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8SY8

Crystal structure of TsaC

Summary for 8SY8
Entry DOI10.2210/pdb8sy8/pdb
Descriptor4-formylbenzenesulfonate dehydrogenase TsaC (2 entities in total)
Functional Keywordsshort chain dehydrogenase/reductase, rossmann fold, nad(h)binding, oxidoreductase
Biological sourceComamonas testosteroni
Total number of polymer chains2
Total formula weight57479.00
Authors
Boggs, D.G.,Tian, J.,Bridwell-Rabb, J. (deposition date: 2023-05-24, release date: 2023-09-20, Last modification date: 2023-10-25)
Primary citationTian, 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
Cited by
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
PDB entries with the same primary citation
Experimental method
X-RAY DIFFRACTION (2.18 Å)
Structure validation

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