6NET

FAD-dependent monooxygenase TropB from T. stipitatus substrate complex

Summary for 6NET

• Entry DOI: 10.2210/pdb6net/pdb
• Descriptor: FAD-dependent monooxygenase tropB, 2,4-dihydroxy-3,6-dimethylbenzaldehyde, FLAVIN-ADENINE DINUCLEOTIDE, ... (6 entities in total)
• Functional Keywords: oxidative dearomatization, flavoprotein
• Biological source: Talaromyces stipitatus (strain ATCC 10500 / CBS 375.48 / QM 6759 / NRRL 1006) (Penicillium stipitatum)
• Total number of polymer chains: 2
• Total formula weight: 102116.27

Authors

Rodriguez Benitez, A.,Tweedy, S.E.,Baker Dockrey, S.A.,Lukowski, A.L.,Wymore, T.,Khare, D.,Brooks, C.L.,Palfey, B.A.,Smith, J.L.,Narayan, A.R.H. (deposition date: 2018-12-18, release date: 2019-08-14, Last modification date: 2023-10-11)

Primary citation

Rodriguez Benitez, A.,Tweedy, S.E.,Baker Dockrey, S.A.,Lukowski, A.L.,Wymore, T.,Khare, D.,Brooks 3rd, C.L.,Palfey, B.A.,Smith, J.L.,Narayan, A.R.H.
Structural basis for selectivity in flavin-dependent monooxygenase-catalyzed oxidative dearomatization.
Acs Catalysis, 9:3633-3640, 2019

PubMed Abstract: Biocatalytic reactions embody many features of ideal chemical transformations, including the potential for impeccable selectivity, high catalytic efficiency, mild reaction conditions and the use of environmentally benign reagents. These advantages have created a demand for biocatalysts that expand the portfolio of complexity-generating reactions available to synthetic chemists. However, the tradeoff that often exists between the substrate scope of a biocatalyst and its selectivity limits the application of enzymes in synthesis. We recently demonstrated that a flavin-dependent monooxygenase, TropB, maintains high levels of site- and stereoselectivity across a range of structurally diverse substrates. Herein, we disclose the structural basis for substrate binding in TropB, which performs a synthetically challenging asymmetric oxidative dearomatization reaction with exquisite site- and stereoselectivity across a range of phenol substrates, providing a foundation for future protein engineering and reaction development efforts. Our hypothesis for substrate binding is informed by a crystal structure of TropB and molecular dynamics simulations with the corresponding computational TropB model and is supported by experimental data. In contrast to canonical class A FAD-dependent monooxygenases in which substrates bind in a protonated form, our data indicate that the phenolate form of the substrate binds in the active site. Furthermore, the substrate position is controlled through twopoint binding of the phenolate oxygen to Arg206 and Tyr239, which are shown to have distinct and essential roles in catalysis. Arg206 is involved in the reduction of the flavin cofactor, suggesting a role in flavin dynamics. Further, QM/MM simulations reveal the interactions that govern the facial selectivity that leads to a highly enantioselective transformation. Thus, the structural origins of the high levels of site-and stereoselectivity observed in reactions of TropB across a range of substrates are elucidated, providing a foundation for future protein engineering and reaction development efforts.

PubMed: 31346489
DOI: 10.1021/acscatal.8b04575

Experimental method

X-RAY DIFFRACTION (2.25 Å)