Multiple Turnover Research Articles

Methylene blue and ascorbate interfere with the accurate determination of the kinetic properties of IDO2

Indoleamine 2,3-dioxygenases (IDOs) catalyze the oxidative cleavage of L-tryptophan (Trp) to N-formylkynurenine. Two IDOs, IDO1 and IDO2, are present in vertebrates. IDO1 is a high-affinity Trp-degrading enzyme involved in several physiological processes. By comparison, IDO2 generally has been reported to have low affinity (high Km -value) for Trp, and the enzyme's invivo function remains unclear. Using IDOs from different species, we show that compared with ferrous-oxy (Fe2+ -O2 ) IDO1, Fe2+ -O2 IDO2 is substantially more stable and engages in multiple turnovers of the reaction in the absence of a reductant. Without reductant, Fe2+ -O2 IDO2 showed Km -values in the range of 80-356μM, that is, values substantially lower than reported previously and close to the physiological concentrations of Trp. Methylene blue and ascorbate (Asc), used commonly as the reducing system for IDO activity determination, significantly affected the enzymatic activity of IDO2: In combination, the two reductants increased the apparent Km - and kcat -values 8- to 117-fold and 2-fold, respectively. Asc alone both activated and inhibited IDO2 by acting as a source of electrons and as a weak competitive inhibitor, respectively. In addition, ferric (Fe3+ ) IDO1 and IDO2 exhibited weak dioxygenase activity, similar to tryptophan 2,3-dioxygenase. Our results shed new light in the enzymatic activity of IDO2, and they support the view that this isoform of IDO also participates in the metabolism of Trp invivo.

Characterization and Mechanistic Study of the Radical SAM Enzyme ArsS Involved in Arsenosugar Biosynthesis

AbstractArsenosugars are a group of arsenic‐containing ribosides that are found predominantly in marine algae but also in terrestrial organisms. It has been proposed that arsenosugar biosynthesis involves a key intermediate 5′‐deoxy‐5′‐dimethylarsinoyl‐adenosine (DDMAA), but how DDMAA is produced remains elusive. Now, we report characterization of ArsS as a DDMAA synthase, which catalyzes a radical S‐adenosylmethionine (SAM)‐mediated alkylation (adenosylation) of dimethylarsenite (DMAsIII) to produce DDMAA. This radical‐mediated reaction is redox neutral, and multiple turnover can be achieved without external reductant. Phylogenomic and biochemical analyses revealed that DDMAA synthases are widespread in distinct bacterial phyla with similar catalytic efficiencies; these enzymes likely originated from cyanobacteria. This study reveals a key step in arsenosugar biosynthesis and also a new paradigm in radical SAM chemistry, highlighting the catalytic diversity of this superfamily of enzymes.

Characterization and Mechanistic Study of the Radical SAM Enzyme ArsS Involved in Arsenosugar Biosynthesis.

Arsenosugars are a group of arsenic-containing ribosides that are found predominantly in marine algae but also in terrestrial organisms. It has been proposed that arsenosugar biosynthesis involves a key intermediate 5'-deoxy-5'-dimethylarsinoyl-adenosine (DDMAA), but how DDMAA is produced remains elusive. Now, we report characterization of ArsS as a DDMAA synthase, which catalyzes a radical S-adenosylmethionine (SAM)-mediated alkylation (adenosylation) of dimethylarsenite (DMAsIII ) to produce DDMAA. This radical-mediated reaction is redox neutral, and multiple turnover can be achieved without external reductant. Phylogenomic and biochemical analyses revealed that DDMAA synthases are widespread in distinct bacterial phyla with similar catalytic efficiencies; these enzymes likely originated from cyanobacteria. This study reveals a key step in arsenosugar biosynthesis and also a new paradigm in radical SAM chemistry, highlighting the catalytic diversity of this superfamily of enzymes.

