The broad and vague phenotypic definition allowed the genus Pseudomonas to become a dumping ground for incompletely characterized polarly flagellated, Gram-negative, rod-shaped, aerobic bacteria, and a large number of species have been accommodated in the genus Pseudomonas. The 16S rRNA sequences of 128 valid and invalid Pseudomonas species, which included almost valid species of the genus Pseudomonas listed in the Approved Lists of Bacterial Names, were obtained : sequences of 59 species were determined and those of 69 species were obtained from the GenBank/EMBL/DD BJ databases. These sequences were compared with the sequences of other species of the Proteobacteria. Fifty-seven valid or invalid species including Pseudomonas aeruginosa (type species of the genus Pseudomonas Migula 1894) belonged to the genus Pseudomonas (sensu stricto). Seven subclusters were formed in the cluster of the genus Pseudomonas (sensu stricto), and the resulting clusters conformed well to the rRNA-DNA hybridization study by Palleroni (1984). The other species did not belong to the genus Pseudomonas (sensu stricto) and were related to other genera, which were placed in four subclasses of the

The 16s rRNA sequences of Chryseomonas Zuteola, the type species of the genus Chryseomonas, and Flavimonas oryzihabitans, the type species of the genus Flavimonas, were determined. These sequences were compared with the sequences of 27 representative strains of the genus Pseudomonus. C. Zuteola and F. oryzihabitans were located in the cluster that contains Pseudomonas aemginosa, the type species of genus Pseudomonas Migula 1894, and the levels of 16s rRNA sequence homology between P. aemginosa and the other two species were more than 93.9%. All of the strains of the genus Pseudomonas sensu stricto whose sequences have been determined were included in the P. aemginosa cluster. These results suggested that Chryseomonas, Flavimonas, and Pseudomonas are synonymous, and we concluded that Chryseomonas and Flavimonas are junior subjective synonyms of Pseudomonas.

Elucidation of natural product biosynthetic pathways provides important insights about the assembly of potent bioactive molecules, and expands access to unique enzymes able to selectively modify complex substrates. Here we show full reconstitution in vitro of an unusual multi-step oxidative cascade for post-assembly line tailoring of tirandamycin antibiotics. This pathway involves a remarkably versatile and iterative cytochrome P450 monooxygenase (TamI) and an FAD-dependent oxidase (TamL), which act co-dependently through repeated exchange of substrates. TamI hydroxylates tirandamycin C (TirC) to generate tirandamycin E (TirE), a heretofore unidentified tirandamycin intermediate. TirE is subsequently oxidized by TamL, giving rise to the ketone of tirandamycin D (TirD), after which a unique exchange back to TamI enables successive epoxidation and hydroxylation to afford, respectively, the final products tirandamycin A (TirA) and tirandamycin B (TirB). Ligand-free, substrate- and product-bound crystal structures of bicovalently flavinylated TamL oxidase reveal a likely mechanism for the C-10 oxidation of TirE.

The hierarchical system of the 'Alphaproteobacteria': description of Hyphomonadaceae fam. nov., Xanthobacteraceae fam. nov. and Erythrobacteraceae fam. nov.

SUMMARY Macrolide antibiotics are a class of valuable anti-infective agents that include a macrolactone ring, at least one appended sugar unit, and in most cases, additional functionalization in the form of hydroxyl and/or epoxide groups. There is a significant body of work to understand assembly of the polyketide derived aglycone for this class of compounds, particularly for erythromycin and pikromycin that are generated through the action of a modular polyketide synthases. In addition, the mode of assembly of deoxysugars and aminosugars as well as their transfer to the aglycone has been reported over the past decade. However, much less information exists for the final tailoring reactions, typically mediated by P450 enzymes that are capable of regio- and stereospecific oxidations to add hydroxyl or epoxide functionality. Herein, we have characterized in vitro two P450 enzymes from the mycinamicin biosynthetic gene cluster of Micromonospora griseorubida. Cloning, overexpression and purification of MycCI revealed its selectivity for C21 methyl group hydroxylation. The natural substrate for this P450 enzyme is mycinamicin VIII, the earliest macrolide form in the post-PKS tailoring pathway appended with desosamine at C5 OH. Moreover, we found the optimal activity of MycCI is dependent on the native ferredoxin MycCII. The second and third oxidation reactions including hydroxylation and epoxidation respectively are mediated by MycG with mycinamicin IV as initial substrate. This reaction requires prior dimethylation of the second deoxysugar residue (e.g., 6-deoxyallose to mycinose) for effective conversion by the dual function MycG P450, the first natural product biosynthetic monooxygenase characterized with an ability to catalyze both hydroxylation and epoxidation steps.

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Background: A hierarchy of catalytic steps characterizes multifunctional cytochrome P450 enzymes. Results: In the post-polyketide oxidative tailoring of mycinamicins by MycG, the two methoxy groups of mycinose are sensors that mediate initial recognition and discriminate between closely related molecules. Conclusion: Bulky and conformationally restrained macrolide substrates advance to the catalytically productive mode through multiple steps. Significance: Protein engineering facilitating substrate progression may enhance catalysis.

Cytochrome P450 enzymes are capable of catalyzing a great variety of synthetically useful reactions such as selective C-H functionalization. Surrogate redox partners are widely used for reconstitution of P450 activity based on the assumption that the choice of these auxiliary proteins or their mode of action does not affect the type and selectivity of reactions catalyzed by P450s. Herein, we present an exceptional example to challenge this postulate. MycG, a multifunctional biosynthetic P450 monooxygenase responsible for hydroxylation and epoxidation of 16-membered ring macrolide mycinamicins, is shown to catalyze the unnatural N-demethylation(s) of a range of mycinamicin substrates when partnered with the free Rhodococcus reductase domain RhFRED or the engineered Rhodococcus-spinach hybrid reductase RhFRED-Fdx. By contrast, MycG fused with the RhFRED or RhFRED-Fdx reductase domain mediates only physiological oxidations. This finding highlights the larger potential role of variant redox partner protein-protein interactions in modulating the catalytic activity of P450 enzymes.