The NADH recycling enzymes TsaC and TsaD regenerate reducing equivalents for Rieske oxygenase chemistry.

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.

Leveraging a Structural Blueprint to Rationally Engineer the Rieske Oxygenase TsaM.

Rieske nonheme iron oxygenases use two metallocenters, a Rieske-type [2Fe-2S] cluster and a mononuclear iron center, to catalyze oxidation reactions on a broad range of substrates. These enzymes are widely used by microorganisms to degrade environmental pollutants and to build complexity in a myriad of biosynthetic pathways that are industrially interesting. However, despite the value of this chemistry, there is a dearth of understanding regarding the structure-function relationships in this enzyme class, which limits our ability to rationally redesign, optimize, and ultimately exploit the chemistry of these enzymes. Therefore, in this work, by leveraging a combination of available structural information and state-of-the-art protein modeling tools, we show that three "hotspot" regions can be targeted to alter the site selectivity, substrate preference, and substrate scope of the Rieske oxygenase p-toluenesulfonate methyl monooxygenase (TsaM). Through mutation of six to 10 residues distributed between three protein regions, TsaM was engineered to behave as either vanillate monooxygenase (VanA) or dicamba monooxygenase (DdmC). This engineering feat means that TsaM was rationally engineered to catalyze an oxidation reaction at the meta and ortho positions of an aromatic substrate, rather than its favored native para position, and that TsaM was redesigned to perform chemistry on dicamba, a substrate that is not natively accepted by the enzyme. This work thus contributes to unlocking our understanding of structure-function relationships in the Rieske oxygenase enzyme class and expands foundational principles for future engineering of these metalloenzymes.

Engineering Rieske oxygenase activity one piece at a time.

Enzyme engineering plays a central role in the development of biocatalysts for biotechnology, chemical and pharmaceutical manufacturing, and environmental remediation. Rational design of proteins has historically relied on targeting active site residues to confer a protein with desirable catalytic properties. However, additional "hotspots" are also known to exist beyond the active site. Structural elements such as subunit-subunit interactions, entrance tunnels, and flexible loops influence enzyme catalysis and serve as potential "hotspots" for engineering. For the Rieske oxygenases, which use a Rieske cluster and mononuclear iron center to catalyze a challenging set of reactions, these outside of the active site regions are increasingly being shown to drive catalytic outcomes. Therefore, here, we highlight recent work on structurally characterized Rieske oxygenases that implicates architectural pieces inside and outside of the active site as key dictators of catalysis, and we suggest that these features may warrant attention in efforts aimed at Rieske oxygenase engineering.