Retron-Eco1 assembles NAD+-hydrolyzing filaments that provide immunity against bacteriophages.

Retrons are toxin-antitoxin systems protecting bacteria against bacteriophages via abortive infection. The Retron-Eco1 antitoxin is formed by a reverse transcriptase (RT) and a non-coding RNA (ncRNA)/multi-copy single-stranded DNA (msDNA) hybrid that neutralizes an uncharacterized toxic effector. Yet, the molecular mechanisms underlying phage defense remain unknown. Here, we show that the N-glycosidase effector, which belongs to the STIR superfamily, hydrolyzes NAD+ during infection. Cryoelectron microscopy (cryo-EM) analysis shows that the msDNA stabilizes a filament that cages the effector in a low-activity state in which ADPr, a NAD+ hydrolysis product, is covalently linked to the catalytic E106 residue. Mutations shortening the msDNA induce filament disassembly and the effector's toxicity, underscoring the msDNA role in immunity. Furthermore, we discovered a phage-encoded Retron-Eco1 inhibitor (U56) that binds ADPr, highlighting the intricate interplay between retron systems and phage evolution. Our work outlines the structural basis of Retron-Eco1 defense, uncovering ADPr's pivotal role in immunity.

The multistep oxidation of cholesterol to pregnenolone by human cytochrome P450 11A1 is highly processive.

Cytochrome P450 (P450, CYP) 11A1 is the classical cholesterol side chain cleavage enzyme (P450scc) that removes six carbons of the side chain, the first and rate-limiting step in the synthesis of all mammalian steroids. The reaction is a 3-step, 6-electron oxidation that proceeds via formation of 22R-hydroxy (OH) and 20R,22R-(OH)2 cholesterol, yielding pregnenolone. We expressed human P450 11A1 in bacteria, purified the enzyme in the absence of nonionic detergents, and assayed pregnenolone formation by HPLC-mass spectrometry of the dansyl hydrazone. The reaction was inhibited by the nonionic detergent Tween 20, and several lipids did not enhance enzymatic activity. The 22R-OH and 20R,22R-(OH)2 cholesterol intermediates were bound to P450 11A1 relatively tightly, as judged by steady-state optical titrations and koff rates. The electron donor adrenodoxin had little effect on binding; the substrate cholesterol showed a ∼5-fold stimulatory effect on the binding of adrenodoxin to P450 11A1. Presteady-state single-turnover kinetic analysis was consistent with a highly processive reaction with rates of intermediate oxidation steps far exceeding dissociation rates for products and substrates. The presteady-state kinetic analysis revealed a second di-OH cholesterol product, separable by HPLC, in addition to 20R,22R-(OH)2 cholesterol, which we characterized as a rotamer that was also converted to pregnenolone at a similar rate. The first oxidation step (at C-22) is the slowest, limiting the overall rate of cleavage. d3-Cholesterol showed no kinetic deuterium isotope effect on C-22, indicating that C-H bond cleavage is not rate-limiting in the first hydroxylation step.

Ninety-eight semesters of cytochrome P450 enzymes and related topics-What have I taught and learned?

This Reflection article begins with my family background and traces my career through elementary and high school, followed by time at the University of Illinois, Vanderbilt University, the University of Michigan, and then for 98 semesters as a Vanderbilt University faculty member. My research career has dealt with aspects of cytochrome P450 enzymes, and the basic biochemistry has had applications in fields as diverse as drug metabolism, toxicology, medicinal chemistry, pharmacogenetics, biological engineering, and bioremediation. I am grateful for the opportunity to work with the Journal of Biological Chemistry not only as an author but also for 34 years as an Editorial Board Member, Associate Editor, Deputy Editor, and interim Editor-in-Chief. Thanks are extended to my family and my mentors, particularly Profs. Harry Broquist and Minor J. Coon, and the more than 170 people who have trained with me. I have never lost the enthusiasm for research that I learned in the summer of 1968 with Harry Broquist, and I have tried to instill this in the many trainees I have worked with. A sentence I use on closing slides is "It's not just a laboratory-it's a fraternity."

