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1. Evidence for the Chemical Mechanism of RibB (3,4-Dihydroxy-2-butanone 4-phosphate Synthase) of Riboflavin Biosynthesis

   https://doi.org/10.1021/jacs.2c03376  

   Kenjić, Nikola; Meneely, Kathleen M.; Wherritt, Daniel J.; Denler, Melissa C.; Jackson, Timothy A.; Moran, Graham R.; Lamb, Audrey L. (July 2022, Journal of the American Chemical Society)

   RibB (3,4-dihydroxy-2-butanone 4-phosphate synthase) is a magnesium-dependent enzyme that excises the C4 of d-ribulose-5-phosphate (d-Ru5P) as formate. RibB generates the four-carbon substrate for lumazine synthase that is incorporated into the xylene moiety of lumazine and ultimately the riboflavin isoalloxazine. The reaction was first identified by Bacher and co-workers in the 1990s, and their chemical mechanism hypothesis became canonical despite minimal direct evidence. X-ray crystal structures of RibB typically show two metal ions when solved in the presence of non-native metals and/or liganding non-substrate analogues, and the consensus hypothetical mechanism has incorporated this cofactor set. We have used a variety of biochemical approaches to further characterize the chemistry catalyzed by RibB from Vibrio cholera (VcRibB). We show that full activity is achieved at metal ion concentrations equal to the enzyme concentration. This was confirmed by electron paramagnetic resonance of the enzyme reconstituted with manganese and crystal structures liganded with Mn2+ and a variety of sugar phosphates. Two transient species prior to the formation of products were identified using acid quench of single turnover reactions in combination with NMR for singly and fully 13C-labeled d-Ru5P. These data indicate that dehydration of C1 forms the first transient species, which undergoes rearrangement by a 1,2 migration, fusing C5 to C3 and generating a hydrated C4 that is poised for elimination as formate. Structures determined from time-dependent Mn2+ soaks of VcRibB-d-Ru5P crystals show accumulation in crystallo of the same intermediates. Collectively, these data reveal for the first time crucial transient chemical states in the mechanism of RibB. 

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2. Reconstruction and identification of the native PLP synthase complex from Methanosarcina acetivorans lysate

   https://doi.org/10.1101/2024.07.09.602819  

   Agnew, Angela; Humm, Ethan; Zhou, Kang; Gunsalus, Robert P; Zhou, Z Hong (July 2024, bioRxiv)

   Abstract Many protein-protein interactions behave differently in biochemically purified forms as compared to theirin vivostates. As such, determining native protein structures may elucidate structural states previously unknown for even well-characterized proteins. Here we apply the bottom-up structural proteomics method,cryoID, toward a model methanogenic archaeon. While they are keystone organisms in the global carbon cycle and active members of the human microbiome, there is a general lack of characterization of methanogen enzyme structure and function. Through thecryoIDapproach, we successfully reconstructed and identified the nativeMethanosarcina acetivoranspyridoxal 5’-phosphate (PLP) synthase (PdxS) complex directly from cryogenic electron microscopy (cryoEM) images of fractionated cellular lysate. We found that the native PdxS complex exists as a homo-dodecamer of PdxS subunits, and the previously proposed supracomplex containing both the synthase (PdxS) and glutaminase (PdxT) was not observed in cellular lysate. Our structure shows that the native PdxS monomer fashions a single 8α/8β TIM-barrel domain, surrounded by seven additional helices to mediate solvent and interface contacts. A density is present at the active site in the cryoEM map and is interpreted as ribose 5-phosphate. In addition to being the first reconstruction of the PdxS enzyme from a heterogeneous cellular sample, our results reveal a departure from previously published archaeal PdxS crystal structures, lacking the 37 amino acid insertion present in these prior cases. This study demonstrates the potential of applying thecryoIDworkflow to capture native structural states at atomic resolution for archaeal systems, for which traditional biochemical sample preparation is nontrivial. 