Mechanism of Pyrroloquinoline Quinone-Dependent Hydride Transfer Chemistry from Spectroscopic and High-Resolution X-ray Structural Studies of the Methanol Dehydrogenase from Methylococcus capsulatus (Bath).

The active site of methanol dehydrogenase (MDH) contains a rare disulfide bridge between adjacent cysteine residues. As a vicinal disulfide, the structure is highly strained, suggesting it might work together with the pyrroloquinoline quinone (PQQ) prosthetic group and the Ca2+ ion in the catalytic turnover during methanol (CH3OH) oxidation. We purify MDH from Methylococcus capsulatus (Bath) with the disulfide bridge broken into two thiols. Spectroscopic and high-resolution X-ray crystallographic studies of this form of MDH indicate that the disulfide bridge is redox active. We observe an internal redox process within the holo-MDH that produces a disulfide radical anion concomitant with a companion PQQ radical, as evidenced by an optical absorption at 408 nm and a magnetically dipolar-coupled biradical in the EPR spectrum. These observations are corroborated by electron-density changes between the two cysteine sulfurs of the disulfide bridge as well as between the bound Ca2+ ion and the O5-C5 bond of the PQQ in the high-resolution X-ray structure. On the basis of these findings, we propose a mechanism for the controlled redistribution of the two electrons during hydride transfer from the CH3OH in the alcohol oxidation without formation of the reduced PQQ ethenediol, a biradical mechanism that allows for possible recovery of the hydride for transfer to an external NAD+ oxidant in the regeneration of the PQQ cofactor for multiple catalytic turnovers. In support of this mechanism, a steady-state level of the disulfide radical anion is observed during turnover of the MDH in the presence of CH3OH and NAD+.

XNA enzymes by evolution and design

The last decade has witnessed tremendous growth in the field of synthetic genetics, an area of synthetic biology that applies concepts that are commonly associated with the field of genetics, such as heredity and evolution, to artificial genetic polymers with novel backbone structures (XNAs). In addition to the emergence of biologically stable affinity reagents (aptamers), progress in this area has led to the discovery of XNA enzymes (XNAzymes) that are capable of mediating transphosphorylation chemistry with multiple turnover activity. This review explores the evolution and rational design of XNAzymes as well as their potential as reagents in biomedical applications.

CRISPR technology incorporating amplification strategies: molecular assays for nucleic acids, proteins, and small molecules.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) protein systems have transformed the field of genome editing and transcriptional modulation. Progress in CRISPR–Cas technology has also advanced molecular detection of diverse targets, ranging from nucleic acids to proteins. Incorporating CRISPR–Cas systems with various nucleic acid amplification strategies enables the generation of amplified detection signals, enrichment of low-abundance molecular targets, improvements in analytical specificity and sensitivity, and development of point-of-care (POC) diagnostic techniques. These systems take advantage of various Cas proteins for their particular features, including RNA-guided endonuclease activity, sequence-specific recognition, multiple turnover trans-cleavage activity of Cas12 and Cas13, and unwinding and nicking ability of Cas9. Integrating a CRISPR–Cas system after nucleic acid amplification improves detection specificity due to RNA-guided recognition of specific sequences of amplicons. Incorporating CRISPR–Cas before nucleic acid amplification enables enrichment of rare and low-abundance nucleic acid targets and depletion of unwanted abundant nucleic acids. Unwinding of dsDNA to ssDNA using CRISPR–Cas9 at a moderate temperature facilitates techniques for achieving isothermal exponential amplification of nucleic acids. A combination of CRISPR–Cas systems with functional nucleic acids (FNAs) and molecular translators enables the detection of non-nucleic acid targets, such as proteins, metal ions, and small molecules. Successful integrations of CRISPR technology with nucleic acid amplification techniques result in highly sensitive and rapid detection of SARS-CoV-2, the virus that causes the COVID-19 pandemic.

