8SY8

Crystal structure of TsaC

8SY8 の概要

• エントリーDOI: 10.2210/pdb8sy8/pdb
• 分子名称: 4-formylbenzenesulfonate dehydrogenase TsaC (2 entities in total)
• 機能のキーワード: short chain dehydrogenase/reductase, rossmann fold, nad(h)binding, oxidoreductase
• 由来する生物種: Comamonas testosteroni
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 57479.00

構造登録者

Boggs, D.G.,Tian, J.,Bridwell-Rabb, J. (登録日: 2023-05-24, 公開日: 2023-09-20, 最終更新日: 2023-10-25)

主引用文献

Tian, J.,Boggs, D.G.,Donnan, P.H.,Barroso, G.T.,Garcia, A.A.,Dowling, D.P.,Buss, J.A.,Bridwell-Rabb, J.
The NADH recycling enzymes TsaC and TsaD regenerate reducing equivalents for Rieske oxygenase chemistry.
J.Biol.Chem., 299:105222-105222, 2023

PubMed Abstract: Many microorganisms use both biological and nonbiological molecules as sources of carbon and energy. This resourcefulness means that some microorganisms have mechanisms to assimilate pollutants found in the environment. One such organism is Comamonas testosteroni, which metabolizes 4-methylbenzenesulfonate and 4-methylbenzoate using the TsaMBCD pathway. TsaM is a Rieske oxygenase, which in concert with the reductase TsaB consumes a molar equivalent of NADH. Following this step, the annotated short-chain dehydrogenase/reductase and aldehyde dehydrogenase enzymes TsaC and TsaD each regenerate a molar equivalent of NADH. This co-occurrence ameliorates the need for stoichiometric addition of reducing equivalents and thus represents an attractive strategy for integration of Rieske oxygenase chemistry into biocatalytic applications. Therefore, in this work, to overcome the lack of information regarding NADH recycling enzymes that function in partnership with Rieske non-heme iron oxygenases (Rieske oxygenases), we solved the X-ray crystal structure of TsaC to a resolution of 2.18 Å. Using this structure, a series of substrate analog and protein variant combination reactions, and differential scanning fluorimetry experiments, we identified active site features involved in binding NAD and controlling substrate specificity. Further in vitro enzyme cascade experiments demonstrated the efficient TsaC- and TsaD-mediated regeneration of NADH to support Rieske oxygenase chemistry. Finally, through in-depth bioinformatic analyses, we illustrate the widespread co-occurrence of Rieske oxygenases with TsaC-like enzymes. This work thus demonstrates the utility of these NADH recycling enzymes and identifies a library of short-chain dehydrogenase/reductase enzyme prospects that can be used in Rieske oxygenase pathways for in situ regeneration of NADH.

PubMed: 37673337
DOI: 10.1016/j.jbc.2023.105222

実験手法

X-RAY DIFFRACTION (2.18 Å)