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3. Transient-State Analysis of Porcine Dihydropyrimidine Dehydrogenase Reveals Reductive Activation by NADPH

   https://doi.org/10.1021/acs.biochem.0c00223  

   Beaupre, Brett A.; Forouzesh, Dariush C.; Moran, Graham R. (July 2020, Biochemistry)

   Dihydropyrimidine dehydrogenase (DPD) catalyzes the initial step in the catabolism of the pyrimidines uracil and thymine. Crystal structures have revealed an elaborate subunit architecture consisting of two flavin cofactors, apparently linked by four Fe4S4 centers. Analysis of the DPD reaction(s) equilibrium position under anaerobic conditions revealed a reaction that favors dihydropyrimidine formation. Single-turnover analysis shows biphasic kinetics. The serine variant of the candidate general acid, cysteine 671, provided enhanced kinetic resolution for these phases. In the first event, one subunit of the DPD dimer takes up two electrons from NADPH in a reductive activation step. Spectrophotometric deconvolution suggests that thes electrons reside on one of the two flavins. That oxidation of the enzyme by dioxygen can be suppressed by the addition of pyrimidine, is consistent with these electrons residing on the FMN. The second phase involves further oxidation of NADPH and concomitant reduction of the pyrimidine substrate. During this phase no net reduction of DPD cofactors is observed indicating that the entire cofactor set acts as a wire, transmitting electrons from NADPH to the pyrimidine rapidly. This indicates that the availability of the proton from C671 general acid controls the transmittance of electrons from NADPH to the pyrimidine. Acid quench and HPLC product analysis of single-turnover reactions with limiting NADPH confirmed 2:1, NADPH:pyrimidine stoichiometry for the enzyme accounting for successive activation and pyrimidine reduction. These data support an alternating subunit model in which one protomer is activated and turns over before the other subunit can be activated and enter catalysis. 

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4. Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate

   https://doi.org/10.1073/pnas.2109235119  

   Holmes, Jacob B.; Liu, Viktoriia; Caulkins, Bethany G.; Hilario, Eduardo; Ghosh, Rittik K.; Drago, Victoria N.; Young, Robert P.; Romero, Jennifer A.; Gill, Adam D.; Bogie, Paul M.; et al (January 2022, Proceedings of the National Academy of Sciences)

   NMR-assisted crystallography—the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry—holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5′-phosphate–dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate β-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid–base catalytic residue βLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate C β and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does. 

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5. A single diiron enzyme catalyses the oxidative rearrangement of tryptophan to indole nitrile

   https://doi.org/10.1038/s41557-024-01603-z  

   Adak, Sanjoy; Ye, Naike; Calderone, Logan A; Duan, Meng; Lubeck, Wilson; Schäfer, Rebecca_J B; Lukowski, April L; Houk, K N; Pandelia, Maria-Eirini; Drennan, Catherine L; et al (December 2024, Nature Chemistry)

   Abstract Nitriles are uncommon in nature and are typically constructed from oximes through the oxidative decarboxylation of amino acid substrates or from the derivatization of carboxylic acids. Here we report a third nitrile biosynthesis strategy featuring the cyanobacterial nitrile synthase AetD. During the biosynthesis of the eagle-killing neurotoxin, aetokthonotoxin, AetD transforms the 2-aminopropionate portion of 5,7-dibromo-l-tryptophan to a nitrile. Employing a combination of structural, biochemical and biophysical techniques, we characterized AetD as a non-haem diiron enzyme that belongs to the emerging haem-oxygenase-like dimetal oxidase superfamily. High-resolution crystal structures of AetD together with the identification of catalytically relevant products provide mechanistic insights into how AetD affords this unique transformation, which we propose proceeds via an aziridine intermediate. Our work presents a unique template for nitrile biogenesis and portrays a substrate binding and metallocofactor assembly mechanism that may be shared among other haem-oxygenase-like dimetal oxidase enzymes. 

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