The protective antigen component of anthrax toxin forms a homoheptameric pore in the endosomal membrane, creating a narrow passageway for the enzymatic components of the toxin to enter the cytosol. We found that, during conversion of the heptameric precursor to the pore, the seven phenylalanine-427 residues converged within the lumen, generating a radially symmetric heptad of solvent-exposed aromatic rings. This "φ-clamp" structure was required for protein translocation and comprised the major conductance-blocking site for hydrophobic drugs and model cations. We conclude that the φ clamp serves a chaperone-like function, interacting with hydrophobic sequences presented by the protein substrate as it unfolds during translocation.Anthrax toxin is composed of three nontoxic proteins, which combine on eukaryotic cell surfaces to form toxic, noncovalent complexes. [See (1) for a review.] Protective antigen (PA), the protein translocase component, binds to a cellular receptor and is activated by a furin-family protease. The resulting 63-kD receptor-bound fragment, PA 63 , self-assembles into the prepore, which is a ring-shaped homoheptamer (Fig. 1A). The prepore then forms complexes with the two ~90-kD enzymatic components, lethal factor (LF) and edema factor (EF). These complexes are endocytosed and delivered to an acidic compartment (2). There, the prepore undergoes an acidic pH-dependent conformational rearrangement (3) to form an ion-conducting, cationselective, transmembrane pore (4), allowing bound LF and EF to translocate into the cytosol.The PA 63 pore (Fig. 1B) is believed to consist of a mushroom-shaped structure, with a globular cap connected to a β-barrel stem that is ~100 Å long (5, 6). A model of the 14-strand β barrel reveals its lumen, which is ~15 Å wide and can only accommodate structure as wide as an α helix (7). The narrow pore creates a structural bottleneck, requiring that the catalytic factors, LF and EF, unfold in order to be translocated (8, 9). The destabilization energy required to unfold the tertiary structure of LF and EF originates partly from the acidic pH in endosomes, which causes their N-terminal domains (LF N and EF N ) to become molten globules (MG) (7). A positive membrane potential [+Δψ (10)], when coupled with these acidic pH conditions, is sufficient to drive LF N through PA 63 pores formed in planar lipid bilayers (9). To enter the narrow confines of the ~15-Å-wide lumen, LF N must shed its residual tertiary structure and convert from the MG form to an extended, "translocatable" conformation (7). How does a solvent-filled pore mediate the disassembly of an MG protein, packed, albeit loosely, with hydrophobically dense stretches of polypeptide? We surmised that an interaction surface inside the pore might facilitate further unfolding of the MG to the extended, translocatable form. †To whom correspondence should be addressed.
The plant kingdom provides a large resource of natural products and various related enzymes are analyzed. The high catalytic activity and easy genetically modification of microbial enzymes would be beneficial for synthesis of natural products. But the identification of functional genes of target enzymes is time consuming and hampered by many contingencies. The potential to mine microbe-derived glycosyltransferases (GTs) cross the plant kingdom was assessed based on alignment and evolution of the full sequences and key motifs of target enzymes, such as Rhodiola-derived UDP-glycosyltransferase (UGT73B6) using in salidroside synthesis. The GTs from Bacillus licheniformis ZSP01 with high PSPG motif similarity were speculated to catalyze the synthesis of salidroside. The UGTBL1, which had similarity (61.4%) PSPG motif to UGT73B6, displayed efficient activity and similar regioselectivity. Highly efficient glycosylation of tyrosol (1 g/L) was obtained by using engineered E. coli harboring UGTBL1 gene, which generated 1.04 g/L salidroside and 0.99 g/L icariside D2. All glycosides were secreted into the culture medium and beneficial for downstream purification. It was the first report on the genome mining of UGTs from microorganisms cross the plant kingdom. The mining approach may have broader applications in the selection of efficient candidate for making high-value natural products.
Uremic toxins are a class of toxins that accumulate in patients with chronic kidney disease (CKD). Indoxyl sulfate (IS), a typical uremic toxin, is not efficiently removed by hemodialysis. Modulation of IS production in the gut microbiota may be a promising strategy for decreasing IS concentration, thus, delaying CKD progression. In the present study, we identified isoquercitrin (ISO) as a natural product that can perturb microbiota-mediated indole production without directly inhibiting the growth of microbes or the indole-synthesizing enzyme TnaA. ISO inhibits the establishment of H proton potential by regulating the gut bacteria electron transport chain, thereby inhibiting the transport of tryptophan and further reducing indole biosynthesis. This non-microbiocidal mechanism may enable ISO to be used as a therapeutic tool, specifically against pathologies triggered by the accumulation of the microbial-produced toxin IS, as in CKD. Herein, we have shown that it is possible to inhibit gut microbial indole production using natural components. Therefore, targeting the uremic toxin metabolic pathway in gut bacteria may be a promising strategy to control host uremic toxin production.
Heptameric anthrax protective antigen (termed prepore), which assembles at the mammalian cell surface, competitively binds edema factor (EF) and/or lethal factor (LF). It then transports them to an acidic intracellular compartment and mediates their translocation across the membrane to the cytosol. Steric constraints limit to three the number of molecules of EF and/or LF that can bind simultaneously to prepore. To determine whether the number of ligand molecules bound per heptamer affects the efficiency of translocation, we measured the low-pH-triggered translocation of the radiolabeled protective antigen (PA(63))-binding domain of LF ((35)S-LF(N)) across the plasma membrane of CHO-K1 cells as a function of the degree of saturation of the prepore. The fraction translocated remained constant at approximately 0.4 as (35)S-LF(N) was varied from nil through saturating concentrations. The same constant value was observed when we held (35)S-LF(N) at a saturating concentration and varied the number of functional ligand sites per prepore by changing the ratio of wild-type PA to a ligand-binding mutant. Thus, prepore containing only a single ligand-binding site is capable of translocating its cargo as efficiently as one containing multiple binding sites. The results as a whole imply that heptamers with one, two, or three ligands bound translocate their ligands with the same efficiency, indicating that each ligand molecule is translocated independently from the others.
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