Biochemical Characterization Of Enzymes Involved In Santalene Synthesis And Taurine Degradation

Enzymes are macromolecular biological catalysts capable of catalyzing diverse reactions in nature.Amongst the diverse class of enzymes,the ones which play a central role in metabolism are of great interest.In the present study we have focused on such enzymes which are（a）involved in the characterization of santalene synthases involved in the production of santalene,a terpenoid of commercial interest and（b）metabolism of taurine,an amino acid which links human health and disease.Terpenoids are most abundant and diverse class of small natural products.They play a key role in many aspects of our life including flavor,fragrances,healthcare,medicine,and nutrition.Terpene synthases found in all kingdoms of life and are promiscuous in terms of product formation with some extremes capable of producing hundreds of structurally and stereochemically similar but different products simultaneously.Traditional methods to quantify the products of terpene synthases rely heavily on gas chromatography-mass spectrometry（GC-MS）or liquid chromatography-mass spectrometry（LC-MS）however sometimes messy elution profiles of the products and low yield result in losing tracks or mis-assigning of products.Reliable activity assays in GC-MS or LC-MS methods depends on authentic standards but some of standards are commercially not available making the assays difficult and cost-ineffective.Thus,we have developed a colorimetric assay using yeast inorganic pyrophosphatase as a coupling enzyme to measure the activities of terpene synthases.We have taken the advantage of pyrophosphate,a common byproduct of all terpene synthases catalyzed by yeast inorganic pyrophosphatase to generate orthophosphate which can react with ammonium molybdate to form phosphomolybdic acid.The phosphomolybdic acid can then be reduced by ferrous sulfate in a weak acid solution to produce blue color which has a characteristic absorbance at 660 nm.As a proof of the concept,we have used this method to quantitatively characterize three santalene synthases,SaSSy,SspiSSy and SanSyn.SaSSy and SspiSSy come from different sandalwood species,S.album and S.spicatum respectively.On the other hand SanSyn comes from a phylogenetically distant tropical fruit Clausena lansium（Wampee）.Our study measured the kinetic parameters of all three santalene synthases using this colorimetric assay and demonstrated the validity of the enzyme coupled colorimetric assay by the comparison of this assay with the existing GC-MS method.Thus,present colorimetric method can be employed for high-throughput screening for inhibitors of drug targets in this enzyme family.Taurine is one of the most abundant and important amino acids in the human body,it serves a wide variety of functions in the central nervous system,and has close relationship with diabetes,cytoprotection,cardiomyopathy and renal dysfunction.Excess dietary taurine is excreted into the gut as the conjugated bile salt taurocholate,the organosulfonates derived from taurine-conjugated bile salts are dissimilated by the microbial consortium in the human gut.The resulting toxic end-product H₂S provides a major link between high fat diet and disease.Here we report the discovery of a novel O₂-sensitive glycyl radical enzyme（IseG）that catalyzes the radical-mediated C-S bond cleavage of hydroxyethylsulfonate（isethionate）,forming sulfite and acetaldehyde.This enzyme is present in disease-associated sulfite-reducing gut bacteria where it generates electron acceptors for anaerobic respiration,playing an important role in their metabolic niche.We have further described new enzymes-taurine pyruvate transaminase（TPA）and NADH-dependent sulfoacetaldehyde reductase（TauF）present in fermenting gut bacteria that catalyzes the formation of isethionate as a byproduct of nitrogen assimilation from taurine.Together,these enzymes constitute a new pathway for taurine degradation,highlighting the involvement of interspecies cross-feeding in converting a host metabolite into a toxin.Since afore mentioned IseG and TauF enzymes are essential for the respiration and growth of pathogenic bacteria in the human gut and there are no human counterparts of the same,they serve as good drug targets for inhibiting and killing such pathogenic gut bacteria.