Leveraging a Structural Blueprint to Rationally Engineer the Rieske Oxygenase TsaM

Abstract

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.

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

“…5 A , S27 , and S28 ). This result agrees with previous studies that indicate the upstream Rieske oxygenase, TsaM, only performs mono-oxygenation chemistry on 3-methylbenzoate and not 2-methylbenzoate ( 22 , 39 ). In addition, it was determined that the active site of TsaC accommodates aromatic substrates with a variety of functional groups at C1, albeit with a slight preference for 4-aminobenzyl alcohol relative to the other provided options ( Figs.…”

“…As a baseline for these experiments, and for comparison to that previously measured for TsaM (53.7 °C ± 0.5 deg. C), the thermal stability of TsaC was measured ( 22 ). Here, it was determined that wild-type TsaC has a T m of 54.7 °C ± 0.02 deg.…”

“…S49 ). VanB was purified for use in these experiments because we previously discovered that VanB supports a significantly higher level of TsaM activity than the native reductase TsaB ( 22 , 39 ). Here, it was determined that the combination of TsaM, VanB, TsaC, TsaD, NADH, and either 4-methylbenzenesulfonate or 4-methylbenzoate allows for the iterative detection of the expected alcohol (4-(hydroxymethyl)benzenesulfonate or 4-(hydroxymethyl)benzoate), aldehyde (4-formylbenzenesulfonate or 4-formylbenzoate), and carboxylic acid (4-sulfobenzoate or 1,4-benzenedicarboxylate) products using LC–MS ( Fig.…”

“…Accordingly, enzymes like TsaC and TsaD provide an intriguing strategy from nature for applications aimed at using engineered microorganisms for environmental remediation. Our laboratory recently demonstrated the ability to engineer TsaM to catalyze divergent chemical transformations ( 22 ). However, to date, the low sequence identity among annotated SDR enzymes and the dearth of structurally characterized SDR enzymes that work in cooperation with Rieske oxygenases leave open questions regarding the utility of TsaC and TsaD, and other SDR enzymes in these types of Rieske oxygenase centered biocatalytic applications ( 23 , 24 ).…”

Electrostatically Regulated Active Site Assembly Governs Reactivity in Nonheme Iron Halogenases

“…The use of enzymes as industrial catalysts has gained increasing attention in recent years for their ability to achieve complex chemical transformations with a high degree of regio- and stereoselectivity under environment-friendly conditions. − Non-heme iron halogenases (NHFe-Hals), in particular, hold great value as biocatalytic platforms as they are able to install a chloride moiety at an unactivated sp 3 -hybridized carbon in a selective manner (Figure A). − Conforming to the literature on non-heme iron enzymes, we use the term “substrate” in this work to strictly refer to the molecule that is the subject of C–H functionalization. ,− NHFe-Hals enable regio- and stereoselective chlorination of their native substrates using chloride, 2-oxoglutarate (2OG), and oxygen as cosubstrates with desired product ratios as high as 95% . NHFe-Hals have also been shown to enable C–H functionalization with non-native anions including bromide, azide, and nitrite, albeit with significantly reduced yields. ,, A major challenge with NHFe-Hals, however, is that their substrate scope is rather limited as most known NHFe-Hals require their substrates to be attached to a partner protein. ,− Only a handful of identified NHFe-Hals are able to act on freestanding substrates, limiting their substrate scope currently to some amino acids, alkaloids, , and nucleotides (Figure A).…”