Terrequinone A biosynthesis through L-tryptophan oxidation, dimerization and bisprenylation

The antitumor fungal metabolite terrequinone A, identified in extracts of Aspergillus sp., is biosynthesized by the five-gene cluster tdiA-tdiE. In this work, we have overproduced all five proteins (TdiA-TdiE) in the bacterial host Escherichia coli, fully reconstituting the biosynthesis of terrequinone A. This pathway involves aminotransferase activity, head-to-tail dimerization and bisprenylation of the scaffold to yield the benzoquinone natural product. We have established that TdiD is a pyridoxal-5'-phosphate-dependent L-tryptophan aminotransferase that generates indolepyruvate for an unusual nonoxidative coupling by the tridomain nonribosomal peptide synthetase TdiA. TdiC, an NADH-dependent quinone reductase, generates the nucleophilic hydroquinone for two distinct rounds of prenylation by the single prenyltransferase TdiB. TdiE is required to shunt the benzoquinone away from an off-pathway monoprenylated species by an as yet unknown mechanism. Overall, we have biochemically characterized the complete biosynthetic pathway to terrequinone A, highlighting the nonoxidative dimerization pathway and the unique asymmetric prenylation involved in its maturation.     

Anemochory of diapausing stages of microinvertebrates in North American drylands

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Wind turbine noise limits propagation of greater prairie‐chicken boom chorus,but does it matter?

Acoustic signals are often critical elements of mating displays, and lekking male greater prairie‐chickens (Tympanuchus cupido pinnatus) use their boom vocalization for this purpose. We quantified the acoustic characteristics of the boom chorus created by multiple male greater prairie‐chickens vocalizing simultaneously at leks in Brown County, NE, USA, in 2013 and 2014. We used these data to evaluate (a) the role of the boom chorus in prairie‐chicken breeding dynamics and (b) the impact of a wind energy facility on the acoustic signal of the boom chorus. We sampled the chorus using audio recorders placed in transects extending from leks; the chorus exhibited an average peak frequency of 297 ± 13 Hz. The mean chorus signal‐to‐noise ratio declined from 15.7 dB (50 m) to 2.6 dB (800 m), and wind speed and direction, topography, and relative humidity caused variation in signal‐to‐noise ratio at a given distance and location. Chorus recordings from leks within 1,000 m of a wind turbine had lower signal‐to‐noise ratio (β_turbine = ?5.659, SE = 1.289) than leks farther from turbines. The chorus signal‐to‐noise ratio increased slightly with more males present on the lek (~0.1 dB for each additional male; β_males = 0.177; SE = 0.037) and considerably more as more females visited the lek (~1.4 dB for each additional female; β_females = 2.498, SE = 0.235; β_females² = ?0.309, SE = 0.039). Our results provide support for the signal enhancement hypothesis that proposes the boom chorus is influenced notably by male–male competition for females on the lek, rather than functioning solely to advertise the presence of the lek to recruit females. Our results also suggest the choruses emanating from small leks have the greatest potential to be masked by anthropogenic (wind turbine) noise, which may affect the breeding success of male and female prairie‐chickens.     

Isolation and characterization of rabbit gastrin

The heptadecapeptide form the rabbit gastrin was extracted from 16 rabbit antra and purified by a combination of DEAE Sephadex, C-18 SEP PAK cartridges, fast performance liquid chromatography (FPLC) and reverse phase high pressure liquid chromatography (HPLC) steps. After the HPLC purification, a sharp, single peak of gastrin-like immunoreactivity was detected that had the same absorption to immunoreactivity ratio as human gastrin. An amino terminal pyrrolidone carboxylic acid blocking group was removed by incubation with pyrrolidone carboxylic peptidase. The amino acid analysis, microsequence analysis and mass spectrometry all confirmed the structure of rabbit gastrin being pQGPWLQEEEEAYGWMDFamide. This sequence is identical to human gastrin-17 except for glutamine in position 6 which replaces glutamate in human gastrin. Both sulfated and unsulfated rabbit gastrin-17 were characterized by mass spectrometry.     

