TY - JOUR T1 - Molecular basis for metabolite channeling in a ring opening enzyme of the phenylacetate degradation pathway. JF - Nat Commun Y1 - 2019 A1 - Sathyanarayanan, Nitish A1 - Cannone, Giuseppe A1 - Gakhar, Lokesh A1 - Katagihallimath, Nainesh A1 - Sowdhamini, Ramanathan A1 - Ramaswamy, Subramanian A1 - Vinothkumar, Kutti R AB -

Substrate channeling is a mechanism for the internal transfer of hydrophobic, unstable or toxic intermediates from the active site of one enzyme to another. Such transfer has previously been described to be mediated by a hydrophobic tunnel, the use of electrostatic highways or pivoting and by conformational changes. The enzyme PaaZ is used by many bacteria to degrade environmental pollutants. PaaZ is a bifunctional enzyme that catalyzes the ring opening of oxepin-CoA and converts it to 3-oxo-5,6-dehydrosuberyl-CoA. Here we report the structures of PaaZ determined by electron cryomicroscopy with and without bound ligands. The structures reveal that three domain-swapped dimers of the enzyme form a trilobed structure. A combination of small-angle X-ray scattering (SAXS), computational studies, mutagenesis and microbial growth experiments suggests that the key intermediate is transferred from one active site to the other by a mechanism of electrostatic pivoting of the CoA moiety, mediated by a set of conserved positively charged residues.

VL - 10 IS - 1 ER - TY - JOUR T1 - A strategy to identify a ketoreductase that preferentially synthesizes pharmaceutically relevant (S)-alcohols using whole-cell biotransformation. JF - Microb Cell Fact Y1 - 2018 A1 - Haq, Saiful F A1 - Shanbhag, Anirudh P A1 - Karthikeyan, Subbulakshmi A1 - Hassan, Imran A1 - Thanukrishnan, Kannan A1 - Ashok, Abhishek A1 - Sukumaran, Sunilkumar A1 - Ramaswamy, S A1 - Bharatham, Nagakumar A1 - Datta, Santanu A1 - Samant, Shalaka A1 - Katagihallimath, Nainesh KW - Biotransformation KW - Catalysis KW - Ethanol KW - Ketones AB -

INTRODUCTION: Chemical industries are constantly in search of an expeditious and environmentally benign method for producing chiral synthons. Ketoreductases have been used as catalysts for enantioselective conversion of desired prochiral ketones to their corresponding alcohol. We chose reported promiscuous ketoreductases belonging to different protein families and expressed them in E. coli to evaluate their ability as whole-cell catalysts for obtaining chiral alcohol intermediates of pharmaceutical importance. Apart from establishing a method to produce high value (S)-specific alcohols that have not been evaluated before, we propose an in silico analysis procedure to predict product chirality.

RESULTS: Six enzymes originating from Sulfolobus sulfotaricus, Zygosaccharomyces rouxii, Hansenula polymorpha, Corynebacterium sp. ST-10, Synechococcus sp. PCC 7942 and Bacillus sp. ECU0013 with reported efficient activity for dissimilar substrates are compared here to arrive at an optimal enzyme for the method. Whole-cell catalysis of ketone intermediates for drugs like Aprepitant, Sitagliptin and Dolastatin using E. coli over-expressing these enzymes yielded (S)-specific chiral alcohols. We explain this chiral specificity for the best-performing enzyme, i.e., Z. rouxii ketoreductase using in silico modelling and MD simulations. This rationale was applied to five additional ketones that are used in the synthesis of Crizotinib, MA-20565 (an antifungal agent), Sulopenem, Rivastigmine, Talampanel and Barnidipine and predicted the yield of (S) enantiomers. Experimental evaluation matched the in silico analysis wherein ~ 95% (S)-specific alcohol with a chemical yield of 23-79% was obtained through biotransformation. Further, the cofactor re-cycling was optimized by switching the carbon source from glucose to sorbitol that improved the chemical yield to 85-99%.

CONCLUSIONS: Here, we present a strategy to synthesize pharmaceutically relevant chiral alcohols by ketoreductases using a cofactor balanced whole-cell catalysis scheme that is useful for the industry. Based on the results obtained in these trials, Zygosaccharomyces rouxii ketoreductase was identified as a proficient enzyme to obtain (S)-specific alcohols from their respective ketones. The whole-cell catalyst when combined with nutrient modulation of using sorbitol as a carbon source helped obtain high enantiomeric and chemical yield.

VL - 17 IS - 1 ER -