Gene targeting for O -methyltransferase genes, mycE and mycF , on the chromosome of Micromonospora griseorubida producing mycinamicin with a disruption cassette containing the bacteriophage φC31 attB attachment site

Mycinamicin, a 16-membered macrolide antibiotic produced by Micromonospora griseorubida , comprises a macrolactone and two deoxysugars: desosamine and mycinose. Mycinose is synthesized through two modification steps: the methylation of 6-deoxyallose in mycinamicin VI and of javose in mycinamicin III. To confirm the role of mycE and mycF genes in mycinamicin biosynthesis in M. griseorubida , disruption mutants of mycE and mycF were constructed by disruption plasmids containing attB in the disruption cassette FRT -neo-oriT- FRT -attB for the integration of φC31-derivative vector plasmids; the disruption mutants were complemented through the integration of pSET152 derivatives containing intact mycE or mycF into the artificially inserted attB site. These disruption mutants did not produce mycinamicin II, but mainly accumulated mycinamicins VI and III, indicating that MycE and MycF methylated the C2″-OH group of 6-deoxyallose in mycinamicin VI and the C3″-OH group of C2″-methylated 6-deoxyallose in mycinamicin III, respectively. The complemented strains of mycE and mycF recovered the mycinamicin II productivity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79258/1/j.1574-6968.2010.01899.x.pd

Monitoring the colonization and infection of legume nodules by Micromonospora in co-inoculation experiments with rhizobia.

The discovery that the actinobacterium Micromonospora inhabits nitrogen-fixing nodules raised questions as to its potential ecological role. The capacity of two Micromonospora strains to infect legumes other than their original host, Lupinus angustifolius, was investigated using Medicago and Trifolium as test plants. Compatible rhizobial strains were used for coinoculation of the plants because Micromonospora itself does not induce nodulation. Over 50% of nodules from each legume housed Micromonospora, and using 16S rRNA gene sequence identification, we verified that the reisolated strains corresponded to the microorganisms inoculated. Entry of the bacteria and colonization of the plant hosts were monitored using a GFP-tagged Lupac 08 mutant together with rhizobia, and by using immunogold labeling. Strain Lupac 08 was localized in plant tissues, confirming its capacity to enter and colonize all hosts. Based on studying three different plants, our results support a non-specific relationship between Micromonospora and legumes. Micromonospora Lupac 08, originally isolated from Lupinus re-enters root tissue, but only when coinoculated with the corresponding rhizobia. The ability of Micromonospora to infect and colonize different legume species and function as a potential plant-growth promoting bacterium is relevant because this microbe enhances the symbiosis without interfering with the host and its nodulating and nitrogen-fixing microbes

Computational-Based Mechanistic Study and Engineering of Cytochrome P450 MycG for Selective Oxidation of 16-Membered Macrolide Antibiotics

MycG is a cytochrome P450 that performs two sequential oxidation reactions on the 16-membered ring macrolide M-IV. The enzyme evolved to oxidize M-IV preferentially over M-III and M-VI, which differ only by the presence of methoxy vs free hydroxyl groups on one of the macrolide sugar moieties. We utilized a two-pronged computational approach to study both the chemoselective reactivity and substrate specificity of MycG. Density functional theory computations determined that epoxidation of the substrate hampers its ability to undergo C-H abstraction, primarily due to a loss of hyperconjugation in the transition state. Metadynamics and molecular dynamics simulations revealed a hydrophobic sugar-binding pocket that is responsible for substrate recognition/specificity and was not apparent in crystal structures of the enzyme/substrate complex. Computational results also led to the identification of other interactions between the enzyme and its substrates that had not previously been observed in the cocrystal structures. Site-directed mutagenesis was then employed to test the effects of mutations hypothesized to broaden the substrate scope and alter the product profile of MycG. The results of these experiments validated this complementary effort to engineer MycG variants with improved catalytic activity toward earlier stage mycinamicin substrates

New Reactions and Products Resulting from Alternative Interactions between the P450 Enzyme and Redox Partners

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 <i>N</i>-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