The enterococcal cytolysin is a virulence factor consisting of two post-translationally modified peptides that synergistically kill human immune cells. Both peptides are made by CylM, a member of the LanM lanthipeptide synthetases. CylM catalyzes seven dehydrations of Ser and Thr residues and three cyclization reactions during the biosynthesis of the cytolysin large subunit. We present here the 2.2 Å resolution structure of CylM, the first structural information on a LanM. Unexpectedly, the structure reveals that the dehydratase domain of CylM resembles the catalytic core of eukaryotic lipid kinases, despite the absence of clear sequence homology. The kinase and phosphate elimination active sites that affect net dehydration are immediately adjacent to each other. Characterization of mutants provided insights into the mechanism of the dehydration process. The structure is also of interest because of the interactions of human homologs of lanthipeptide cyclases with kinases such as mammalian target of rapamycin.DOI: http://dx.doi.org/10.7554/eLife.07607.001

Although the pimeloyl moiety was long known to be a biotin precursor, the mechanism of assembly of this C7 α,ω-dicarboxylic acid was only recently elucidated. In Escherichia coli, pimelate is made by bypassing the strict specificity of the fatty acid synthetic pathway. BioC methylates the free carboxyl of a malonyl thioester, which replaces the usual acetyl thioester primer. This atypical primer is transformed to pimeloyl-acyl carrier protein (ACP) methyl ester by two cycles of fatty acid synthesis. The question is, what stops this product from undergoing further elongation? Although BioH readily cleaves this product in vitro, the enzyme is nonspecific, which made assignment of its physiological substrate problematical, especially because another enzyme, BioF, could also perform this gatekeeping function. We report the 2.05-Å resolution cocrystal structure of a complex of BioH with pimeloyl-ACP methyl ester and use the structure to demonstrate that BioH is the gatekeeper and its physiological substrate is pimeloyl-ACP methyl ester.cofactor biosynthesis | esterase | protein-protein interaction R ecent work delineated the assembly pathway of the enigmatic pimeloyl moiety of biotin. Labeling studies in Escherichia coli had shown that pimelate, a C7 α,ω-dicarboxylic acid, is made by head-to-tail incorporation of three intact acetate units with one of the carboxyl groups being derived from CO 2 (1, 2). The differing origins of the carboxyl groups indicated that free pimelate was not a synthetic intermediate. The acetate incorporation pattern was consistent with use of the synthetic pathway that produces the usual monocarboxylic fatty acids. However, synthesis of a dicarboxylic acid using the fatty acid synthetic pathway appeared precluded by the strongly hydrophobic active sites of the fatty acid synthetic enzymes (3), which seemed unlikely to tolerate the charged carboxyl group in place of the usual terminal methyl group. The solution to this conundrum was provided by the characterization of two enzymes, BioC and BioH, which do not directly catalyze pimelate synthesis but instead allow fatty acid synthesis to assemble the pimelate moiety (4). Such circumvention of the specificity of normal fatty acid synthesis begins by BioC (an Omethyltransferase) conversion of the ω-carboxyl group of malonylacyl carrier protein (ACP) to a methyl ester using S-adenosyl-Lmethionine (SAM) as a methyl donor (Fig. 1). Conversion to a methyl ester neutralizes the negative charge and provides a methyl carbon that mimics the methyl ends of normal fatty acyl chains. The malonyl-ACP methyl ester can now enter the fatty acid synthetic pathway where it is condensed with malonyl-ACP by a 3-oxoacyl-ACP synthase in a decarboxylating Claisen reaction to give 3-oxoglutaryl-ACP methyl ester. The methyl ester shielding allows the 3-oxo group to be processed to a methylene group by the standard fatty acid reductase-dehydratase-reductase reaction sequence. The resulting glutaryl-ACP methyl ester would then be elongated to the C7 species, and another r...