Temperature-Sensitive Substrate and Product Binding Underlie Temperature-Compensated Phosphorylation in the Clock.

Temperature compensation is a striking feature of the circadian clock. Here we investigate biochemical mechanisms underlying temperature-compensated, CKIδ-dependent multi-site phosphorylation in mammals. We identify two mechanisms for temperature-insensitive phosphorylation at higher temperature: lower substrate affinity to CKIδ-ATP complex and higher product affinity to CKIδ-ADP complex. Inhibitor screening of ADP-dependent phosphatase activity of CKIδ identified aurintricarboxylic acid (ATA) as a temperature-sensitive kinase activator. Docking simulation of ATA and mutagenesis experiment revealed K224D/K224E mutations in CKIδ that impaired product binding and temperature-compensated primed phosphorylation. Importantly, K224D mutation shortens behavioral circadian rhythms and changes the temperature dependency of SCN's circadian period. Interestingly, temperature-compensated phosphorylation was evolutionary conserved in yeast. Molecular dynamics simulation and X-ray crystallography demonstrate that an evolutionally conserved CKI-specific domain around K224 can provide a structural basis for temperature-sensitive substrate and product binding. Surprisingly, this domain can confer temperature compensation on a temperature-sensitive TTBK1. These findings suggest the temperature-sensitive substrate- and product-binding mechanisms underlie temperature compensation.

Murine HSD17ß13 does not control liver steatosis and modestly impacts fibrosis in a sex- and diet-specific manner.

Human genetic studies show that loss of function mutations in 17-Beta hydroxysteroid dehydrogenase (HSD17ß13) are associated with protection from non-alcoholic steatohepatitis (NASH). As a result, therapies that reduce HSD17ß13 are being pursued for the treatment of NASH. However, inconsistent effects on steatosis, inflammation, and fibrosis pathogenesis have been reported in murine Hsd17b13 knockdown or knockout models. To clarify whether murine Hsd17b13 loss regulates liver damage and fibrosis, we characterized Hsd17b13 knockout mice subjected to pro-NASH diets or pro-inflammatory chemical-induced liver injury. There were no effects of Hsd17b13 loss on liver injury, inflammation, fibrosis, or lipids after 28 weeks on the Gubra-Amylin NASH (GAN) diet or 12 weeks on a 45% choline-deficient high-fat diet (CDAHFD). However, AAV-mediated re-expression of murine Hsd17b13 in KO mice increased liver macrophage abundance in both sexes fed the 45% CDAHFD. In contrast, there was a modest reduction in liver fibrosis, but not lipids or inflammation within Hsd17b13 null female, but not male, mice after 12 weeks of a 60% CDAHFD compared to WT littermates. Fibrosis and the abundance of liver macrophages were increased in Hsd17b13 KO females upon adenoviral re-expression of mouse HSD17ß13, but this was not reflected in inflammatory markers. Additionally, we found minimal differences in liver injury, lipids, or inflammatory and fibrotic markers 48 h after acute CCl4 exposure. In summary, murine Hsd17b13 loss has modest diet- and sex-specific effects on liver fibrosis which contrasts with human genetic studies. This suggests a disconnect between the biological function of HSD17ß13 in mice and humans.

The inhibitor of κB kinase ß (IKKß) phosphorylates IκBα twice in a single binding event through a sequential mechanism.

