Facile Production of the Pseudomonas aeruginosa Virulence Factor LasB in Escherichia coli for Structure-Based Drug Design.

The human pathogen Pseudomonas aeruginosa has a number of virulence factors at its disposal that play crucial roles in the progression of infection. LasB is one of the major virulence factors and exerts its effects through elastolytic and proteolytic activities aimed at dissolving connective tissue and inactivating host defense proteins. LasB is of great interest for the development of novel pathoblockers to temper the virulence, but access has thus far largely been limited to protein isolated from Pseudomonas cultures. Here, we describe a new protocol for high-level production of native LasB in Escherichia coli. We demonstrate that this facile approach is suitable for the production of mutant, thus far inaccessible LasB variants, and characterize the proteins biochemically and structurally. We expect that easy access to LasB will accelerate the development of inhibitors for this important virulence factor.

Novel Biosynthetic Route to the Isoquinoline Scaffold.

Isoquinoline alkaloids are a large class of natural products with a broad range of biological activities, including antimicrobial, antitumor, antileukemic and anti-inflammatory properties. Although mostly found in plants, isoquinolines can also be found in the extracts of bacterial and fungal cultures. Regardless of the origin, most of the reported biosynthetic routes for isoquinolines use tyrosine as a main biosynthetic precursor. Here, we report the identification of a new biosynthetic pathway for production of isoquinolinequinone alkaloid mansouramycin D in Streptomyces albus Del14. Using feeding, mass spectrometry, and nuclear magnetic resonance spectroscopy, we demonstrate that tryptophan serves instead of tyrosine as a main mansouramycin biosynthetic precursor. The biosynthetic genes were identified in the chromosome of the strain by using gene inactivation and heterologous expression. Insights into the biosynthesis of mansouramycins are also presented.

A Promiscuous Halogenase for the Derivatization of Flavonoids.

Halogenation often improves the bioactive properties of natural products and is used in pharmaceutical research for the generation of new potential drug leads. High regio- and stereospecificity, simple reaction conditions and straightforward downstream processing are the main advantages of halogenation using enzymatic biocatalysts compared to chemical synthetic approaches. The identification of new promiscuous halogenases for the modification of various natural products is of great interest in modern drug discovery. In this paper, we report the identification of a new promiscuous FAD-dependent halogenase, DklH, from Frankia alni ACN14a. The identified halogenase readily modifies various flavonoid compounds, including those with well-studied biological activities. This halogenase has been demonstrated to modify not only flavones and isoflavones, but also flavonols, flavanones and flavanonols. The structural requirements for DklH substrate recognition were determined using a feeding approach. The homology model of DklH and the mechanism of substrate recognition are also proposed in this paper.