Radical SAM Enzyme Spore Photoproduct Lyase: Properties of the Ω Organometallic Intermediate and Identification of Stable Protein Radicals Formed during Substrate-Free Turnover

Spore photoproduct lyase is a radical S-adenosyl-l-methionine (SAM) enzyme with the unusual property that addition of SAM to the [4Fe-4S]1+ enzyme absent substrate results in rapid electron transfer to SAM with accompanying homolytic S-C5' bond cleavage. Herein, we demonstrate that this unusual reaction forms the organometallic intermediate Ω in which the unique Fe atom of the [4Fe-4S] cluster is bound to C5' of the 5'-deoxyadenosyl radical (5'-dAdo•). During catalysis, homolytic cleavage of the Fe-C5' bond liberates 5'-dAdo• for reaction with substrate, but here, we use Ω formation without substrate to determine the thermal stability of Ω. The reaction of Geobacillus thermodenitrificans SPL (GtSPL) with SAM forms Ω within ∼15 ms after mixing. By monitoring the decay of Ω through rapid freeze-quench trapping at progressively longer times we find an ambient temperature decay time of the Ω Fe-C5' bond of τ ≈ 5-6 s, likely shortened by enzymatic activation as is the case with the Co-C5' bond of B12. We have further used hand quenching at times up to 10 min, and thus with multiple SAM turnovers, to probe the fate of the 5'-dAdo• radical liberated by Ω. In the absence of substrate, Ω undergoes low-probability conversion to a stable protein radical. The WT enzyme with valine at residue 172 accumulates a Val•; mutation of Val172 to isoleucine or cysteine results in accumulation of an Ile• or Cys• radical, respectively. The structures of the radical in WT, V172I, and V172C variants have been established by detailed EPR/DFT analyses.

Single-molecule transport kinetics of a glutamate transporter homolog shows static disorder.

Kinetic properties of membrane transporters are typically poorly defined because high-resolution functional assays analogous to single-channel recordings are lacking. Here, we measure single-molecule transport kinetics of a glutamate transporter homolog from Pyrococcus horikoshii, GltPh, using fluorescently labeled periplasmic amino acid binding protein as a fluorescence resonance energy transfer-based sensor. We show that individual transporters can function at rates varying by at least two orders of magnitude that persist for multiple turnovers. A gain-of-function mutant shows increased population of the fast-acting transporters, leading to a 10-fold increase in the mean transport rate. These findings, which are broadly consistent with earlier single-molecule measurements of GltPh conformational dynamics, suggest that GltPh transport is defined by kinetically distinct populations that exhibit long-lasting "molecular memory."

Marine redox variability from Baltica during extinction events in the latest Ordovician–early Silurian

It is well documented that Upper Ordovician and Silurian successions record multiple marine turnover events – including the second-largest mass extinction in the Phanerozoic – widespread glaciation, and multiple global carbon cycle perturbations. Whereas causal mechanisms for the Late Ordovician major mass extinction event involving climate, paleoceanographic variation, and δ13C records have been published, similar records remain poorly constrained for subsequent extinction events in the early Silurian. Here, we present new organic matter carbon isotope (δ13Corg) chemostratigraphy and corresponding paleoredox proxies (Fe speciation, [Mn, V, Mo], and pyrite sulfur isotopes) from two organic-rich drill core sections in Sweden and Latvia that span the upper Katian through lower Wenlock stages (446–431 Ma). Pyritized Fe and bulk sedimentary Mn concentrations from Upper Ordovician strata in southern Sweden suggest a local redox shift to more reducing conditions in the late Hirnantian, possibly including euxinic (anoxic and sulfidic water column) conditions that coincide with the second mass extinction pulse. The new high-resolution δ13Corg and δ34Spyr datasets from the late Aeronian (early Silurian) interval within both drill cores show positive excursions that are broadly coincident with the associated Sandvika and sedgwickii extinction events. Independently, Fe speciation and bulk sedimentary trace metal data from this late Aeronian interval record locally euxinic conditions in both the deep basinal (Sweden) and distal shelf (Latvia) settings before and during the late Aeronian positive δ13C excursion. This multiproxy paleoredox dataset provides the first direct evidence for local to regional expansion of reducing marine conditions coincident with this early Silurian (late Aeronian) biotic event and positive δ13C excursion. Additionally, new δ34Spyr values spanning the Llandovery/Wenlock boundary interval in the Latvia core show a positive excursion coincident with Fe speciation and trace metal enrichments that imply a local redox perturbation with intermittently euxinic bottom waters during the rising limb of the Ireviken positive δ13C excursion. The combination of these geochemical data for local- to regional-scale (more data required for global interpretations) changes in marine redox conditions with paleobiological records and evidence for eustatic sea-level rise indicate that environmental stresses related to an expansion of anoxic to euxinic conditions were a probable driver for several extinction events during the latest Ordovician–early Silurian.

