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1. Evidence for the chemical mechanism of RibB (3,4-dihydroxy-2-butanone 4-phosphate synthase) of riboflavin biosynthesis.

   Nikola Kenjić, Kathleen M. (July 2022, Journal of the American Chemical Society)

   RibB (3,4-dihydroxy-2-butanone 4-phosphate synthase) is a magnesium-dependent enzyme that excis-es the C4 of D-ribulose-5-phosphate (D-Ru5P) as formate. This chemistry forms the four-carbon sub-strate for RibE (lumazine synthase) that is incorporated into the xylene moiety of lumazine and ulti-mately the riboflavin isoalloxazine. The reaction was first identified in studies by Bacher and cowork-ers in the early 1990s and a chemical mechanism hypothesis offered by these researchers has become the consensus mechanism despite minimal direct evidence. In addition, 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 concensus 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 concentra-tions equal to the enzyme concentration indicating that only one metal ion is required for catalysis. This was confirmed from EPR of the enzyme reconstituted with manganese and crystal structures ob-tained from soaking with the native substrate, D-Ru5P. These data definitively show the involvement of a single active site metal ion. The slow rate of turnover of VcRibB was used to identify two transient species prior to the formation of products using acid quench of single turnover reactions in combina-tion with NMR for singly and fully 13C-labelled D-Ru5P. The data indicate that dehydration of C1 forms the first transient species that then undergoes rearrangement by a 1,2 migration that fuses C5 to C3 and renders C4 hydrated as a gem diol that is poised for elimination as formate. Time-dependent Mn2+ soaks of VcRibB-D-Ru5P co-crystals provided structures that show accumulation in crystallo of the same intermediate states as observed with acid-quench and NMR. 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. PPM1B utilizes a trinuclear metal architecture for phosphatase activity

   https://doi.org/10.64898/2026.04.23.720145  

   Stevens, Reece P; Solodushko, Viktoriya; Wierzbicki, Andrzej; Rich, Thomas C; Alexeyev, Mikael F; Thompson, Marlo K; Stone, Madeline; Hall, Camryn; deWeever, Althea; Sayner, Sarah L; et al (April 2026, bioRxiv)

   Abstract The metal-dependent protein phosphatase (PPM/PP2C) family regulates innate immune and cell death pathways through reversible phosphorylation. Although these enzymes contain a conserved third Mg²⁺/Mn²⁺ion (M3) that is essential for activity, its chemical role in phosphate hydrolysis has remained unclear. Here, we report studies that reveal PPM1B promotes cell death duringPseudomonas aeruginosainfection and utilizes a trinuclear metal center in which M3 directly coordinates the substrate phosphate, positioning it for in-line S_N2 hydrolysis. In addition to substrate orientation, M3 positions a water molecule to protonate the departing alkoxide, stabilizing the leaving-group. Functionally, M3 substitutes for the arginine clamp in phosphoprotein phosphatases (PPP), revealing that these evolutionarily distinct phosphatase families have converged on the same chemical strategy through fundamentally different catalytic architectures. Together, these findings define a three-metal mechanism in PPM phosphatases and identify the M3 site as a rare and potentially druggable feature for immune and infectious diseases. 

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4. Kinetic characterization of methylthio-d-ribose-1-phosphate isomerase

   https://doi.org/10.1016/bs.mie.2023.03.015  

   Veeramachineni, Vamsee M; Ubayawardhana, Subashi T; Murkin, Andrew S (January 2023, Methods in enzymology)

   Methylthio-D-ribose-1-phosphate (MTR1P) isomerase (MtnA) catalyzes the reversible isomerization of the aldose MTR1P into the ketose methylthio-D-ribulose 1-phosphate. It serves as a member of the methionine salvage pathway that many organisms require for recycling methylthio-D-adenosine, a byproduct of S-adenosylmethionine metabolism, back to methionine. MtnA is of mechanistic interest because unlike most other aldose–ketose isomerases, its substrate exists as an anomeric phosphate ester and therefore cannot equilibrate with a ring-opened aldehyde that is otherwise required to promote isomerization. To investigate the mechanism of MtnA, it is necessary to establish reliable methods for determining the concentration of MTR1P and to measure enzyme activity in a continuous assay. This chapter describes several such protocols needed to perform steady-state kinetics measurements. It additionally outlines the preparation of [32P]MTR1P, its use in radioactively labeling the enzyme, and the characterization of the resulting phosphoryl adduct. 

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5. Structural basis for divergent and convergent evolution of catalytic machineries in plant aromatic amino acid decarboxylase proteins

   https://doi.org/10.1073/pnas.1920097117  

   Torrens-Spence, Michael P.; Chiang, Ying-Chih; Smith, Tyler; Vicent, Maria A.; Wang, Yi; Weng, Jing-Ke (May 2020, Proceedings of the National Academy of Sciences)

   Radiation of the plant pyridoxal 5′-phosphate (PLP)-dependent aromatic l -amino acid decarboxylase (AAAD) family has yielded an array of paralogous enzymes exhibiting divergent substrate preferences and catalytic mechanisms. Plant AAADs catalyze either the decarboxylation or decarboxylation-dependent oxidative deamination of aromatic l -amino acids to produce aromatic monoamines or aromatic acetaldehydes, respectively. These compounds serve as key precursors for the biosynthesis of several important classes of plant natural products, including indole alkaloids, benzylisoquinoline alkaloids, hydroxycinnamic acid amides, phenylacetaldehyde-derived floral volatiles, and tyrosol derivatives. Here, we present the crystal structures of four functionally distinct plant AAAD paralogs. Through structural and functional analyses, we identify variable structural features of the substrate-binding pocket that underlie the divergent evolution of substrate selectivity toward indole, phenyl, or hydroxyphenyl amino acids in plant AAADs. Moreover, we describe two mechanistic classes of independently arising mutations in AAAD paralogs leading to the convergent evolution of the derived aldehyde synthase activity. Applying knowledge learned from this study, we successfully engineered a shortened benzylisoquinoline alkaloid pathway to produce (S)-norcoclaurine in yeast. This work highlights the pliability of the AAAD fold that allows change of substrate selectivity and access to alternative catalytic mechanisms with only a few mutations. 

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