In vitro reconstitution of the Pseudomonas aeruginosa nonribosomal peptide synthesis of pyochelin: characterization of backbone tailoring thiazoline reductase and N-methyltransferase activities

During iron starvation the Gram-negative pathogenic bacterium Pseudomonas aeruginosa makes the nonribosomal peptide siderophore pyochelin by a four protein, 11 domain assembly line, involving a cascade of acyl-S-enzyme intermediates on the PchE and PchF subunits that are elongated, heterocyclized, reduced, and N-methylated before release. Purified PchG is shown to be an NADPH-dependent reductase for the hydroxyphenylbisthiazoline-S-PchF acyl enzyme, regiospecifically converting one of the dihydroheterocyclic thiazoline rings to a thiazolidine. The K(m) for the PchG protein is 1 microM, and the k(cat) for throughput to pyochelin is 2 min(-1). The nitrogen of the newly generated thiazolidine ring can be N-methylated upon addition of SAM, to yield the mature pyochelin chain still tethered as a pyochelinyl-S-PchF at the PCP domain. A presumed methyltransferase (MT) domain embedded in the PchF subunit catalyzes this N-methylation. Mutation of a conserved G to R in the MT core motif abolishes MT activity and subsequent chain release from PchF. The thioesterase (TE) domain of PchF catalyzes hydrolytic release of the fully mature pyochelinyl chain to produce the pyochelin siderophore at a rate of 2 min(-1), at least 30-40-fold faster than in the absence of hydroxyphenylbisthiazolinyl-COOH (HPTT-COOH) chain reduction and N-methylation. A mutation in the PchF TE domain does not catalyze autodeacylation and release of the pyochelinyl-S-enzyme. Thus, full reconstitution of the nonribosomal peptide synthetase assembly line by purified protein components has been obtained for production of this tandem bisheterocyclic siderophore.     

Differential activation of the Rac pathway by Ha-Ras and K-Ras

Ras proteins are key regulators of cell growth and differentiation. Mammalian cells express three closely related Ras proteins: Ha-Ras, K-Ras, and N-Ras. We have compared the abilities of the Ha-Ras and K-Ras isoforms to activate the Rac effector pathway, using three Rac-dependent readouts: induction of membrane ruffling and pinocytosis, stimulation of cell motility, and Pak binding. The total surface area of membrane ruffles induced by K-RasV12 was 2-fold greater than that induced by Ha-RasV12. Likewise, the number of K-RasV12-induced pinocytic vesicles per cell was approximately 2-fold greater than that induced by Ha-RasV12. In a wound healing assay, K-RasV12-injected cells migrated twice as fast as Ha-RasV12-injected cells. Moreover, the Pak binding activity of Rac, which is indicative of the amount of GTP-bound Rac, was higher in K-RasV12-expressing cells than Ha-RasV12-expressing cells. These results suggest that K-Ras activates Rac more efficiently than Ha-Ras. The preferential activation of Rac by K-Ras is dependent on the mode of membrane anchoring and impacts on the ability of K-Ras to regulate cell survival.     

Estimating the time to the most recent common ancestor for the Y chromosome or mitochondrial DNA for a pair of individuals

Bayesian posterior distributions are obtained for the time to the most recent common ancestor (MRCA) for a nonrecombining segment of DNA (such as the nonpseudoautosomal arm of the Y chromosome or the mitochondrial genome) for two individuals given that they match at k out of n scored markers. We argue that the distribution of the time t to the MRCA is the most natural measure of relatedness for such nonrecombining regions. Both an infinite-alleles (no recurring mutants) and stepwise mutation model are examined, and these agree well when n is moderate to large and k/n is close to one. As expected, the infinite alleles model underestimates t relative to the stepwise model. Using a modest number (20) of microsatellite markers is sufficient to obtain reasonably precise estimates of t for individuals separated by 200 or less generations. Hence, the multilocus haplotypes of two individuals can be used not only to date very deep ancestry but also rather recent ancestry as well. Finally, our results have forensic implications in that a complete match at all markers between a suspect and a sample excludes only a modest subset of the population unless a very large number of markers (>500 microsatellites) are used.