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Abstract: Human apical sodium-dependent bile acid transporter (ASBT) and Na+-dependent co-transporting polypeptide (NTCP) are secondary transporters from the SLC10 family. These 9- transmembrane-helix proteins play critical roles in enterohepatic recycling of bile acids. Here we solve the crystal structure of a bacterial homolog, also with nine transmembrane helices and high sequence homology to human ASBT. We report two different structures of the bacterial homolog in the inward-facing state and a humanized version in the outward-facing state, with and without bile acid bound. Structures of NTCP show a pore through the protein in violation of the classical alternating access mechanism. In our structures, the flexible TM6 seals this pore, showing that an outward-closed structure is not necessarily precluded in this 9-transmembrane-helix protein family. Molecular dynamics simulations of bacterial and human proteins highlight that while the bile acid substrate is anchored to residues at the center of the transporter, lipids from the surrounding membrane interact with the hydrophobic sterol group.

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Abstract: Toxin–antitoxin (TA) systems use diverse strategies to control bacterial growth and represent attractive therapeutic targets to fight pathogens. Mycobacterium tuberculosis, the bacterium responsible for human tuberculosis, encodes one of the largest repertoires of TA systems. Here, we applied a bioinformatic pipeline to predict candidate TA systems in mycobacterial genomes and identified Rv2663–Rv2664 (RelS–RelI) as a previously undetected system in M. tuberculosis. We show that the RelS toxin is highly toxic and is inhibited by a unique antitoxin, RelI. The 1.70 Å X-ray crystallographic structure of RelS:RelI shows an unprecedented heterooctameric quaternary TA complex formed by paired tetramers. In each tetramer, RelS toxins are held at each end of a RelI antitoxin dimer. RelI binds across the putative catalytic center of RelS, resulting in occlusion of essential putative target-binding residues. Investigation of the toxic mechanism revealed that RelS is an atypical RelE/ParE-like RNase toxin that inhibits translation by targeting the 30S ribosomal subunit, specifically cleaving the 16S ribosomal RNA between positions C1520 and U1521, a unique site within the anti-Shine–Dalgarno (anti-SD) core region. This work further highlights the anti-SD region as a hot spot for RNase toxins and extends the arsenal of TA systems harnessed by this major pathogen.

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Abstract: Copper-containing nitrite reductases (CuNiRs) catalyse the reduction of nitrite to nitric oxide and are a key enzyme in the anaerobic ammonium oxidation and denitrification steps of the nitro­gen cycle. The recent recognition of the widespread distribution of three-domain CuNiRs where cognate redox partners are fused to the core NiR enzyme offered the possibility of studying coordinated events (e.g. proton-coupled electron transfer) in a conformationally stable donor–acceptor complex. The C-terminal cytochrome c tethered domain of the CuNiR from Ralstonia pickettii (RpNiR) has been well studied. Reverse engineering of RpNiR undertaken to remove the cognate partner domain showed that the presence of the additional domain resulted in significant differences in the apparent Km for nitrite and the reduction potentials of the Cu centres when compared with the core enzyme. The oxidation state of the haem centre and the position of the tethering linker have also been shown to control access of substrate to the active site. A key feature of this control is a conserved tyrosine residue (Tyr323 in RpNiR) located in the tethering linker between the fused domain and the core enzyme. To gain insight into this control, we have undertaken targeted mutations of RpNiR to probe the so-called primary proton channel and perturb putative electron transfer routes from the haem to the `gatekeeper' Tyr323 and to the T1Cu centre. The resolution of our crystallographic data to better than 1.2 Å enabled us to apply unrestrained SHELXL refinement of the structures. Our data provide a significant advance in our understanding of catalysis and modulation of electron transfer in these tethered systems, with wider implications for these fundamental processes in other protein complexes.

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Abstract: Excessive foraging of colonic mucin glycans by gut bacteria is associated with diseases such as inflammatory bowel disease. Although Akkermansia muciniphila is an important mucin degrader, the role of carbohydrate sulfatases that facilitate digestion of these heavily sulfated glycans remains unclear. Combining in vitro digestion assays, proteomics and structural biology, we show that A. muciniphila sulfatases, such as Amuc1755 and Amuc0953, have rare adaptations targeted towards known sulfated mucin structures. They show larger degrees of modularity, including a previously unknown mucin-binding domain. When grown on colonic mucin substrates, glycoproteins of reduced size were important for the growth of A. muciniphila. Further mutational analysis and localization studies revealed that desulfation of N-acetyl-d-glucosamine was periplasmic, while desulfation of d-galactose occurred extracellularly and in the periplasm. These data improve our understanding of contexts for the positive health correlations of A. muciniphila while metabolizing colonic mucin as its sole carbon source.

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Abstract: Chlorothricin is a polyketide-derived natural product isolated from Streptomyces antibioticus. It possesses an elaborate pentacyclic aglycone core which incorporates a spirotetronic acid moiety, linked to a trans-decalin system, embedded within a macrocycle. Using synthetic substrate analogues and purified recombinant proteins, here we demonstrate that assembly of this scaffold proceeds via sequential biocatalytic Diels–Alder reactions, promoted by the enzymes ChlE3 and ChlL. Both Diels–Alderases exhibit sufficiently relaxed substrate selectivity to facilitate access to non-natural chlorothricin analogues via biotransformations. The X-ray crystal structure of ChlE3 reveals the molecular basis of decalin formation by this enzyme. Harnessing this enzymatic cascade in biocatalysis could provide a valuable biomimetic route to both natural and non-natural spirotetronates, and the work described herein lays the foundation for application of these enzymes in chemoenzymatic syntheses of complex products.