Complex carbapenems are important clinical antibiotics used to treat recalcitrant infections. Their biosynthetic gene clusters contain three essential B 12 -dependent radical S -adenosylmethionine (rSAM) enzymes. The majority of characterized enzymes in this subfamily catalyze methyl transfer, but only one is required to sequentially install all methionine-derived carbons in complex carbapenems. Therefore, it is probable that the other two rSAM enzymes have noncanonical functions. Through a series of fermentation and in vitro experiments, we show that ThnL uses radical SAM chemistry to catalyze thioether bond formation between C2 of a carbapenam precursor and pantetheine, uniting initial bicycle assembly common to all carbapenems with later tailoring events unique to complex carbapenems. ThnL also catalyzes reversible thiol/disulfide redox on pantetheine. Neither of these functions has been observed previously in a B 12 -dependent radical SAM enzyme. ThnL expands the known activity of this subclass of enzymes beyond carbon–carbon bond formation or rearrangement. It is also the only radical SAM enzyme currently known to catalyze carbon–sulfur bond formation with only an rSAM Fe–S cluster and no additional auxiliary clusters.

While bioinformatic evidence of cobalamin-dependent radical S-adenosylmethionine (SAM) enzymes has existed since the naming of the radical SAM superfamily in 2001, none were biochemically characterized until 2011. In the past decade, the field has flourished as methodological advances have facilitated study of the subfamily. Because of the ingenuity and perseverance of researchers in this field, we now have functional, mechanistic, and structural insight into how this class of enzymes harnesses the power of both the cobalamin and radical SAM cofactors to achieve catalysis. All of the early characterized enzymes in this subfamily were methylases, but the activity of these enzymes has recently been expanded beyond methylation. We anticipate that the characterized functions of these enzymes will become both better understood and increasingly diverse with continued study.

Tumor cell invasion is one result of the bidirectional interactions occurring between tumor cells and the surrounding milieu. The ability of tumor cells to invade through the extracellular matrix is in part regulated by the formation of a class of protease-loaded extracellular vesicles, called tumor microvesicles (TMVs), which are released directly from the cell surface. Here we show that the actin bundling protein, fascin, redistributes to the cell periphery in a ternary complex with podocalyxin and ezrin, where it promotes TMV release. The peripheral localization of fascin is prompted by the loss of Rab35 signaling, which in turn unleashes ARF6 activation. The result is a mechanism through which Rab35 and ARF6 cooperatively and simultaneously regulate the distribution and localization of fascin and promote oncogenic signaling, which leads to TMV release while inhibiting invadopodium formation. These studies are clinically significant as fascin-loaded TMVs can be detected in bodily fluids and elevated fascin expression coupled with low Rab35 levels correlates with poor overall survival in some cancers.

Complex carbapenem β-lactam antibiotics contain diverse C6 alkyl substituents constructed by cobalamin-dependent radical SAM enzymes. TokK installs the C6 isopropyl chain found in asparenomycin. Time-course analyses of TokK and its ortholog ThnK, which forms the C6 ethyl chain of thienamycin, indicate that catalysis occurs through a sequence of discrete, non-processive methyl transfers.

Lytic transglycosylases (LTs) catalyze the non-hydrolytic cleavage of the bacterial cell wall by an intramolecular transacetalization reaction. This reaction is critically and broadly important in modifications of the bacterial cell wall in the course of its biosynthesis, recycling, manifestation of virulence, insertion of structural entities such as the flagellum and the pili, among others. The first QM/MM analysis of the mechanism of reaction of an LT, that for the Escherichia coli MltE, is undertaken. The study reveals a conformational itinerary consistent with an oxocarbenium-like transition state, characterized by a pivotal role for the active-site glutamic acid in proton transfer. Notably, an oxazolinium intermediate, as a potential intermediate, is absent. Rather, substrate-assisted catalysis is observed through a favorable dipole provided by the N-acetyl carbonyl group of MurNAc saccharide. This interaction stabilizes the incipient positive charge development in the transition state. This mechanism coincides with near-synchronous acetal cleavage and acetal formation.

Lytic transglycosylases (LTs) are bacterial enzymes that catalyze the cleavage of the glycan strands of the bacterial cell wall. The mechanism of this cleavage is a remarkable intramolecular transacetalization reaction, accomplished by an ensemble of active-site residues. Because the LT reaction occurs in parallel with the cell wall bond-forming reactions catalyzed by the penicillin-binding proteins, simultaneous inhibition of both enzymes can be particularly bactericidal to Gram-negative bacteria. The MltE lytic transglycosylase is the smallest of the eight LTs encoded by the Escherichia coli genome. Prior crystallographic and computational studies identified four active-site residues-E64, S73, S75, and Y192-as playing roles in catalysis. Each of these four residues was individually altered by mutation to give four variant enzymes (E64Q, S73A, S75A, and Y192F). All four variants showed reduced catalytic activity [soluble wild type (100%) > soluble Y192F and S75A (both 40%) > S73A (4%) > E64Q (≤1%)]. The crystal structure of each variant protein was determined at the resolution of 2.12 Å for E64Q, 2.33 Å for Y192F, 1.38 Å for S73A, and 1.35 Å for S75A. These variants show alteration of the hydrogen-bond interactions of the active site. Within the framework of a prior computational study of the LT mechanism, we suggest the mechanistic role of these four active-site residues in MltE catalysis.

