Title: A structure-function analysis of chlorophyllase reveals a mechanism for activity regulation dependent on disulfide bonds Jo M, Knapp M, Boggs DG, Brimberry M, Donnan PH, Bridwell-Rabb J Ref: Journal of Biological Chemistry, :102958, 2023 : PubMed
Chlorophyll (Chl) 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 alpha/beta hydrolase that hydrolyzes the phytol tail of Chl pigments to produce chlorophyllide (Chlide) 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- 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.
The metabolic serine hydrolase family is, arguably, one of the largest functional enzyme classes in mammals, including humans, comprising 1-2% of the total proteome. This enzyme family uses a conserved nucleophilic serine residue in the active site to perform diverse hydrolytic reactions and consists of proteases, lipases, esterases, amidases, and transacylases, which are prototypical members of this family. In humans, this enzyme family consists of >250, of which approximately 40% members remain unannotated, in terms of both their endogenous substrates and the biological pathways that they regulate. The enzyme ABHD14B, an outlying member of this family, is also known as CCG1/TAFII250-interacting factor B, as it was found to be associated with transcription initiation factor TFIID. The crystal structure of human ABHD14B was determined more than a decade ago; however, its endogenous substrates remain elusive. In this paper, we annotate ABHD14B as a lysine deacetylase (KDAC), showing this enzyme's ability to transfer an acetyl group from a post-translationally acetylated lysine to coenzyme A (CoA), to yield acetyl-CoA, while regenerating the free amine of protein lysine residues. We validate these findings by in vitro biochemical assays using recombinantly purified human ABHD14B in conjunction with cellular studies in a mammalian cell line by knocking down ABHD14B and by identification of a putative substrate binding site. Finally, we report the development and characterization of a much-needed, exquisitely selective ABHD14B antibody, and using it, we map the cellular and tissue distribution of ABHD14B and prospective metabolic pathways that this enzyme might biologically regulate.