Self‐limited Phosphatase‐mimicking CeO2 Nanozymes

AbstractCeO2 nanoparticles or nanoceria is a very interesting enzyme mimic (nanozyme) with a diverse range of catalytic activities. Most of the previous studies focused on its redox chemistry for mimicking oxidase, peroxidase, and catalase‐like activities, and as a scavenger of reactive oxygen species. Considering CeO2 has both Ce(III) and Ce(IV) species and both interact strongly with inorganic phosphate, nanoceria has also been studied for its phosphatase‐like activity. We herein compared these species along with alkaline phosphatase (ALP). First, CeO2 and Ce(IV) have good activity using p‐nitrophenylphosphate (p‐NPP) as a substrate, while other metal ions failed to show this activity. Since a reaction product is inorganic phosphate and we observed an interesting enzyme kinetic profile, the effect of phosphate was studied, which inhibited both CeO2 and ALP. The inhibition constant was about 5‐fold smaller for CeO2. The inhibition effect of polyphosphates was even stronger. Due to the product inhibition effect, CeO2 is unlikely to be a typical multiple turnover nanozyme, and the system is self‐limited. For the other anions, only F− showed a moderate inhibition effect on the cerium species, while adsorption of DNA did not inhibit the activity. Finally, heat treated ALP lost the activity, but CeO2 and Ce(IV) remained active. This study has deepened our understanding of CeO2 as a phosphatase mimicking nanozyme, which could be useful for biosensing and chemical biology applications.

Repair of 1, N2‐ethenoguanine by Alkyladenine DNA Glycosylase

Human DNA can be damaged by numerous endogenous processes as well as by diverse exogenous factors. The exocyclic amine groups of adenine, cytosine, and guanine can be alkylated by the reactive products of lipid peroxidation or by exposure to vinyl chloride pollutants, leading to the formation of harmful etheno adducts. These etheno lesions can be both mutagenic and cytotoxic, necessitating timely repair. Alkyladenine DNA glycosylase (AAG) has been proposed to initiate the base excision repair of both 1, N6‐ethenoadenine (ɛA) and 1, N2‐ethenoguanine (ɛG). However, examination of the structure of AAG in complex with ɛA‐DNA raises questions about how ɛG could be recognized in an active site optimized for recognition of ɛA. Systematic single and multiple turnover kinetic assays of ɛG excision by AAG reveal that ɛG is recognized far less readily than ɛA in both the natural ɛG:C context as well as in bulges and mispairs. Moreover, AAG is unable to repair ɛG in competition with a large excess of undamaged bases, calling into question how the lesion could be identified in genomic DNA. I hypothesized that the conserved active site residue, Asn169, may play a role in discriminating against the ɛG lesion. Indeed, mutation of Asn169 to Ser results in 7‐fold greater activity toward ɛG. Thus, the conserved active site residues are not optimized for recognition and repair of eG. Together these findings indicate that eG is not a preferred substrate of AAG, and other mechanisms are likely to contribute to eG repair in human cells.Support or Funding InformationFunding was provided by a graduate student award from the Department of Biological Chemistry at the University of Michigan, an NSF award MCB‐1615586 (P.J.O), and a fellowship from the Chemistry/Biology Interface Training Program supported by the NIGMS of the NIH under Grant T32GM008597 (A.Z.T.) and R01GM108022 (P.J.O.).