The lytic transglycosylases (LTs) are bacterial enzymes that catalyze the non-hydrolytic cleavage of the peptidoglycan structures of the bacterial cell wall. They are not catalysts of glycan synthesis as might be surmised from their name. Notwithstanding the seemingly mundane reaction catalyzed by the LTs, their lytic reactions serve bacteria for a series of astonishingly diverse purposes. These purposes include cell-wall synthesis, remodeling, and degradation; for the detection of cell-wall-acting antibiotics; for the expression of the mechanism of cell-wall-acting antibiotics; for the insertion of secretion systems and flagellar assemblies into the cell wall; as a virulence mechanism during infection by certain Gram-negative bacteria; and in the sporulation and germination of Gram-positive spores. Significant advances in the mechanistic understanding of each of these processes have coincided with the successive discovery of new LTs structures. In this review, we provide a systematic perspective on what is known on the structure-function correlations for the LTs, while simultaneously identifying numerous opportunities for the future study of these enigmatic enzymes.

We have produced draft whole-genome sequences for two bacterial strains reported to produce the bulgecins as well as NRPS-derived monobactam β-lactam antibiotics. We propose classification of ATCC 31363 as Paraburkholderia acidophila. We further reaffirm that ATCC 31433 (Burkholderia ubonensis subsp. mesacidophila) is a taxonomically distinct producer of bulgecins with notable gene regions shared with Paraburkholderia acidophila. We use RAST multiple-gene comparison and MASH distancing with published genomes to order the draft contigs and identify unique gene regions for characterization. Forty-eight natural-product gene clusters are presented from PATRIC (RASTtk) and antiSMASH annotations. We present evidence that the 10 genes that follow the sulfazecin and isosulfazecin pathways in both species are likely involved in bulgecin A biosynthesis.

Significance The antibacterial effectiveness of penicillins and cephalosporins is increasingly challenged by bacterial resistance, highlighting the increasing clinical importance of the carbapenems as the most potent, broad-spectrum class of β-lactam antibiotics. The carbapenems are produced commercially by total synthesis, rather than by lower-cost fermentation methods. The development of chemoenzymatic or semisynthetic means of manufacture requires elucidation of at least four central, undefined steps in the biosynthesis of thienamycin. The cobalamin-dependent radical S -adenosylmethionine (RS) superfamily enzyme ThnK performs two sequential methyl transfers in a stereodefined manner. This enzyme activity identifies critical intermediates in carbapenem biosynthesis, more clearly defining the enigmatic central steps of the pathway and exemplifying an unprecedented reaction sequence among RS enzymes.

A phenomenological mixture model is presented for interactions between biosynthesis of extracellular matrix (ECM) constituents and ECM linking in a scaffold seeded with chondrocytes. A system of three ordinary differential equations for average apparent densities of unlinked ECM, linked ECM and scaffold is developed along with associated initial conditions for scaffold material properties. Equations for unlinked ECM synthesis and ECM linking include an inhibitory mechanism where associated rates decrease as unlinked ECM concentration in the interstitial fluid increases. Linking rates are proposed to depend on average porosity in the evolving tissue construct. The resulting initial value problem contains nine independent parameters that account for scaffold biomaterial properties and interacting mechanisms in the engineered system. Effects of parameter variations on model variables are analyzed relative to a baseline case with emphasis on the evolution of solid phase apparent density, which is often correlated with the compressive elastic modulus of the tissue construct. The new model provides an additional quantitative framework for assessing and optimizing the design of engineered cell-scaffold systems and guiding strategies for articular cartilage tissue engineering.

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Positive behavioral responses to 2,4-dichlorophenol, 2,6-dichlorophenol, methyl salicylate and squalene individually are reported for Rhipicephalus sanguineus (Latreille), domestic pest and vector of Rocky Mountain spotted fever. Phenolics acted as attractants by recruiting 60-67% of ticks and squalene acted as an arrestant by causing up to 77% of ticks to cluster on treated surfaces in short-range petri dish bioassays. Compound concentrations were consistently most effective at 0.01M and 0.05M without a dose-response. Other known tick attractants 2,4,6-trichlorophenol and o-nitrophenol, however, did not elicit a response. Of interest is that squalene elicited a different set of behavioral responses (arrestment instead of attraction) and more heightened activity than did the phenolics.