Phosphorylation of Inhibitor of κB (IκB) proteins by IκB Kinase ß (IKKß) leads to IκB degradation and subsequent activation of nuclear factor κB transcription factors. Of particular interest is the IKKß-catalyzed phosphorylation of IκBα residues Ser32 and Ser36 within a conserved destruction box motif. To investigate the catalytic mechanism of IKKß, we performed pre-steady-state kinetic analysis of the phosphorylation of IκBα protein substrates catalyzed by constitutively active, human IKKß. Phosphorylation of full-length IκBα catalyzed by IKKß was characterized by a fast exponential phase followed by a slower linear phase. The maximum observed rate (kp) of IKKß-catalyzed phosphorylation of IκBα was 0.32 s-1 and the binding affinity of ATP for the IKKß•IκBα complex (Kd) was 12 µM. Substitution of either Ser32 or Ser36 with Ala, Asp, or Cys reduced the amplitude of the exponential phase by approximately 2-fold. Thus, the exponential phase was attributed to phosphorylation of IκBα at Ser32 and Ser36, whereas the slower linear phase was attributed to phosphorylation of other residues. Interestingly, the exponential rate of phosphorylation of the IκBα(S32D) phosphomimetic amino acid substitution mutant was nearly twice that of WT IκBα and 4-fold faster than any of the other IκBα amino acid substitution mutants, suggesting that phosphorylation of Ser32 increases the phosphorylation rate of Ser36. These conclusions were supported by parallel experiments using GST-IκBα(1-54) fusion protein substrates bearing the first 54 residues of IκBα. Our data suggest a model wherein, IKKß phosphorylates IκBα at Ser32 followed by Ser36 within a single binding event.

The Stereochemical Course of DmdC, an Enzyme Involved in the Degradation of Dimethylsulfoniopropionate.

The acyl-CoA dehydrogenase DmdC is involved in the degradation of the marine sulfur metabolite dimethylsulfonio propionate (DMSP) through the demethylation pathway. The stereochemical course of this reaction was investigated through the synthesis of four stereoselectively deuterated substrate surrogates carrying stereoselective deuterations at the α- or the ß-carbon. Analysis of the products revealed a specific abstraction of the 2-pro-R proton and of the 3-pro-S hydride, establishing an anti elimination for the DmdC reaction.

Enzymatic Synthesis of Diterpenoids from iso-GGPP†III: A Geranylgeranyl Diphosphate Analog with a Shifted Double Bond.

The analog of the diterpene precursor geranylgeranyl diphosphate with a double bond shifted from C14=C15 to C15=C16 (named iso-GGPP III) has been synthesized and enzymatically converted with six bacterial diterpene synthases; this allowed the isolation of nine unnatural diterpenes. For some of the enzyme-substrate combinations, the different reactivity implemented in the substrate analog iso-GGPP III opened reaction pathways that are not observed with natural GGPP, resulting in the formation of diterpenes with novel skeletons. A stereoselective deuteration strategy was used to assign the absolute configurations of the isolated diterpenes.

Biocatalytic Ether Lipid Synthesis by an Archaeal Glycerolprenylase.

Although ethers are common in secondary natural products, they are an underrepresented functional group in primary metabolism. As such, there are comparably few enzymes capable of constructing ether bonds in a general fashion. However, such enzymes are highly sought after for synthetic applications as they typically operate with higher regioselectivity and under milder conditions than traditional organochemical approaches. To expand the repertoire of well characterized ether synthases, we herein report on a promiscuous archaeal prenyltransferase from the scarcely researched family of geranylgeranylglyceryl phosphate synthases (GGGPSs or G3PSs). We show that the ultrastable Archaeoglobus fulgidus G3PS makes various (E)- and (Z)-configured prenyl glycerol ethers from the corresponding pyrophosphates while exerting perfect control over the configuration at the glycerol unit. Based on experimental and computational data, we propose a mechanism for this enzyme which involves an intermediary prenyl carbocation equivalent. As such, this study provides the fundamental understanding and methods to introduce G3PSs into the biocatalytic alkylation toolbox.

Common Biosynthesis of Non-Canonical C16 Terpenes through a Fragmentation-Recombination Mechanism.

