Search

Type IV pilins are proteins which form polymers that extend from the surface of the bacterial cell; they are involved in mediating a wide variety of functions, including adhesion, motility and natural competence. Here we describe the determination of the crystal structures of three type IVa pilins proteins from the thermophile Thermus thermophilus. They form part of a cluster of pilus-like proteins within the genome; our results show that one, Tt1222, is very closely related to the main structural type IV pilin, PilA4. The other two, Tt1218 and Tt1219, also adopt canonical pilin-like folds but, interestingly, are most closely related to the structures of the type II secretion system pseudopilins, EpsI/GspI and XcpW/GspJ. GspI and GspJ have been shown to form a complex with another pseudopilin, GspK, and this heterotrimeric complex is known to play a key role in initiating assembly of a pseudopilus which is thought to drive the secretion process. The structural similarity of Tt1218 and Tt1219 to GspI and GspJ suggests that they might work in a similar way, to deliver functions associated with type IV pili in T. thermophilus, such as natural competence.

Natural competence is the term used to describe the uptake of “naked” extracellular DNA by bacteria; it plays a significant role in horizontal genetic exchange. It is associated with type IV pili, and specialized competence pili mediate DNA uptake. Here, we show that the crystal structure of a competence-associated protein from Thermus thermophilus, ComZ, consists of a type II secretion pseudopilin-like domain, with a large β-solenoid domain inserted into the β-sheet of the pilin-like fold. ComZ binds with high affinity to another competence-associated pilin, PilA2, which lies adjacent to the comZ gene in the genome. The crystal structure of PilA2 revealed a similar type II secretion pseudopilin-like fold, with a small subdomain; docking simulations predicted that PilA2 binds between the pseudopilin-like and β-solenoid domains of ComZ. Electrophoretic shift analysis and DNase protection studies were used to show that ComZ alone and the ComZ/PilA2 complex are able to bind DNA. Protection against reductive dimethylation was used in combination with mass spectrometry and site-directed mutagenesis to identify two lysine residues in ComZ which are involved in DNA binding. They are located between the two domains in ComZ, on the opposite side from the predicted PilA2 binding site. These results suggest a model in which PilA2 assists ComZ in forming the competence pilus tip and DNA binds to the side of the fiber. The results demonstrate how a type IV pilin can be adapted to a specific function by domain insertion and provide the first structural insights into a tip-located competence pilin.

Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS), and we show that these are active biocatalysts for monoterpene production using biocatalysis and metabolic engineering platforms. In metabolically engineered monoterpene-producing E. coli strains, use of bLinS leads to 300-fold higher linalool production compared with the corresponding plant monoterpene synthase. With bCinS, 1,8-cineole is produced with 96% purity compared to 67% from plant species. Structures of bLinS and bCinS, and their complexes with fluorinated substrate analogues, show that these bacterial monoterpene synthases are similar to previously characterized sesquiterpene synthases. Molecular dynamics simulations suggest that these monoterpene synthases do not undergo large-scale conformational changes during the reaction cycle, making them attractive targets for structured-based protein engineering to expand the catalytic scope of these enzymes toward alternative monoterpene scaffolds. Comparison of the bLinS and bCinS structures indicates how their active sites steer reactive carbocation intermediates to the desired acyclic linalool (bLinS) or bicyclic 1,8-cineole (bCinS) products. The work reported here provides the analysis of structures for this important class of monoterpene synthase. This should now guide exploitation of the bacterial enzymes as gateway biocatalysts for the production of other monoterpenes and monoterpenoids.

Reduction of double bonds of α,β-­‐unsaturated carboxylic acids and esters by ene-­‐reductases remains challenging and it typically requires activation by a second electron-­‐withdrawing moiety, such as a halide or second carboxylate group. We showed that profen precursors, 2-­‐arylpropenoic acids and their esters, were efficiently reduced by Old Yellow Enzymes (OYEs). The XenA and GYE enzymes showed activity towards acids, while a wider range of enzymes were active towards the equivalent methyl esters. Comparative co-­‐crystal structural analysis of profen-­‐bound OYEs highlighted key interactions important in determining substrate binding in a catalytically active conformation. The general utility of ene reductases for the synthesis of (R)-­‐profens was establishedand this work will now drive future mutagenesis studies to screen for the production of pharmaceutically-­‐active (S)-­‐profens.

Type IV pili are expressed from a wide variety of Gram-negative bacteria and play a major role in host cell adhesion and bacterial motility. PilC is one of at least a dozen different proteins that are implicated in Type IV pilus assembly in Thermus thermophilus and a member of a conserved family of integral inner membrane proteins which are components of the Type II secretion system (GspF) and the archeal flagellum. PilC/ GspF family members contain repeats of a conserved helix-rich domain of around 100 residues in length. Here, we describe the crystal structure of one of these domains, derived from the N-terminal domain of Thermus thermophilus PilC. The N-domain forms a dimer, adopting a six helix bundle structure with an up-down-up-down-updown topology. The monomers are related by a rotation of 170 °, followed by a translation along the axis of the final α-helix of approximately one helical turn. This means that the regions of contact on helices 5 and 6 in each monomer are overlapping, but different. Contact between the two monomers is mediated by a network of hydrophobic residues which are highly conserved in PilC homologs from other Gram-negative bacteria. Site-directed mutagenesis of residues at the dimer interface resulted in a change in oligomeric state of PilC from tetramers to dimers, providing evidence that this interface is also found in the intact membrane protein and suggesting that it is important to its function. © 2010 Wiley-Liss, Inc.