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The hexameric low-pH stress response enzyme oxalate decarboxylase catalyzes the decarboxylation of the oxalate mono-anion in the soil bacterium Bacillus subtilis. A single protein subunit contains two Mn-binding cupin domains, and catalysis depends on Mn(III) at the N-terminal site. The present study suggests a mechanistic function for the C-terminal Mn as an electron hole donor for the N-terminal Mn. The resulting spatial separation of the radical intermediates directs the chemistry toward decarboxylation of the substrate. A π-stacked tryptophan pair (W96/W274) links two neighboring protein subunits together, thus reducing the Mn-to-Mn distance from 25.9 Å (intrasubunit) to 21.5 Å (intersubunit). Here, we used theoretical analysis of electron hole-hopping paths through redox-active sites in the enzyme combined with site-directed mutagenesis and X-ray crystallography to demonstrate that this tryptophan pair supports effective electron hole hopping between the C-terminal Mn of one subunit and the N-terminal Mn of the other subunit through two short hops of ∼8.5 Å. Replacement of W96, W274, or both with phenylalanine led to a large reduction in catalytic efficiency, whereas replacement with tyrosine led to recovery of most of this activity. W96F and W96Y mutants share the wildtype tertiary structure. Two additional hole-hopping networks were identified leading from the Mn ions to the protein surface, potentially protecting the enzyme from high Mn oxidation states during turnover. Our findings strongly suggest that multistep hole-hopping transport between the two Mn ions is required for enzymatic function, adding to the growing examples of proteins that employ aromatic residues as hopping stations., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Thiamin is essential for plant growth but is short-lived in vivo and energetically very costly to produce - a combination that makes thiamin biosynthesis a prime target for improvement by redesign. Thiamin consists of thiazole and pyrimidine moieties. Its high biosynthetic cost stems from use of the suicide enzyme THI4 to form the thiazole and the near-suicide enzyme THIC to form the pyrimidine. These energetic costs lower biomass yield potential and are likely compounded by environmental stresses that destroy thiamin and hence increase the rate at which it must be made. The energy costs could be slashed by refactoring the thiamin biosynthesis pathway to eliminate the suicidal THI4 and THIC reactions. To substantiate this design concept, we first document the energetic costs of the THI4 and THIC steps in the pathway and explain how cutting these costs could substantially increase crop biomass and grain yields. We then show that a refactored pathway must produce thiamin itself rather than a stripped-down analog because the thiamin molecule cannot be simplified without losing biological activity. Lastly, we consider possible energy-efficient alternatives to the inefficient natural THI4- and THIC-mediated steps., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The enzyme DpgC is included in the small family of cofactor-independent dioxygenases. The chemistry of DpgC is uncommon as the protein binds and utilizes dioxygen without the aid of a metal or organic cofactor. Previous structural and biochemical studies identified the substrate-binding mode and the components of the active site that are important in the catalytic mechanism. In addition, the results delineated a putative binding pocket and migration pathway for the co-substrate dioxygen. Here, structural biology is utilized, along with site-directed mutagenesis, to probe the assigned dioxygen-binding pocket. The key residues implicated in dioxygen trafficking were studied to probe the process of binding, activation and chemistry. The results support the proposed chemistry and provide insight into the general mechanism of dioxygen binding and activation.

Optical methods for O2 determination based on dynamic fluorescence quenching have been applied to measure oxygen uptake rates in cell culture and to determine intracellular oxygen levels. Here we demonstrate the applicability of fluorescence-based probes in determining kinetic parameters for O2 using as an example catalysis by a cofactor-independent oxygenase (DpgC). Fluorescence-based sensors provide a direct assessment of enzyme-catalyzed O2 consumption using commercially available, low-cost instrumentation that is easily customizable and, thus, constitutes a convenient alternative to the widely used Clark-type electrode, especially in cases where chemical interference is expected to be problematic., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Enzyme-catalysed oxidations are some of the most common transformations in primary and secondary metabolism. The vancomycin biosynthetic enzyme DpgC belongs to a small class of oxygenation enzymes that are not dependent on an accessory cofactor or metal ion. The detailed mechanism of cofactor-independent oxygenases has not been established. Here we report the first structure of an enzyme of this oxygenase class in complex with a bound substrate mimic. The use of a designed, synthetic substrate analogue allows unique insights into the chemistry of oxygen activation. The structure confirms the absence of cofactors, and electron density consistent with molecular oxygen is present adjacent to the site of oxidation on the substrate. Molecular oxygen is bound in a small hydrophobic pocket and the substrate provides the reducing power to activate oxygen for downstream chemical steps. Our results resolve the unique and complex chemistry of DpgC, a key enzyme in the biosynthetic pathway of an important class of antibiotics. Furthermore, mechanistic parallels exist between DpgC and cofactor-dependent flavoenzymes, providing information regarding the general mechanism of enzymatic oxygen activation.