Laccases (EC 1.10.3.2) are multicopper oxidases that can oxidize a range of substrates, including phenols, aromatic amines, and nonphenolic substrates. To investigate the involvement of the small Streptomyces laccases in lignin degradation, we generated acid-precipitable polymeric lignin obtained in the presence of wild-type Streptomyces coelicolor A3(2) (SCWT) and its laccase-less mutant (SCΔLAC) in the presence of Miscanthus x giganteus lignocellulose. The results showed that strain SCΔLAC was inefficient in degrading lignin compared to strain SCWT, thereby supporting the importance of laccase for lignin degradation by S. coelicolor A3(2). We also studied the lignin degradation activity of laccases from S. coelicolor A3(2), Streptomyces lividans TK24, Streptomyces viridosporus T7A, and Amycolatopsis sp. 75iv2 using both lignin model compounds and ethanosolv lignin. All four laccases degraded a phenolic model compound (LM-OH) but were able to oxidize a nonphenolic model compound only in the presence of redox mediators. Their activities are highest at pH 8.0 with a low krel/Kapp for LM-OH, suggesting that the enzymes’ natural substrates must be different in shape or chemical nature. Crystal structures of the laccases from S. viridosporus T7A (SVLAC) and Amycolatopsis sp. 75iv2 were determined both with and without bound substrate. This is the first report of a crystal structure for any laccase bound to a nonphenolic β-O-4 lignin model compound. An additional zinc metal binding site in SVLAC was also identified. The ability to oxidize and/or rearrange ethanosolv lignin provides further evidence of the utility of laccase activity for lignin degradation and/or modification.

The determination of structural models of the various stable states of an ion channel is a key step toward the characterization of its conformational dynamics. In the case of nicotinic-type receptors, different structures have been solved but, thus far, these different models have been obtained from different members of the superfamily. In the case of the bacterial member ELIC, a cysteamine-gated channel from Erwinia chrisanthemi, a structural model of the protein in the absence of activating ligand (and thus, conceivably corresponding to the closed state of this channel) has been previously generated. In this article, electrophysiological characterization of ELIC mutants allowed us to identify pore mutations that slow down the time course of desensitization to the extent that the channel seems not to desensitize at all for the duration of the agonist applications (>20 min). Thus, it seems reasonable to conclude that the probability of ELIC occupying the closed state is much lower for the ligand-bound mutants than for the unliganded wild-type channel. To gain insight into the conformation adopted by ELIC under these conditions, we solved the crystal structures of two of these mutants in the presence of a concentration of cysteamine that elicits an intracluster open probability of >0.9. Curiously, the obtained structural models turned out to be nearly indistinguishable from the model of the wild-type channel in the absence of bound agonist. Overall, our findings bring to light the limited power of functional studies in intact membranes when it comes to inferring the functional state of a channel in a crystal, at least in the case of the nicotinicreceptor superfamily.electrophysiology | structure-function T he use of bacterial and archaeal ion channels as models of the structure and function of their eukaryotic counterparts has become one of the mainstays of ion-channel biophysics. It seems to us that this practice is particularly well justified whenever the use of noneukaryotic channels facilitates experiments that are otherwise too difficult or too cumbersome to perform. A clear case in point is the determination of structural models of these membrane proteins using X-ray crystallography.One of the main goals of direct structural approaches as applied to ion channels is to determine what these proteins look like in their different functional states. Assuming that all of these conformations can form well-diffracting crystals, the problem is reduced to finding the conditions under which the occupancy of each particular state is maximized. In general, for well-characterized ion channels, the experimental maneuvers that are needed to favor or disfavor at least some of these different conformations are known. For example, in the case of (wild-type) neurotransmitter-gated channels, the binding of the natural neurotransmitter strongly stabilizes the desensitized state (e.g., ref. 1), whereas the absence of neurotransmitter or the binding of a competitive antagonist keeps the channel mostly closed (2, 3). Similarly, mut...

Human monocyte differentiation antigen CD14 is a pattern recognition receptor that enhances innate immune responses to infection by sensitizing host cells to bacterial lipopolysaccharide (LPS; endotoxin), lipoproteins, lipoteichoic acid and other acylated microbial products. CD14 physically delivers these lipidated microbial products to various Toll-like receptor signaling complexes that subsequently induce intracellular proinflammatory signaling cascades upon ligand binding. The ensuing cellular responses are usually protective to the host, but can also result in host fatality through sepsis. In this work, we have determined the X-ray crystal structure of human CD14. The structure reveals a bent solenoid typical of leucine rich repeat proteins with an amino terminal pocket that presumably binds acylated ligands including LPS. Comparison of human and mouse CD14 structures show great similarity in overall protein fold. However, compared to mouse CD14, human CD14 contains an expanded pocket and alternative rim residues that are likely to be important for LPS binding and cell activation. The X-ray crystal structure of human CD14 presented herein may foster additional ligand bound structural studies, virtual docking studies, and drug design efforts to mitigate LPS induced sepsis and other inflammatory diseases.