Crystallographic Evidence of Drastic Conformational Changes in the Active Site of a Flavin-Dependent

The soil actinomycete Kutzneria sp. 744 produces a class of highly decorated hexadepsipeptides, which represent a new chemical scaffold that has both antimicrobial and antifungal properties. These natural products, known as kutznerides, are created via nonribosomal peptide synthesis using various derivatized amino acids. The piperazic acid moiety contained in the kutzneride scaffold, which is vital for its antibiotic activity, has been shown to derive from the hydroxylated product of l-ornithine, l-N5-hydroxyornithine. The production of this hydroxylated species is catalyzed by the action of an FAD- and NAD(P)H-dependent N-hydroxylase known as KtzI. We have been able to structurally characterize KtzI in several states along its catalytic trajectory, and by pairing these snapshots with the biochemical and structural data already available for this enzyme class, we propose a structurally based reaction mechanism that includes novel conformational changes of both the protein backbone and the flavin cofactor. Further, we were able to recapitulate these conformational changes in the protein crystal, displaying their chemical competence. Our series of structures, with corroborating biochemical and spectroscopic data collected by us and others, affords mechanistic insight into this relatively new class of flavin-dependent hydroxylases and adds another layer to the complexity of flavoenzymes.National Center for Research Resources (U.S.) (P41RR012408)National Institute of General Medical Sciences (U.S.) (P41GM103473

Potential therapeutic applications of microbial surface-activecompounds

Numerous investigations of microbial surface-active compounds or biosurfactants over the past two decades have led to the discovery of many interesting physicochemical and biological properties including antimicrobial, anti-biofilm and therapeutic among many other pharmaceutical and medical applications. Microbial control and inhibition strategies involving the use of antibiotics are becoming continually challenged due to the emergence of resistant strains mostly embedded within biofilm formations that are difficult to eradicate. Different aspects of antimicrobial and anti-biofilm control are becoming issues of increasing importance in clinical, hygiene, therapeutic and other applications. Biosurfactants research has resulted in increasing interest into their ability to inhibit microbial activity and disperse microbial biofilms in addition to being mostly nontoxic and stable at extremes conditions. Some biosurfactants are now in use in clinical, food and environmental fields, whilst others remain under investigation and development. The dispersal properties of biosurfactants have been shown to rival that of conventional inhibitory agents against bacterial, fungal and yeast biofilms as well as viral membrane structures. This presents them as potential candidates for future uses in new generations of antimicrobial agents or as adjuvants to other antibiotics and use as preservatives for microbial suppression and eradication strategies

Reconstruction of the Saccharopolyspora erythraea genome-scale model and its use for enhancing erythromycin production

Genome-scale metabolic reconstructions are routinely used for the analysis and design of metabolic engineering strategies for production of primary metabolites. The use of such reconstructions for metabolic engineering of antibiotic production is not common due to the lack of simple design algorithms in the absence of a cellular growth objective function. Here, we present the metabolic network reconstruction for the erythromycin producer Saccharopolyspora erythraea NRRL23338. The model was manually curated for primary and secondary metabolism pathways and consists of 1,482 reactions (2,075 genes) and 1,646 metabolites. As part of the model validation, we explored the potential benefits of supplying amino acids and identified five amino acids "compatible" with erythromycin production, whereby if glucose is supplemented with this amino acid on a carbon mole basis, the in silico model predicts that high erythromycin yield is possible without lowering biomass yield. Increased erythromycin titre was confirmed for four of the five amino acids, namely valine, isoleucine, threonine and proline. In bioreactor experiments, supplementation with 2.5 % carbon mole of valine increased the growth rate by 20 % and simultaneously the erythromycin yield on biomass by 50 %. The model presented here can be used as a framework for the future integration of high-throughput biological data sets in S. erythraea and ultimately to realise strain designs capable of increasing erythromycin production closer to the theoretical yield

Isolation and structure determination of new siderophore albachelin from Amycolatopsis alba

autho

Bactericidal synergy of lysostaphin in combination with antimicrobial peptides

Drug-resistant staphylococci constitute a serious problem that urgently requires the discovery of new therapeutic agents. There has been a resurgence in interest in using lysostaphin (a specific anti-staphylococcal enzyme) as a treatment for infections caused by these important pathogens. However, bacterial resistance to lysostaphin is a problem, but the use of a combination treatment may surmount this issue. In this present study, using viable counts from suspension incubations, lysostaphin is shown to be synergistically bactericidal in combination with various conventional antimicrobial peptides, the antimicrobial protein bovine lactoferrin, a lantibiotic (nisin), and certain lipopeptides used clinically (colistin, daptomycin and polymyxin B). Combinations that act in synergy are of clinical importance as these reduce the doses of the compounds needed for effective treatments and decrease the chances of resistance being selected. The use of lysostaphin in combination with a peptide may represent a new avenue in tackling drug-resistant staphylococci