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Engineering of lysine cyclodeaminse conformational dynamics for relieving substrate and product inhibitions in the biosynthesis of L-pipecolic acid

Abstract

Substrate and product inhibition in enzyme-catalyzed reactions is a major limitation in the preparative biosynthesis of valuable chemicals. In the present study, we described a modulation of conformational dynamics of lysine cyclodeaminase from Streptomyces pristinaespiralis (SpLCD) for synchronously reducing substrate and product inhibitions. LCD is the key enzyme in the biosynthesis of piperidine derivatives, but incurs both severe substrate and product inhibitions due to steric hindrance by the narrow delivery tunnels of substrate and product. Conformational dynamics studies via molecular simulations, which revealed the detailed atomic structures of both substrate and product delivery processes of SpLCD, indicated two separate intrinsic motions affected by the position of NAD+. Two key residues, Ile61 and Ile94 was observed to play a key role in regulating the shape of the substrate and product delivery processes. Via saturation mutagenesis studies, Val61-Val94-SpLCD variant which improved the KM/kcat, Ki-lys and Ki-LPA by 3.6, 19.4 and 9.2 times, respectively, was obtained. The structures analysis showed that the superior catalytic performance of of Val61-Val94-SpLCD variant is mainly owing to the expanded substrate and product delivery tunnels when compared with the wild-type enzyme. By using recombinant Escherichia coli containing Val61-Val94-SpLCD as the whole-cell biocatalyst, a 2.5-fold higher substrate loading concentration with a total space-time yield of 0.83 g L-1 h-1 was achieved. Moreover, L-pipecolic acid titer increased to 73.4 g/L without a decrease in yield, which was 4.2 folds higher than that achieved with the original recombinant whole-cell biocatalyst. The results of this study provide insights into the application of conformational dynamics of protein substrate and product delivery processes for simultaneously reducing both substrate and product inhibitions.

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Publication details

The article was received on 07 Nov 2018, accepted on 30 Nov 2018 and first published on 04 Dec 2018


Article type: Paper
DOI: 10.1039/C8CY02301H
Citation: Catal. Sci. Technol., 2019, Accepted Manuscript
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    Engineering of lysine cyclodeaminse conformational dynamics for relieving substrate and product inhibitions in the biosynthesis of L-pipecolic acid

    H. Ying, J. Wang, T. Shi, Y. Zhao, P. Ouyang and K. Chen, Catal. Sci. Technol., 2019, Accepted Manuscript , DOI: 10.1039/C8CY02301H

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