Subunit Interaction Dynamics of Class Ia Ribonucleotide Reductases: In Search of a Robust Assay.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides (NDP) to deoxynucleotides (dNDP), in part, by controlling the ratios and quantities of dNTPs available for DNA replication and repair. The active form of Escherichia coli class Ia RNR is an asymmetric α2β2 complex in which α2 contains the active site and β2 contains the stable diferric-tyrosyl radical cofactor responsible for initiating the reduction chemistry. Each dNDP is accompanied by disulfide bond formation. We now report that, under in vitro conditions, β2 can initiate turnover in α2 catalytically under both "one" turnover (no external reductant, though producing two dCDPs) and multiple turnover (with an external reductant) assay conditions. In the absence of reductant, rapid chemical quench analysis of a reaction of α2, substrate, and effector with variable amounts of β2 (1-, 10-, and 100-fold less than α2) yields 3 dCDP/α2 at all ratios of α2:β2 with a rate constant of 8-9 s-1, associated with a rate-limiting conformational change. Stopped-flow fluorescence spectroscopy with a fluorophore-labeled β reveals that the rate constants for subunit association (163 ± 7 μM-1 s-1) and dissociation (75 ± 10 s-1) are fast relative to turnover, consistent with catalytic β2. When assaying in the presence of an external reducing system, the turnover number is dictated by the ratio of α2:β2, their concentrations, and the concentration and nature of the reducing system; the rate-limiting step can change from the conformational gating to a step or steps involving disulfide rereduction, dissociation of the inhibited α4β4 state, or both. The issues encountered with E. coli RNR are likely of importance in all class I RNRs and are central to understanding the development of screening assays for inhibitors of these enzymes.

Synthesis of phosphoramidate-linked DNA by a modified DNA polymerase

All known polymerases copy genetic material by catalyzing phosphodiester bond formation. This highly conserved activity proceeds by a common mechanism, such that incorporated nucleoside analogs terminate chain elongation if the resulting primer strand lacks a terminal hydroxyl group. Even conservatively substituted 3'-amino nucleotides generally act as chain terminators, and no enzymatic pathway for their polymerization has yet been found. Although 3'-amino nucleotides can be chemically coupled to yield stable oligonucleotides containing N3'→P5' phosphoramidate (NP) bonds, no such internucleotide linkages are known to occur in nature. Here, we report that 3'-amino terminated primers are, in fact, slowly extended by the DNA polymerase from B. stearothermophilus in a template-directed manner. When its cofactor is Ca2+ rather than Mg2+, the reaction is fivefold faster, permitting multiple turnover NP bond formation to yield NP-DNA strands from the corresponding 3'-amino-2',3'-dideoxynucleoside 5'-triphosphates. A single active site mutation further enhances the rate of NP-DNA synthesis by an additional 21-fold. We show that DNA-dependent NP-DNA polymerase activity depends on conserved active site residues and propose a likely mechanism for this activity based on a series of crystal structures of bound complexes. Our results significantly broaden the catalytic scope of polymerase activity and suggest the feasibility of a genetic transition between native nucleic acids and NP-DNA.

