in the formation of substrate binding channel and determine the substrate specifity and binding modes.Comparison structures of the PcP4H1 with the other P4Hs reveal that:the active pocket of PcP4H1 is deeper and bigger,might lead to PcP4H1 bind to more complexed cofactors.PcP4H1 contains extended C-terminal?-helices,which might involve in substrate binding and other biological function.3.PcP4H1-cofactor binary complex structure:we selected three different inhibitor molecules similar to 2-oxoglutarate:N-oxalylglycine,pyridine-2,4-dicarboxylic acid and dimethyloxalylglycine to prepare the PcP4H1-cofactor complex crystals.We used two different methods:PcP4H1 protein-cofactor mixed together to screen crystals and PcP4H1crystals directly socked into cryoprotection solution containing of these molecules to get potential protein-cofactors crystals.These crytals were examined by X-ray diffraction,the diffraction data were collected and the co-factors binding modes were analyzed.We obtained the PcP4H1-NOG binary complex structure:NOG interacts with the ferrous iron via a bidentate coordination through its 1-carboxylate moiety and forms direct hydrogen bond with the side chain of Thr227.Hydrogen bond also be formed between a water molecule and the side chain of Arg223.All these interactions can make the NOG inhibitor bind to the actove pocket stably.4.We obtained two PcP4H1 silencing transformants,SiPcP4H1-1 and SiPcP4H1-2.The mycelia of SiPcP4H1-1 and SiPcP4H1-2 grew slowly,and the morphology of colony,mycelia and sporangia did not change significantly.The shape of sporangia was spherical in silencing transformants that was so different from the wild-typed pear sporangia.The effects of SiPcP4H1-1 and SiPcP4H1-2 on host infection stage are being tested.PcP4H1 may be involved in the growth and development of hyphae and sporangia.In conclusion,we got five prolyl 4-hydroxylase genes from P.capsici,PcP4H1-5.This PcP4H1 may be involved in development and interaction process with host.The PcP4H1crystal structure consists of the classical DSBH core fold and ferrous iron and co-factor are present into the bigger active pocket.The flexible?2-?3 loop,C-terminal?9-?10 loop and??-??thumb loop might participate in the formation of the substrate binding channeland determine the substrate specifity.Based on activity analysis,PcP4H1 can not or can only weakly hydroxylate the existing P4Hs polypeptide substrates.Further studies on PcP4H1 have been initiated to characterize the specific substrates,biological function of PcP4H1 in P.capsici and the precise mode of peptide-substrate binding.Chracterization of the PcP4H1structure may provide further insights into the inhibitors screen for P4Hs.Structurally informed phylogenetic analyses suggest that the role of prolyl-hydroxylation from different organisms may have ancient origins.