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. 2020 Jan 8:16:50-59.
doi: 10.3762/bjoc.16.7. eCollection 2020.

Understanding the role of active site residues in CotB2 catalysis using a cluster model

Affiliations

Understanding the role of active site residues in CotB2 catalysis using a cluster model

Keren Raz et al. Beilstein J Org Chem. .

Abstract

Terpene cyclases are responsible for the initial cyclization cascade in the multistep synthesis of a large number of terpenes. CotB2 is a diterpene cyclase from Streptomyces melanosporofaciens, which catalyzes the formation of cycloocta-9-en-7-ol, a precursor to the next-generation anti-inflammatory drug cyclooctatin. In this work, we present evidence for the significant role of the active site's residues in CotB2 on the reaction energetics using quantum mechanical calculations in an active site cluster model. The results revealed the significant effect of the active site residues on the relative electronic energy of the intermediates and transition state structures with respect to gas phase data. A detailed understanding of the role of the enzyme environment on the CotB2 reaction cascade can provide important information towards a biosynthetic strategy for cyclooctatin and the biomanufacturing of related terpene structures.

Keywords: CotB2 cyclase; active site; diterpene; mechanism; quantum mechanics.

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Figures

Scheme 1
Scheme 1
Mechanism for formation of cyclooctat-9-en-7-ol, published similarly in [42].
Figure 1
Figure 1
Computed electronic energy profiles (kcal/mol) for the CotB2 cyclase mechanism. The calculations used M062X/6-31+G(d,p).
Figure 2
Figure 2
Intermediates AI in the active site model. Interactions are marked by dashed orange lines, the interacting residues are labeled in black, the non-interacting residues are labeled in grey, and plus signs note location of the cations.
Figure 3
Figure 3
TS structures TS_A_B–TS_G/H_I in the active site model. Interactions are marked by dashed orange lines, the interacting residues are labeled in black, the non-interacting residues are labeled in grey, and the plus signs note the location of the cations.
Figure 4
Figure 4
Comparison between gas phase and active site model conformations. A) Intermediate D. B) Intermediate E.

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