The biosynthesis of six recently reported non-canonical C16 sesquiterpenoids named after ancient Greek philosophers, archimedene, aristotelene, eratosthenene, pythagorene, α-democritene and anaximandrene, was investigated through density functional theory (DFT) calculations and isotopic labeling experiments. The results revealed for all compounds except archimedene a unique fragmentation-recombination mechanism as previously demonstrated for sodorifen biosynthesis, in addition to a remarkable "dancing" mechanism for anaximandrene biosynthesis.

A Functional Switch Between Asperfumene and Fusicoccadiene Synthase and Entrance to Asperfumene Biosynthesis through a Vicinal Deprotonation-Reprotonation Process.

The diterpene synthase AfAS was identified from Aspergillus fumigatiaffinis. Its amino acid sequence and-according to a structural model-active site architecture are highly similar to those of the fusicocca-2,10(14)-diene synthase PaFS, but AfAS produces a structurally much more complex diterpene with a novel 6-5-5-5 tetracyclic skeleton called asperfumene. The cyclisation mechanism of AfAS was elucidated through isotopic labelling experiments and DFT calculations. The reaction cascade proceeds in its initial steps through similar intermediates as for the PaFS cascade, but then diverges through an unusual vicinal deprotonation-reprotonation process that triggers a skeletal rearrangement at the entrance to the steps leading to the unique asperfumene skeleton. The structural model revealed only one major difference between the active sites: The PaFS residue F65 is substituted by I65 in AfAS. Intriguingly, site-directed mutagenesis experiments with both diterpene synthases revealed that position 65 serves as a bidirectional functional switch for the biosynthesis of tetracyclic asperfumene versus structurally less complex diterpenes.

Biosynthesis of the Non-Canonical C17 Sesquiterpenoids Chlororaphen A and B from Pseudomonas Chlororaphis.

Chlororaphens A and B are structurally unique non-canonical C17 sesquiterpenoids from Pseudomonas chlororaphis that are made by two SAM-dependent methyltransferases and a type I terpene synthase. This study addresses the mechanism of their formation in isotopic labelling experiments and DFT calculations. The results demonstrate an astonishing complexity with distribution of labellings within a cyclopentane core that is reversely connected to two acyclic fragments in chlororaphen A and B. In addition, the uptake of up to 14 deuterium atoms from D2O was observed. These findings are explainable by a repeated late stage multistep rearrangement sequence. The absolute configurations of the chlororaphens and their biosynthetic intermediates were elucidated in stereoselective labelling experiments.

Oxygen-18 Labeling Reveals a Mixed Fe-O Mechanism in the Last Step of Cytochrome P450 51 Sterol 14α-Demethylation.

The 14α-demethylation step is critical in eukaryotic sterol biosynthesis, catalyzed by cytochrome P450 (P450) Family 51 enzymes, for example, with lanosterol in mammals. This conserved three-step reaction terminates in a C-C cleavage step that generates formic acid, the nature of which has been controversial. Proposed mechanisms involve roles of P450 Compound 0 (ferric peroxide anion, FeO2 - ) or Compound I (perferryl oxygen, FeO3+ ) reacting with either the aldehyde or its hydrate, respectively. Analysis of 18 O incorporation into formic acid from 18 O2 provides a means of distinguishing the two mechanisms. Human P450 51A1 incorporated 88 % 18 O (one atom) into formic acid, consistent with a major but not exclusive FeO2 - mechanism. Two P450 51 orthologs from amoeba and yeast showed similar results, while two orthologs from pathogenic trypanosomes showed roughly equal contributions of both mechanisms. An X-ray crystal structure of the human enzyme showed the aldehyde oxygen atom 3.5 Šaway from the heme iron atom. Experiments with human P450 51A1 and H2 18 O yielded primarily one 18 O atom but 14 % of the formic acid product with two 18 O atoms, indicative of a minor contribution of a Compound I mechanism. LC-MS evidence for a Compound 0-derived Baeyer-Villiger reaction product (a 14α-formyl ester) was also found.

