The Structure And Activity Characterization Of A Novel ThDP- Dependent Enzyme From Phytophthora Capsici

Pepper Blight is an notorious disease with extensive infection in the worldwide,and have a serious influence on the quality and quantity of crops,caused a grievous economic losses.In1922,Leon Leonian described a blight of peppers in New Mexico and identified the pathogen as P.capsici.P.capsici has the extensive hosts,including pepper,tomato,eggplant,cucumber and other crops.So far,the chemical control is still the cheif control method to prevent the infection of P.capsici.The prevention and controling of pepper disease is becoming more and more difficult because of the single-agent and drug resistance.P.capsici is morphologically very similar to true fungi.Yet its evolutionary history is quite disitanct.In contrast to fungi,P.capsici is classified into oomycota that is more closely related to algae,and have interspecific divergences in metabolic regulation.Select of specific genes with important function in metabolic regulation,screening new drug targets,design efficient and low toxic drugs,seek a new way for the prevention and control of Pepper Blight.Thiamine diphosphate(Th DP)is the functional derivative of vitamin B1,widely employed as a cofactor by enzymes that catalyse anabolism and catabolism.The representative Th DP-dependent enzyme-Acetohydroxyacid synthase(AHAs),catalyses the condensation of either two molecules of pyruvate,or of one molecule of pyruvate with one molecule of 2-ketobutyrate(2KB)to form2-aceto-2-hydroxybutyrate as the first step in the branched chain amino acid biosynthesis(BCAAs).Because BCAAs only exist in plants,fungi and bacterium,not found in animals,therefore,AHAs generally used as a target of herbicides.Herein,we report a novel Th DP-dependent enzymes,Pc UE1,that firstly found in oomycetes.It is similar to the NAD(P)~+-dependent aldehyde dehydrogenase(ADH)on sequence and secondary structure.However,there are some characteries in the coenzyme species and binding modes difference from ADH,and possess obvious specificity in the phylogeny compared with homologous genes.We gained the three-dimensional structure of Pc UE1 by X-ray diffraction and conduct a preliminary study of catalytic mechanism,giving the results as follows:(1)We successfully clone the gene of Pc UE1 from the genome of P.capsici,Pc UE1has only<27%sequence identity with NAD(P)~+-dependent aldehyde dehydrogenase,and posess an inserted motif containing 22 amino acids and a conserved NAD(P)~+binding domain at the N-terminus.Phylogenetic analysis showed that Pc UE1 was significantly different from other species,but keep a identity of>86%in Phytophthora.(2)PcUE1 is successfully expressed,purified and crystallized.The recombinant proteins is purified by Ni-NTA affinity chromatography,Ion exchange chromatography,Gel filtration,obtained crystals complexed with Th DP diffracted to a resolution of 2.20?.The structure of Pc UE1 was solved by molecular replacement.The structural data demonstrates that Pc UE1 form a homodimer consisting of two subunits with a total buried surface area of4395?~2.The interface of the dimer is mainly composed of a six-stranded parallelβ-sheet from each subunit and has a total buried surface area of 1780?~2.The six-stranded parallelβ-sheet constract the core of overall structure with a shape of inverted“L”.(3)The structural properties of Pc UE1.Although Pc UE1 is similar to the aldehyde dehydrogenase from bacteries and animals in the secondary structure arrangement and the overall conformation,neither of them is common with active sites.The structures of ADH exist two firmly bound zinc ions in per subunit.One zinc ion,the so-called structural zinc,is the key factor to stabilize the structure.The other zinc ion,called the catalytic zinc,is the site for substrate binding and catalysis.However,One molecule Th DP and one ion zinc is found in per subunit of Pc UE1.Similar to the Th DP-dependent enzymes,the Th DP bind to Pc UE1via PRY(Thiazole ring binding domain)and PP(Pyrophosphate binding domain).In addition,Pc UE1 was verified by mass spectrometry after apo-,it was found that Pc UE1 was combined with the molecular of NAD~+,and have a similar bind moldular with FAD~+in Th DP-dependent enzymes.(4)The enzymatic activity of Pc UE1 towards the substrates is measured.Then pyruvate is defined as the substrate of Pc UE1,and the result of High Performance Liquid Chromatography(HPLC)indicate that the pyruvate was completely degradation in the reaction system,and further proved that the substrate specificity of Pc UE1 to pyruvate.To ensure the binding site of the pyruvate,acetyl phosphate,a analogue of pyruvate is used to cocrystallization with Pc UE1,and we have gained the compound crystals.