Biochemical And Structural Investigation Of Taurine:2-oxoglutarate Aminotransferase From Bifidobacterium Kashiwanohense

Taurine is one of the most abundant amino acids in the human body,where it has various important physiological functions such as regulating osmotic pressure and neural transmission.Its bile acid conjugates are secreted into the intestines to emulsify dietary lipids.Taurine is richly present in meat and animal fat,and some energy drinks also contain high levels of taurine.Microbial metabolites in the human intestines have great impact on human health.Taurocholate is hydrolyzed by gut bacteria releasing taurine,which is then further metabolized by the gut microbiome via pathways whose details remain unknown.Our group recently identified a novel oxygen-sensitive glycyl radical enzyme（GRE）that is present in all five sulfate and sulfite reducing bacteria（SSRB）with genome sequences available at the NIH（National Institute of Health）,including Desulfovibrio piger and the disease-linked Bilophila wadsworthia.This GRE（named by us Ise G）catalyzes the cleavage of the C-S bond in taurine-derived isethionate,forming sulfite,which can be used as the terminal electron acceptor for the respiration of SSRB.Such anaerobic respiration in SSRB results in the reduction of sulfite to H₂S,a gaseous toxin implicated in inflammation and colorectal cancer.The sequential action of a taurine:pyruvate aminotransferase（Tpa）,a sulfoacetaldehyde reductase（Tau F）,and Ise G in B.wadsworthia degrades taurine and generates sulfite.B.wadsworthia is also capable of directly importing isethionate from outside of the cell and utilizing it via Ise G,generating sulfite,whereas D.piger can only use isethionate and not taurine.Therefore,this bacterium is dependent on other gut bacteria to provide isethionate.It has been known since 1980 that a high concentration of isethionate is present in the human gut as a byproduct of microbial taurine metabolism.However,the exact bacterial strain responsible for the production of isethionate has yet to be identified and the underlying biochemical process is largely unknown.To solve this decades-old mystery,this thesis project was initiated with bioinformatics studies that revealed a putative pathway for nitrogen assimilation from taurine in a human gut bacterium,Bifidobacterium kashiwanohense.Genome neighborhood analysis suggested that nitrogen assimilation from taurine requires the sequential action of a taurine ABC transporter,a taurine:2-oxoglutarate aminotransferase（Bk Toa）,a sulfoacetaldehyde reductase（Bk Tau F）,and an isethionate exporter（Isf E）.This pathway may result in nitrogen assimilation and formation of isethionate as a byproduct,which is excreted into the human gut environment.To test the hypothesized pathway,we optimized the expression of Bk Toa gene using an E.coli expression vector.The purified recombinant protein was confirmed to be a taurine:2-oxoglutarate aminotransferase using two different enzyme-coupled activity assays and LC-MS analysis.We also tested other amine acceptors including pyruvate and oxaloacetate and found that 2-oxoglutarate is indeed the preferred substrate of this enzyme.When 2-oxoglutarate is used as amine acceptor,glutamate is formed.Glutamate serves as a common nitrogen source in cell metabolism.Thus,our finding of the specificity of this enzyme for this universal amine acceptor further suggested that this is a dedicated pathway for the assimilation of nitrogen from taurine.We next characterized the Bk Toa enzyme in detail.The optimal p H of this enzyme was found to be 8.0.The enzyme kinetic parameters were also measured,revealing a k_cat of 3.7 s^-1,a K_M（taurine）of 1.1 m M,and K_M（2-KG）of 1.8 m M under optimal conditions.These parameters are consistent with Bk Toa being involved in primary metabolism.Using the sitting-drop vapor diffusion method,we carried out a high-throughput screening for Bk Toa protein crystals in complex with its cofactor 5’-pyridoxal phosphate（PLP）and glutamate.X-ray diffraction was used to solve the crystal complex structure at 2.7?resolution.Similar to the structures of otherω-aminotransferases deposited in the protein databank（PDB）,the Bk Toa structure shares the common PLP type-I fold and active site residues interacting with PLP.Importantly,in our complex structure,two glutamate molecules were bound in sites near the predicted active site,and may occupy a path for substrate entry and product release.We also used Schr?dinger software to perform induced fit docking of taurine-PLP,the transamination reaction intermediate,to the Bk Toa protein structure.Our molecular docking revealed a role for active site residues Trp 21 and Arg 156 in the interaction with the sulfonate group of taurine.Asp 154 forms a salt bridge with Arg 156,stabilizing the interaction between Arg 156 and the taurine sulfonate.Sequence alignments demonstrated that Trp 21,Asp 154,and Arg 156 are highly conserved among Toa enzymes studied to date,but not in otherω-aminotransferases.This further confirmed that these residues contribute to the substrate specificity of Toa.Site-directed mutagenesis was carried out and the W21A,D154A and R156A mutants of Bk Toa were tested for catalytic activity.Complete loss of activity in these mutants was consistent with our hypothesis that Trp 21,Asp 154 and Arg 156 are involved in taurine binding.These residues might further help identify Toa enzymes in the aminotransferase family and also identifying organisms that can metabolize taurine.Bioinformatics analysis showed that close homologs of Bk Toa are also present in other anaerobic gut bacteria.