Flavin-N5OOH Functions as both a Powerful Nucleophile and a Base in the Superfamily of Flavoenzymes.

Flavoenzymes can mediate a large variety of oxidation reactions through the activation of oxygen. However, the O2 activation chemistry of flavin enzymes is not yet fully exploited. Normally, the O2 activation occurs at the C4a site of the flavin cofactor, yielding the flavin C4a-(hydro)hydroperoxyl species in monooxygenases or oxidases. Using extensive MD simulations, QM/MM calculations and QM calculations, our studies reveal the formation of the common nucleophilic species, Flavin-N5OOH, in two distinct flavoenzymes (RutA and EncM). Our studies show that Flavin-N5OOH acts as a powerful nucleophile that promotes C-N cleavage of uracil in RutA, and a powerful base in the deprotonation of substrates in EncM. We reason that Flavin-N5OOH can be a common reactive species in the superfamily of flavoenzymes, which accomplish generally selective general base catalysis and C-X (X=N, S, Cl, O) cleavage reactions that are otherwise challenging with solvated hydroxide ion base. These results expand our understanding of the chemistry and catalysis of flavoenzymes.

Molecular and Structural Basis for Cγ-C Bond Formation by PLP-Dependent Enzyme Fub7.

Pyridoxal 5'-phosphate (PLP)-dependent enzymes that catalyze γ-replacement reactions are prevalent, yet their utilization of carbon nucleophile substrates is rare. The recent discovery of two PLP-dependent enzymes, CndF and Fub7, has unveiled unique C-C bond forming capabilities, enabling the biocatalytic synthesis of alkyl- substituted pipecolic acids from O-acetyl-L-homoserine and ß-keto acid or aldehyde derived enolates. This breakthrough presents fresh avenues for the biosynthesis of pipecolic acid derivatives. However, the catalytic mechanisms of these enzymes remain elusive, and a dearth of structural information hampers their extensive application. Here, we have broadened the catalytic scope of Fub7 by employing ketone-derived enolates as carbon nucleophiles, revealing Fub7's capacity for substrate-dependent regioselective α-alkylation of unsymmetrical ketones. Through an integrated approach combining X-ray crystallography, spectroscopy, mutagenesis, and computational docking studies, we offer a detailed mechanistic insight into Fub7 catalysis. Our findings elucidate the structural basis for its substrate specificity, stereoselectivity, and regioselectivity. Our work sets the stage ready for subsequent protein engineering effort aimed at expanding the synthetic utility of Fub7, potentially unlocking novel methods to access a broader array of noncanonical amino acids.

Roles of Ferric Peroxide Anion Intermediates (Fe3+O2 -, Compound 0) in Cytochrome P450 19A1 Steroid Aromatization and a Cytochrome P450 2B4 Secosteroid Oxidation Model.

Cytochrome P450 (P450, CYP) 19A1 is the steroid aromatase, the enzyme responsible for the 3-step conversion of androgens (androstenedione or testosterone) to estrogens. The final step is C-C bond scission (removing the 19-oxo group as formic acid) that proceeds via a historically controversial reaction mechanism. The two competing mechanistic possibilities involve a ferric peroxide anion (Fe3+O2 -, Compound 0) and a perferryl oxy species (FeO3+, Compound I). One approach to discern the role of each species in the reaction is with the use of oxygen-18 labeling, i.e., from 18O2 and H2 18O of the reaction product formic acid. We applied this approach, using several technical improvements, to study the deformylation of 19-oxo-androstenedione by human P450 19A1 and of a model secosteroid, 3-oxodecaline-4-ene-10-carboxaldehyde (ODEC), by rabbit P450 2B4. Both aldehyde substrates were sensitive to non-enzymatic acid-catalyzed deformylation, yielding 19-norsteroids, and conditions were established to avoid issues with artifactual generation of formic acid. The Compound 0 reaction pathway predominated (i.e., Fe3+O2 -) in both P450 19A1 oxidation of 19-oxo-androstenedione and P450 2B4 oxidation of ODEC. The P450 19A1 results contrast with our prior conclusions (J. Am. Chem. Soc. 2014, 136, 15016-16025), attributed to several technical modifications.

