8sy8

Publication Abstract from PubMed

Many microorganisms use both biological and non-biological 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 (SDR) 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 oxygenases, we solved the X-ray crystal structure of TsaC to 2.18-A resolution. 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 analysis 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 SDR enzyme prospects that can be used in Rieske oxygenase pathways for in situ regeneration of NADH.

The NADH recycling enzymes TsaC and TsaD regenerate reducing equivalents for Rieske oxygenase chemistry.,Tian J, Boggs DG, Donnan PH, Barroso GT, Garcia AA, Dowling DP, Buss JA, Bridwell-Rabb J J Biol Chem. 2023 Sep 4:105222. doi: 10.1016/j.jbc.2023.105222. PMID:37673337^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.