Study On The Catalytic Mechanism Of Brucella Type Ⅱ MEP Synthase

With the emergence of bacterial resistance,people must constantly develop new antibiotics and look for new antibacterial targets.Terpenoids are the most widely distributed products in nature.They play an extremely important role in the growth and development of many living organisms such as microorganisms,animals and plants.Studies have found that most bacteria and malaria parasites synthesize various terpenoids being necessary for their lives through the 2-methyl-D-erythritol-4-phosphate（MEP）pathway,while animals are through mevalonate（MVA）pathway.This phenomenon suggests that the MEP pathway can be used as a target for screening antibacterial compounds.Thus exploration of the mechanism of the key enzymes in MEP pathway and screening of their inhibitors have become a research hotspot in recent years.1-Deoxy-D-xylulose-5-phosphate reductoisomerase（DXR）,also referred to as MEP synthase catalyzes the rearrangement-reduction reaction of the substrate 1-deoxy-D-xylulose-5-phosphate（DXP）with the aid of divalent metal ions and NADPH to form MEP.Up to date,the in-depth studies of the catalytic mechanism of DXR have led to the elucidation of a series of DXR inhibitors,most of which are structural analogs of the DXR substrate DXP or reaction intermediates,such as fosmidomycin and its analog FR900098,etc.These inhibitors exhibit very strong antibacterial/antimalarial activity both in vitro and in vivo.Recent genomewide sequencing and comparative genomics studies have indicated that pathogenic microorganisms such as Brucella and Bartonella synthesize terpenoid precursors via the MEP pathway,but there is no a gene that can encode DXR in the genomes of the strains.An in-depth analysis pointed out that the synthesis of MEP in vivo is catalyzed by another NADPH-dependent oxidoreductase.Since this newly discovered MEP synthase differs greatly from DXR in terms of primary structures and spatial structures,it is called Type II MEP synthase or DRL（DXR-like）.Although both DRL and DXR can catalyze the same reaction,DRL-targeted inhibitors can only kill DRL containing strains without affecting the beneficial bacteria utilizing DXR（such as beneficial bacteria in the intestine）due to the unique active central region of DRL.This indicates that the antibacterial spectrum of the inhibitors screened by DRL will be narrower.Thus,elucidation of the catalytic mechanism of DRL is of great importance because it can provide a solid ground for screening antibacterial compounds using the protein as a target.Kinetic isotope effects（KIEs）can often have a multiplier effect in predicting chemical mechanisms of related reactions,studying chemical kinetics,and bioenzyme catalysis.Our team has successfully prepared the DRL protein（BaDRL）derived from B.abortus,and analyzed the factors affecting the activity of the enzyme.On this basis,this study intends to investigate the mechanism of the DXP rearrangement step catalyzed by DRL by detecting the KIEs of BaDRL,and to determine the possible intermediates in BaDRL reaction as well by a pre-column derivatization-HPLC-MS method.First,the unlabeled and C-13labeled substrate DXP were synthesized enzymatically,including DXP,[1-¹³C]DXP,[3-¹³C]DXP,[4-¹³C]DXP,[3,4-¹³C₂]DXP,then the first-order KIE in the BaDRL catalytic reaction was determined by MS.The results showed that the use of[1-¹³C]DXP as the substrate had no KIE.When[3-¹³C]DXP and[4-¹³C]DXP were used as the substrates,respectively,reasonable KIEs were detected.In addition,when[3,4-¹³C₂]DXP was used as the substrate,almost doubled KIE could be determined.The above data indicate that BaDRL causes changes in the hybrid state of both the C3 and C4 atoms of the substrate during the rearrangement,while the C1 atom does not change its hybrid state when the substrate is rearranged.Combining with the study of the catalytic mechanism of DXR,we would speculate that the rearrangement mechanism of BaDRL-catalyzed reaction is Retro-aldol/aldol,just same as that of DXR.That is,both atoms C3 and C4 in DXP undergo changes in the hybrid state,being from sp³ to sp².The mechanism of DRL-catalyzed substrate rearrangement is the same as that of DXR,suggesting that the intermediates formed during the BaDRL catalytic reaction should be consistent with those of DXR.According to this rearrangement mechanism,three intermediates would form during the catalytic reaction of DXR or DRL,namely enolized hydroxyacetone,hydroxyacetaldehyde phosphate and 2-methyl-D-erythrose-4-phosphate（MEsP）.These intermediates are highly water-soluble and do not contain chromophore groups and therefore are difficult to be detected directly.However,since they all contain a carbonyl group in their structures which can be derivatized and then detected.In this thesis,a pre-column-HPLC method for the determination of hydroxyacetone and hydroxyl-acetaldehyde with 2,4-dinitrophenylhydrazine（DNPH）as a derivatization reagent was established and based on the process we have also tried to capture the intermediates.Furthermore,preliminary exploration of employing O-（2,3,4,5,6-pentafluorobenzyl）-hydroxylamine（PFBHA）,p-aminobenzoic acid（PABA）,and N-propyl-4-mercapto-1,8-Naphthalimide（NPHNA）as derivatization reagents for detecting hydroxyacetone and hydroxyacetaldehyde phosphate were also carried out.The above results laid solid foundation for the revealing of the catalytic mechanism of DRL and the detection of its intermediates,and will provide new ideas for screening antibacterial compounds with DRL as a target.