Substrate specificity of a ketosynthase domain involved in bacillaene biosynthesis.

An isotopic labelling method was developed to investigate substrate binding by ketosynthases, exemplified by the second ketosynthase of the polyketide synthase BaeJ involved in bacillaene biosynthesis (BaeJ-KS2). For this purpose, both enantiomers of a 13C-labelled N-acetylcysteamine thioester (SNAC ester) surrogate of the proposed natural intermediate of BaeJ-KS2 were synthesised, including an enzymatic step with glutamate decarboxylase, and incubated with BaeJ-KS2. Substrate binding was demonstrated through 13C NMR analysis of the products against the background of various control experiments.

A Case of Convergent Evolution: The Bacterial Sesquiterpene Synthase for 1-epi-Cubenol from Nonomuraea coxensis.

A terpene synthase from Nonomuraea coxensis was identified as (+)-1-epi-cubenol synthase. The enzyme is phylogenetically unrelated to the known enzyme of the same function that is widespread in streptomycetes. Isotopic labelling experiments were performed to unambiguously assign the NMR data and to investigate hydrogen migrations during terpene cyclisations. Epoxidations of (+)-1-epi-cubenol and of the plant derived compounds (-)-cubenol and (-)-1-epi-cubenol confirmed the structure of a natural product isolated from the brown alga Dictyopteris divaricata and allowed to conclude on its absolute configuration. The crystal structures of the epoxides from (+)- and (-)-1-epi-cubenol and the acid catalysed conversion into an isomeric ketone are reported.

Mechanistic Characterisation of Collinodiene Synthase, a Diterpene Synthase from Streptomyces collinus.

Two homologs of the diterpene synthase CotB2 from Streptomyces collinus (ScCotB2) and Streptomyces iakyrus (SiCotB2) were investigated for their products by in vitro incubations of the recombinant enzymes with geranylgeranyl pyrophosphate, followed by compound isolation and structure elucidation by NMR. ScCotB2 produced the new compound collinodiene, besides the canonical CotB2 product cyclooctat-9-en-7-ol, dolabella-3,7,18-triene and dolabella-3,7,12-triene, while SiCotB2 gave mainly cyclooctat-9-en-7-ol and only traces of dolabella-3,7,18-triene. The cyclisation mechanism towards the ScCotB2 products and their absolute configurations were investigated through isotopic labelling experiments.

Diverse enzymatic chemistry for propionate side chain cleavages in tetrapyrrole biosynthesis.

Tetrapyrroles represent a unique class of natural products that possess diverse chemical architectures and exhibit a broad range of biological functions. Accordingly, they attract keen attention from the natural product community. Many metal-chelating tetrapyrroles serve as enzyme cofactors essential for life, while certain organisms produce metal-free porphyrin metabolites with biological activities potentially beneficial for the producing organisms and for human use. The unique properties of tetrapyrrole natural products derive from their extensively modified and highly conjugated macrocyclic core structures. Most of these various tetrapyrrole natural products biosynthetically originate from a branching point precursor, uroporphyrinogen III, which contains propionate and acetate side chains on its macrocycle. Over the past few decades, many modification enzymes with unique catalytic activities, and the diverse enzymatic chemistries employed to cleave the propionate side chains from the macrocycles, have been identified. In this review, we highlight the tetrapyrrole biosynthetic enzymes required for the propionate side chain removal processes and discuss their various chemical mechanisms. ONE-SENTENCE SUMMARY: This mini-review describes various enzymes involved in the propionate side chain cleavages during the biosynthesis of tetrapyrrole cofactors and secondary metabolites.