A Cambrian age for the upper Bambuí Group, Brazil, supported by the first U-Pb dating of volcaniclastic bed

The Serra da Saudade Formation is part of the Bambuí Group, a cratonic cover from the western São Francisco craton. It is composed by siliciclastic sediments deposited in a foreland basin during a transgressive event. The Serra da Saudade Formation is mainly represented by rhythmites, siltstones (locally glauconitic) and rare sandstones. The glauconitic siltstone was deposited during a maximum flooding surface and sediment starvation, which allowed high concentrations of probable volcanic-sourced potassium. The interpretation of the depositional age of the Bambui Group had multiple turnovers in the last decades. Finding and dating synsedimentary volcanic rocks is essential to solve this conundrum. This paper presents the first U-Pb direct dating of the Bambuí Group from zircon grains recovered from a volcaniclastic layer interbedded with glauconitic siltstones from the upper Serra da Saudade Formation. A total of 107 U-Pb ages were obtained by LA-ICP-MS and plotted into a Concordia diagram. The provenance signature comprises a wide range of detrital zircon ages (1.0–2.8 Ga). These data represent inherited zircon grains incorporated into the magma during magmatic activity that produced the volcanic sediment, or to dilution and reworking of volcanic ashes that mixed to fine-grained clastic sediments prior to deposition. A significant amount of 10 highly concordant, prismatic zircon grains clustered into a well constrained age of 520.2 ± 5.3 Ma, which represent the explosive volcanic eruption age, and by extent, the depositional age of the upper Serra da Saudade Formation. Since the currently final thrust stacking ages from the Brasilia Belt and the Araçuaí Belt are dated ca. 600 and 540 Ma, respectively, one must carefully look for the real age of the deformation event that affected the Bambuí Group over the São Francisco craton.

Catalytic Knockdown of miR-21 by Artificial Ribonuclease: Biological Performance in Tumor Model.

Control of the expression of oncogenic small non-coding RNAs, notably microRNAs (miRNAs), is an attractive therapeutic approach. We report a design platform for catalytic knockdown of miRNA targets with artificial, sequence-specific ribonucleases. miRNases comprise a peptide [(LeuArg)2Gly]2 capable of RNA cleavage conjugated to the miRNA-targeted oligodeoxyribonucleotide, which becomes nuclease-resistant within the conjugate design, without resort to chemically modified nucleotides. Our data presented here showed for the first time a truly catalytic character of our miR-21-miRNase and its ability to cleave miR-21 in a multiple catalytic turnover mode. We demonstrate that miRNase targeted to miR-21 (miR-21-miRNase) knocked down malignant behavior of tumor cells, including induction of apoptosis, inhibition of cell invasiveness, and retardation of tumor growth, which persisted on transplantation into mice of tumor cells treated once with miR-21-miRNase. Crucially, we discover that the high biological activity of miR-21-miRNase can be directly related not only to its truly catalytic sequence-specific cleavage of miRNA but also to its ability to recruit the non-sequence specific RNase H found in most cells to elevate catalytic turnover further. miR-21-miRNase worked synergistically even with low levels of RNase H. Estimated degradation in the presence of RNase H exceeded 103 miRNA target molecules per hour for each miR-21-miRNase molecule, which provides the potency to minimize delivery requirements to a few molecules per cell. In contrast to the comparatively high doses required for the simple steric block of antisense oligonucleotides, truly catalytic inactivation of miRNA offers more effective, irreversible, and persistent suppression of many copy target sequences. miRNase design can be readily adapted to target other pathogenic microRNAs overexpressed in many disease states.

Understanding the role of electron donors in the reaction catalyzed by Tsrm, a cobalamin-dependent radical S-adenosylmethionine methylase.

