A gene cluster for taurine sulfur assimilation in an anaerobic human gut bacterium.

Aminoethylsulfonate (taurine) is widespread in the environment and highly abundant in the human body. Taurine and other aliphatic sulfonates serve as sulfur sources for diverse aerobic bacteria, which carry out cleavage of the inert sulfonate C-S bond through various O2-dependent mechanisms. Taurine also serves as a sulfur source for certain strict anaerobic fermenting bacteria. However, the mechanism of C-S cleavage by these bacteria has long been a mystery. Here we report the biochemical characterization of an anaerobic pathway for taurine sulfur assimilation in a strain of Clostridium butyricum from the human gut. In this pathway, taurine is first converted to hydroxyethylsulfonate (isethionate), followed by C-S cleavage by the O2-sensitive isethionate sulfo-lyase IseG, recently identified in sulfate- and sulfite-reducing bacteria. Homologs of the enzymes described in this study have a sporadic distribution in diverse strict and facultative anaerobic bacteria, from both the environment and the taurine-rich human gut, and may enable sulfonate sulfur acquisition in certain nutrient limiting conditions.

Biochemical and structural investigation of taurine:2-oxoglutarate aminotransferase from Bifidobacterium kashiwanohense.

Taurine aminotransferases catalyze the first step in taurine catabolism in many taurine-degrading bacteria and play an important role in bacterial taurine metabolism in the mammalian gut. Here, we report the biochemical and structural characterization of a new taurine:2-oxoglutarate aminotransferase from the human gut bacterium Bifidobacterium kashiwanohense (BkToa). Biochemical assays revealed high specificity of BkToa for 2-oxoglutarate as the amine acceptor. The crystal structure of BkToa in complex with pyridoxal 5'-phosphate (PLP) and glutamate was determined at 2.7 Šresolution. The enzyme forms a homodimer, with each monomer exhibiting a typical type I PLP-enzyme fold and conserved PLP-coordinating residues interacting with the PLP molecule. Two glutamate molecules are bound in sites near the predicted active site and they may occupy a path for substrate entry and product release. Molecular docking reveals a role for active site residues Trp21 and Arg156, conserved in Toa enzymes studied to date, in interacting with the sulfonate group of taurine. Bioinformatics analysis shows that the close homologs of BkToa are also present in other anaerobic gut bacteria.

Radical-mediated C-S bond cleavage in C2 sulfonate degradation by anaerobic bacteria.

Bacterial degradation of organosulfonates plays an important role in sulfur recycling, and has been extensively studied. However, this process in anaerobic bacteria especially gut bacteria is little known despite of its potential significant impact on human health with the production of toxic H2S. Here, we describe the structural and biochemical characterization of an oxygen-sensitive enzyme that catalyzes the radical-mediated C-S bond cleavage of isethionate to form sulfite and acetaldehyde. We demonstrate its involvement in pathways that enables C2 sulfonates to be used as terminal electron acceptors for anaerobic respiration in sulfate- and sulfite-reducing bacteria. Furthermore, it plays a key role in converting bile salt-derived taurine into H2S in the disease-associated gut bacterium Bilophila wadsworthia. The enzymes and transporters in these anaerobic pathways expand our understanding of microbial sulfur metabolism, and help deciphering the complex web of microbial pathways involved in the transformation of sulfur compounds in the gut.

Structure of glycerol dehydrogenase (GldA) from Escherichia coli.

Escherichia coli (strain K-12, substrain MG1655) glycerol dehydrogenase (GldA) is required to catalyze the first step in fermentative glycerol metabolism. The protein was expressed and purified to homogeneity using a simple combination of heat-shock and chromatographic methods. The high yield of the protein (∼250 mg per litre of culture) allows large-scale production for potential industrial applications. Purified GldA exhibited a homogeneous tetrameric state (∼161 kDa) in solution and relatively high thermostability (Tm = 65.6°C). Sitting-drop sparse-matrix screens were used for protein crystallization. An optimized condition with ammonium sulfate (2 M) provided crystals suitable for diffraction, and a binary structure containing glycerol in the active site was solved at 2.8 Šresolution. Each GldA monomer consists of nine ß-strands, thirteen α-helices, two 310-helices and several loops organized into two domains, the N- and C-terminal domains; the active site is located in a deep cleft between the two domains. The N-terminal domain contains a classic Rossmann fold for NAD+ binding. The O1 and O2 atoms of glycerol serve as ligands for the tetrahedrally coordinated Zn2+ ion. The orientation of the glycerol within the active site is mainly stabilized by van der Waals and electrostatic interactions with the benzyl ring of Phe245. Computer modeling suggests that the glycerol molecule is sandwiched by the Zn2+ and NAD+ ions. Based on this, the mechanism for the relaxed substrate specificity of this enzyme is also discussed.

Characterization of santalene synthases using an inorganic pyrophosphatase coupled colorimetric assay.

We developed a colorimetric assay using yeast inorganic pyrophosphatase (IPP1) as a coupling enzyme to measure the activities of terpene synthases. IPP1 hydrolyzes pyrophosphate, the byproduct of terpene synthase catalyzed reactions, into orthophosphate, which can then be quantitated by reacting with molybdic acid to form a blue color compound. As a proof of concept, this method was used to quantitatively characterize three santalene synthases, SaSSy and SspiSSy involved in sandalwood oil biosynthesis, and a phylogenetically distant SanSyn from Clausena lansium. Our study provided the kinetic parameters of all three santalene synthases and demonstrated the validity of the enzyme couple colorimetric assay by the comparison of this assay with the existing GC-MS (Gas Chromatography-Mass Spectrometry) method.