Complex polyketides are characterized by multiple chiral centers harboring hydroxyl and alkyl substituents. To investigate the mechanisms by which these stereocenters are set, several high-resolution structures of the ketoreductase (KR) domain from the second module of the amphotericin modular polyketide synthase (PKS) were solved. This first structural analysis of an A-type KR helps reveal how these KRs direct polyketide intermediates into their active sites from the side opposite that used by B-type KRs, resulting in a beta-hydroxyl group of opposite stereochemistry. A comparison of structures obtained in the absence and presence of ligands reveals an induced fit mechanism that is important for catalysis. Activity assays of mutants of KRs from the first and second modules of the amphotericin PKS reveal the relative contributions of several active site residues toward catalysis and stereocontrol. Together, these results highlight the possibility of region-specific modification of polyketides through active site engineering of KRs.

Chiral building blocks are valuable intermediates in the syntheses of natural products and pharmaceuticals. A scalable chemoenzymatic route to chiral diketides has been developed that includes the general synthesis of α-substituted, β-ketoacyl N-acetylcysteamine thioesters followed by a biocatalytic cycle in which a glucose-fueled NADPH-regeneration system drives reductions catalyzed by isolated modular polyketide synthase (PKS) ketoreductases (KRs). To identify KRs that operate as active, stereospecific biocatalysts, 11 isolated KRs were incubated with 5 diketides and their products were analyzed by chiral chromatography. KRs that naturally reduce small polyketide intermediates were the most active and stereospecific toward the panel of diketides. Several biocatalytic reactions were scaled up to yield more than 100 mg of product. These syntheses demonstrate the ability of PKS enzymes to economically and greenly generate diverse chiral building blocks on a preparative scale.

Modular polyketide synthase ketoreductases often set two stereocenters when reducing intermediates in the biosynthesis of a complex polyketide. Here we report the 2.60 Å-resolution structure of an A2-type ketoreductase from the eleventh module of the amphotericin polyketide synthase that sets a combination of l-α-methyl and l-β-hydroxyl stereochemistries and represents the final catalytically-competent ketoreductase type to be structurally elucidated. Through structure-guided mutagenesis a double mutant of an A1-type ketoreductase was generated that functions as an A2-type ketoreductase on a diketide substrate analog, setting an α-alkyl substituent in an l- orientation rather than in the d-orientation set by the unmutated ketoreductase. When the activity of the double-mutant was examined in the context of an engineered triketide lactone synthase, the anticipated triketide lactone was not produced even though the ketoreductase-containing module still reduced the diketide substrate analog as expected. These findings suggest that re-engineered ketoreductases may be catalytically outcompeted within engineered polyketide synthase assembly lines.

While the cis-acyltransferase modular polyketide synthase assembly lines have largely been structurally dissected, enzymes from within the recently discovered trans-acyltransferase polyketide synthase assembly lines are just starting to be observed crystallographically. Here we examine the ketoreductase from the first polyketide synthase module of the bacillaene nonribosomal peptide synthetase/polyketide synthase at 2.35-Å resolution. This ketoreductase naturally reduces both α- and β-keto groups and is the only ketoreductase known to do so during the biosynthesis of a polyketide. The isolated ketoreductase not only reduced an N-acetylcysteamine-bound β-keto substrate to a D-β-hydroxy product, but also an N-acetylcysteamine- bound α-keto substrate to an L-α-hydroxy product. That the substrates must enter the active site from opposite directions to generate these stereochemistries suggests that the acyl-phosphopantetheine moiety is capable of accessing very different conformations despite being anchored to a serine residue of a docked acyl carrier protein. The features enabling stereocontrolled α-ketoreduction may not be extensive since a β-ketoreductase from a cis-acyltransferase polyketide synthase was identified that performs a completely stereoselective reduction of the same α-keto substrate to generate the D-α-hydroxy product. A sequence analysis of trans-acyltransferase ketoreductases reveals that a single residue, rather than a three-residue motif found in cis-acyltransferase ketoreductases, is predictive of the orientation of the resulting β-hydroxyl group.

Background: There are an overwhelming number of patients suffering from low back pain (LBP) resulting from disc pathology. Although several strategies are being developed pre-clinically, simple strategies to treat the large number of patients currently affected is still needed. One option is to use concentrated bone marrow aspirate (cBMA), which may be effective due to its intrinsic stem cells and growth factors. Methods: Thirty-three patients who received intradiscal injections of cBMA to relieve LBP were followed up based on Numeric Rating Scale (NRS), Oswestry Low Back Pain Disability Index (ODI), and Short Form-36 Health Survey (SF-36) scores. Patients were also subdivided into those with a pre-injection NRS > 5 and pre-injection NRS ≤ 5. The proportion of patients demonstrating at least 50% improvement (and 95% confidence intervals) from baseline at five follow-up visits for each outcome was evaluated. Results: At least 50% improvement in NRS was observed for 13.8, 45.8, 41.1, 23.5, and 38.9% of patients across five follow-up visits, out to 1 year. When stratified by high (> 5) versus low (≤ 5) baseline NRS scores, the values were 14.3, 45.5, 71.4, 22.2, and 44.4% among those with high baseline pain, and 13.3, 46.2, 20.0, 25.0, and 33.3% among those with low baseline pain. The 50% improvement rates across visits were 4.3, 28.6, 30.0, 22.2, and 30.8% for SF-36, and 4.2, 26.7, 36.4, 55.6, and 30.8% for ODI. Conclusions: Intradiscal cBMA injections may be effective to reduce pain and improve function. Patients with relatively higher initial pain may have potential for greatest improvement.