The cobalamin-dependent radical S-adenosylmethionine (SAM) enzyme TsrM catalyzes the methylation of C2 of L-tryptophan to form 2-methyltryptophan during the biosynthesis of thiostrepton A. Although TsrM is a member of the radical SAM superfamily, unlike all other annotated members, it does not catalyze a reductive cleavage of SAM to a 5'-deoxyadenosyl 5'-radical intermediate. In fact, it has been proposed that TsrM catalyzes its reaction through two polar nucleophilic displacements, with its cobalamin cofactor cycling directly between methylcobalamin (MeCbl) and cob(I)alamin. Nevertheless, the enzyme has been stated to require the action of a reductant, which can be satisfied by dithiothreitol. By contrast, all other annotated RS enzymes require a reductant that exhibits a much lower reduction potential, which is necessary for the reductive cleavage of SAM. Herein, we show that TsrM can catalyze multiple turnovers in the absence of any reducing agent, but only when it is pre-loaded with MeCbl. When hydroxocobalamin (OHCbl) or cob(II)alamin is bound to TsrM, a reductant is required to convert it to cob(I)alamin, which can acquire a methyl group directly from SAM. Our studies suggest that TsrM uses an external reductant to prime its reaction by converting bound OHCbl or cob(II)alamin to MeCbl, and to regenerate the MeCbl form of the cofactor upon adventitious oxidation of the cob(I)alamin intermediate to cob(II)alamin.

Carbon-Nanotube-Appended PAMAM Dendrimers Bearing Iron(II) α-Keto Acid Complexes: Catalytic Non-Heme Oxygenase Models.

Poly(amidoamine) dendrimers grafted on carbon nanotubes have been appended with iron(II)-α-keto acid (benzoylformate) complex of polypyridyl ligand to design artificial non-heme oxygenase model. This nano-enzyme was applied for selective catalytic oxidation of organic molecules. Although the carbon nanotubes serve as a robust heterogeneous platform, the amine terminals of dendrimers provide catalysts binding sites and the amide bonds provide a necessary second coordination sphere similar to the enzymatic polypeptide chains. Such a hybrid design prevented the deactivation of the primary active sites leading to 8 times faster oxidative decarboxylation rates than those of its homogeneous analogue. An electrophilic iron(IV)-oxo intermediate has been intercepted, which catalyzes the selective oxidation of alcohols to aldehydes and incorporates single oxygen atoms into sulfides and olefins by using aerial oxygen with multiple turnover numbers. The catalyst was consecutively regenerated three times by mild chemical treatment and showed negligible loss of activity.

PGAM5 is a key driver of mitochondrial dysfunction in experimental lung fibrosis.

Mitochondrial homeostasis has recently emerged as a focal point in the pathophysiology of idiopathic pulmonary fibrosis (IPF), but conflicting data have been reported regarding its regulation. We speculated that phosphoglycerate mutase family member 5 (PGAM5), a mitochondrial protein at the intersection of multiple cell death and mitochondrial turnover pathways, might be involved inthe pathogenesis of IPF. PGAM5-deficient mice and human pulmonary epithelial cells were analyzed comparatively with PGAM5-proficient controls in a bleomycin-based model of pulmonary fibrogenesis. Mitochondria were visualized by confocal and transmission electron microscopy. Mitochondrial homeostasis was assessed using JC1 (ΔΨ) and flow cytometry. PGAM5 plays an important role in pulmonary fibrogenesis. Pgam5-/- mice displayed significantly attenuated lung fibrosis compared to controls. Complementary, in vitro studies demonstrated that PGAM5 impaired mitochondrial integrity on a functional and structural level independently of mtROS-production. On a molecular level, reduced mitophagy caused by PGAM5 deficiency improved mitochondrial homeostasis. Our study identifies PGAM5 as an important regulator of mitochondrial homeostasis in pulmonary fibrosis. Our data further indicate PGAM5-mediated mitophagy itself as a pivotal gateway event in the mediation of self-sustaining mitochondrial damage and membrane depolarization. Our work hereby highlights the importance of mitochondrial dynamics and identifies a potential therapeutic target that warrants further studies. Toxic agents lead to mitochondrial damage resulting in depolarization of the mitochondrial membrane potential (ΔΨ) which is a gateway event for the initiation of PGAM5-mediated mitophagy. PGAM5-mediated mitophagy in turn leads to a self-perpetuating escalation of ΔΨ depolarization. Loss of the mitophagy-based damage-enhancing loop under PGAM5-deficient conditions breaks this vicious cycle, leading to improved mitochondrial homeostasis.

