A structure-function analysis of chlorophyllase reveals a mechanism for activity regulation dependent on disulfide bonds.

Chlorophyll pigments are used by photosynthetic organisms to facilitate light capture and mediate the conversion of sunlight into chemical energy. Due to the indispensable nature of this pigment and its propensity to form reactive oxygen species, organisms heavily invest in its biosynthesis, recycling, and degradation. One key enzyme implicated in these processes is chlorophyllase, an α/ß hydrolase that hydrolyzes the phytol tail of chlorophyll pigments to produce chlorophyllide molecules. This enzyme was discovered a century ago, but despite its importance to diverse photosynthetic organisms, there are still many missing biochemical details regarding how chlorophyllase functions. Here, we present the 4.46-Å resolution crystal structure of chlorophyllase from Triticum aestivum. This structure reveals the dimeric architecture of chlorophyllase, the arrangement of catalytic residues, an unexpected divalent metal ion-binding site, and a substrate-binding site that can accommodate a diverse range of pigments. Further, this structure exhibits the existence of both intermolecular and intramolecular disulfide bonds. We investigated the importance of these architectural features using enzyme kinetics, mass spectrometry, and thermal shift assays. Through this work, we demonstrated that the oxidation state of the Cys residues is imperative to the activity and stability of chlorophyllase, illuminating a biochemical trigger for responding to environmental stress. Additional bioinformatics analysis of the chlorophyllase enzyme family reveals widespread conservation of key catalytic residues and the identified "redox switch" among other plant chlorophyllase homologs, thus revealing key details regarding the structure-function relationships in chlorophyllase.

Rieske Oxygenase Catalyzed C-H Bond Functionalization Reactions in Chlorophyll b Biosynthesis.

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.

Structural and mechanistic basis for redox sensing by the cyanobacterial transcription regulator RexT.

Organisms have a myriad of strategies for sensing, responding to, and combating reactive oxygen species, which are unavoidable consequences of aerobic life. In the heterocystous cyanobacterium Nostoc sp. PCC 7120, one such strategy is the use of an ArsR-SmtB transcriptional regulator RexT that senses H2O2 and upregulates expression of thioredoxin to maintain cellular redox homeostasis. Different from many other members of the ArsR-SmtB family which bind metal ions, RexT has been proposed to use disulfide bond formation as a trigger to bind and release DNA. Here, we present high-resolution crystal structures of RexT in the reduced and H2O2-treated states. These structures reveal that RexT showcases the ArsR-SmtB winged-helix-turn-helix fold and forms a vicinal disulfide bond to orchestrate a response to H2O2. The importance of the disulfide-forming Cys residues was corroborated using site-directed mutagenesis, mass spectrometry, and H2O2-consumption assays. Furthermore, an entrance channel for H2O2 was identified and key residues implicated in H2O2 activation were pinpointed. Finally, bioinformatics analysis of the ArsR-SmtB family indicates that the vicinal disulfide "redox switch" is a unique feature of cyanobacteria in the Nostocales order, presenting an interesting case where an ArsR-SmtB protein scaffold has been evolved to showcase peroxidatic activity and facilitate redox-based regulation.