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.

Chronic consumption of excess ethanol increases the risk of colorectal cancer. The pathogenesis of ethanol-related colorectal cancer (ER-CRC) is thought to be partly mediated by gut microbes. Specifically, bacteria in the colon and rectum convert ethanol to acetaldehyde (AcH), which is carcinogenic. However, the effects of chronic ethanol consumption on the human gut microbiome are poorly understood, and the role of gut microbes in the proposed AcH-mediated pathogenesis of ER-CRC remains to be elaborated. Here we analyse and compare the gut microbiota structures of non-alcoholics and alcoholics. The gut microbiotas of alcoholics were diminished in dominant obligate anaerobes (e.g., Bacteroides and Ruminococcus) and enriched in Streptococcus and other minor species. This alteration might be exacerbated by habitual smoking. These observations could at least partly be explained by the susceptibility of obligate anaerobes to reactive oxygen species, which are increased by chronic exposure of the gut mucosa to ethanol. The AcH productivity from ethanol was much lower in the faeces of alcoholic patients than in faeces of non-alcoholic subjects. The faecal phenotype of the alcoholics could be rationalised based on their gut microbiota structures and the ability of gut bacteria to accumulate AcH from ethanol.

This study demonstrates that ATO is metabolized when administered intravenously to APL patients and methylated metabolites are promptly eliminated from the blood and excreted into urine after completion of administration, indicating no measurable accumulation of ATO in the blood.

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The beta-ketoacyl-acyl carrier protein synthase (KS) domain of the modular 6-deoxyerythronolide B synthase (DEBS) catalyzes the fundamental chain building reaction of polyketide biosynthesis. The KS-catalyzed reaction involves two discrete steps consisting of formation of an acyl-enzyme intermediate generated from the incoming acylthioester substrate and an active site cysteine residue, and the conversion of this intermediate to the beta-ketoacyl-acyl carrier protein product by a decarboxylative condensation with a paired methylmalonyl-SACP. We have determined the rate constants for the individual biochemical steps by a combination of protein acylation and transthioesterification experiments. The first-order rate constant (k(2)) for formation of the acyl-enzyme intermediate from [1-(14)C]-(2S,3R)-2-methyl-3-hydroxypentanoyl-SNAC (2) and recombinant DEBS module 2 is 5.8 +/- 2.6 min(-)(1), with a dissociation constant (K(S)) of 3.5 +/- 2.8 mM. The acyl-enzyme adduct was formed at a near-stoichiometric ratio of approximately 0.8:1. Transthioesterification between unlabeled diketide-SNAC 2 and N-[1-(14)C-acetyl]cysteamine gave a k(exch) of 0.15 +/- 0.06 min(-)(1), with a K(m) for HSNAC of 5.7 +/- 4.9 mM and a K(m) for 2 of 5.3 +/- 0.9 mM. Under the conditions that were used, k(exch) was equal to k(-)(2), the first-order rate constant for reversal of the acyl-enzyme-forming reaction. Since the rate of the decarboxylative condensation is much greater that the rate of reversion to the starting material (k(3) >> k(-)(2)), formation of the acyl-enzyme adduct is effectively irreversible, thereby establishing that the observed value of the specificity constant (k(cat)/K(m)) is solely a reflection of the intrinsic substrate specificity of the KS-catalyzed acyl-enzyme-forming reaction. These findings were also extended to a panel of diketide- and triketide-SNAC analogues, revealing that some substrate analogues that are not converted to product by DEBS module 2 form dead-end acyl-enzyme intermediates.