Biocatalytic Carbon-Hydrogen and Carbon-Fluorine Bond Cleavage through Hydroxylation Promoted by a Histidyl-Ligated Heme Enzyme.

LmbB2 is a peroxygenase-like enzyme that hydroxylates L-tyrosine to L-3,4-dihydroxyphenylalanine (DOPA) in the presence of hydrogen peroxide. However, its heme cofactor is ligated by a proximal histidine, not cysteine. We show that LmbB2 can oxidize L-tyrosine analogs with ring-deactivated substituents such as 3-nitro-, fluoro-, chloro-, iodo-L-tyrosine. We also found that the 4-hydroxyl group of the substrate is essential for reacting with the heme-based oxidant and activating the aromatic C-H bond. The most interesting observation of this study was obtained with 3-fluoro-L-tyrosine as a substrate and mechanistic probe. The LmbB2-mediated catalytic reaction yielded two hydroxylated products with comparable populations, i.e., oxidative C-H bond cleavage at C5 to generate 3-fluoro-5-hydroxyl-L-tyrosine and oxygenation at C3 concomitant with a carbon-fluorine bond cleavage to yield DOPA and fluoride. An iron protein-mediated hydroxylation on both C-H and C-F bonds with multiple turnovers is unprecedented. Thus, this finding reveals a significant potential of biocatalysis in C-H/C-X bond (X = halogen) cleavage. Further 18O-labeling results suggest that the source of oxygen for hydroxylation is a peroxide, and that a commonly expected oxidation by a high-valent iron intermediate followed by hydrolysis is not supported for the C-F bond cleavage. Instead, the C-F bond cleavage is proposed to be initiated by a nucleophilic aromatic substitution mediated by the iron-hydroperoxo species. Based on the experimental results, two mechanisms are proposed to explain how LmbB2 hydroxylates the substrate and cleaves C-H/C-F bond. This study broadens the understanding of heme enzyme catalysis and sheds light on enzymatic applications in medicinal and environmental fields.

Probing the Cys-Tyr Cofactor Biogenesis in Cysteine Dioxygenase by the Genetic Incorporation of Fluorotyrosine.

Cysteine dioxygenase (CDO) is a nonheme iron enzyme that adds two oxygen atoms from dioxygen to the sulfur atom of l-cysteine. Adjacent to the iron site of mammalian CDO, there is a post-translationally generated Cys-Tyr cofactor, whose presence substantially enhances the oxygenase activity. The formation of the Cys-Tyr cofactor in CDO is an autocatalytic process, and it is challenging to study by traditional techniques because the cross-linking reaction is a side, uncoupled, single-turnover oxidation buried among multiple turnovers of l-cysteine oxygenation. Here, we take advantage of our recent success in obtaining a purely uncross-linked human CDO due to site-specific incorporation of 3,5-difluoro-l-tyrosine (F2-Tyr) at the cross-linking site through the genetic code expansion strategy. Using EPR spectroscopy, we show that nitric oxide (•NO), an oxygen surrogate, similarly binds to uncross-linked F2-Tyr157 CDO as in wild-type human CDO. We determined X-ray crystal structures of uncross-linked F2-Tyr157 CDO and mature wild-type CDO in complex with both l-cysteine and •NO. These structural data reveal that the active site cysteine (Cys93 in the human enzyme), rather than the generally expected tyrosine (i.e., Tyr157), is well-aligned to be oxidized should the normal oxidation reaction uncouple. This structure-based understanding is further supported by a computational study with models built on the uncross-linked ternary complex structure. Together, these results strongly suggest that the first target to oxidize during the iron-assisted Cys-Tyr cofactor biogenesis is Cys93. Based on these data, a plausible reaction mechanism implementing a cysteine radical involved in the cross-link formation is proposed.