Rieske oxygenases perform precise C−H bond functionalization reactions in anabolic and catabolic pathways. These reactions are typically characterized as monooxygenation or dioxygenation reactions, but other divergent reactions are also catalyzed by Rieske oxygenases. Chlorophyll(ide) a oxygenase (CAO), for example is proposed to catalyze two monooxygenation reactions to transform a methyl-group into the formyl-group of Chlorophyll b. This formyl group, like the formyl groups found in other chlorophyll pigments, tunes the absorption spectra of chlorophyllb and supports the ability of several photosynthetic organisms to adapt to environmental light. Despite the importance of this reaction, CAO has never been studied in vitro with purified protein, leaving many open questions regarding whether CAO can facilitate both oxygenation reactions using just the Rieske oxygenase machinery. In this study, we demonstrated that four CAO homologues in partnership with a non-native reductase convert a Chlorophyll a precursor, chlorophyllidea, into chlorophyllideb in vitro. Analysis of this reaction confirmed the existence of the proposed intermediate, highlighted the stereospecificity of the reaction, and revealed the potential of CAO as a tool for synthesizing custom-tuned natural and unnatural chlorophyll pigments. This work thus adds to our fundamental understanding of chlorophyll biosynthesis and Rieske oxygenase chemistry.

The lipoyl cofactor plays an integral role in several essential biological processes. The last step in its de novo biosynthetic pathway, the attachment of two sulfur atoms at C6 and C8 of an n-octanoyllysyl chain, is catalyzed by lipoyl synthase (LipA), a member of the radical SAM superfamily. In addition to the [4Fe-4S] cluster common to all radical SAM enzymes, LipA contains a second [4Fe-4S] auxiliary cluster, which is sacrificed during catalysis to supply the requisite sulfur atoms, rendering the protein inactive for further turnovers. Recently, it was shown that the Fe-S cluster carrier protein NfuA from Escherichia coli can regenerate the auxiliary cluster of E. coli LipA after each turnover, but the molecular mechanism is incompletely understood. Herein, using protein-protein interaction and kinetic assays as well as sitedirected mutagenesis, we provide further insight into the mechanism of NfuA-mediated cluster regeneration. In particular, we show that the N-terminal A-type domain of E. coli NfuA is essential for its tight interaction with LipA. Further, we demonstrate that NfuA from Mycobacterium tuberculosis can also regenerate the auxiliary cluster of E. coli LipA. However, an Nfu protein from Staphylococcus aureus, which lacks the A-type domain, was severely diminished in facilitating cluster regeneration. Of note, addition of the N-terminal domain of E. coli NfuA to S. aureus Nfu, fully restored cluster-regenerating activity. These results expand our understanding of the newly discovered mechanism by which the auxiliary cluster of LipA is restored after each turnover.

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Staphylococcus aureus (S. aureus) is the leading cause of infections acquired in a hospital setting. In recent years, the rise and spread of methicillin‐resistant S. aureus (MRSA) that has evolved resistance to antibiotics previously successful at eradicating the infection has become a major global health concern. Thus, the search for novel antibiotic targets has been the focus of many research efforts. One potential new target is the biosynthesis of the lipoyl cofactor, which plays an essential role in the oxidative decarboxylation of various α‐keto acids and breakdown of glycine. The second step of the de novo pathway for the biosynthesis of the lipoyl cofactor, the attachment of two sulfur atoms to C6 and C8 of an n‐octanoyl chain connected to a target lysyl residue on a lipoyl carrier protein, is catalyzed by lipoyl synthase (LipA), a member of the radical S‐adenosylmethionine (SAM) superfamily. In addition to its [4Fe–4S] radical SAM cluster, LipA contains a second [4Fe–4S] ‘auxiliary cluster’ which is sacrificed during catalysis to supply the requisite sulfur atoms. The destruction of its auxiliary cluster renders LipA inactive in the absence of a system to restore it. Further studies have identified that Escherichia coli (E. coli) has an additional protein, NfuA, an iron‐sulfur cluster carrier protein, that can target LipA and regenerate its auxiliary cluster to reactivate it for catalysis. However, while E. coli LipA has been extensively studied, the S. aureus LipA homolog has not been fully characterized. Interestingly, in vitro studies have shown that the activity of S. aureus LipA is unaffected by S. aureus Nfu alone, indicating that a second factor may be involved. Recent in vivo studies have reported that an S. aureus protein, SufT, is involved in iron‐sulfur cluster assembly and is implicated in lipoic acid biosynthesis; however, the role of SufT is unknown. We will, therefore, use a combination of genetic, biochemical, and spectroscopic approaches to functionally characterize Staphylococcus aureus LipA and understand the potential roles of Staphylococcus aureus Nfu and SufT in the regeneration of LipA's auxiliary cluster. Ultimately, this information could be used to design antibiotics that target this pathway and obstruct the essential biosynthesis of lipoic acid, inducing death to pathogenic bacteria.Support or Funding InformationI acknowledge support from the HHMI EXROP program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rieske Oxygenases are enzymes that showcase the remarkable ability to functionalize an inert C–H bond. These enzymes use a Rieske [2Fe‐2S] cluster and a mononuclear iron center to bind and activate molecular oxygen. Using these metallocenters, Rieske Oxygenases have been shown to catalyze a variety of reactions, including one, two, and sequential hydroxylation reactions. This chemistry is important to the biosynthetic pathways of multiple organisms and is thus attractive for industrially building and tailoring natural product scaffolds. Similarly, the chemistry of the Rieske Oxygenases is abundant in degradative pathways and therefore offers compelling strategies for degrading environmental pollutants. However, a traditional lack of structure–function information for this expansive class of enzymes has restricted our ability to capitalize on these important and diverse catalytic strategies. Therefore, our laboratory is working to understand the architectural features of these enzymes that allow them to control and facilitate catalysis. Here, we present our current understanding of how these enzymes selectively catalyze reactions and use structure to guide chemical outcome.