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1.
J Am Chem Soc ; 144(27): 11991-12006, 2022 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-35786920

RESUMO

Traditionally, the ferritin-like superfamily of proteins was thought to exclusively use a diiron active site in catalyzing a diverse array of oxygen-dependent reactions. In recent years, novel redox-active cofactors featuring heterobimetallic Mn/Fe active sites have been discovered in both the radical-generating R2 subunit of class Ic (R2c) ribonucleotide reductases (RNRs) and the related R2-like ligand-binding oxidases (R2lox). However, the protein-specific factors that differentiate the radical reactivity of R2c from the C-H activation reactions of R2lox remain unknown. In this work, multifrequency pulsed electron paramagnetic resonance (EPR) spectroscopy and ligand hyperfine techniques in conjunction with broken-symmetry density functional theory calculations are used to characterize the molecular and electronic structures of two EPR-active intermediates trapped during aerobic assembly of the R2lox Mn/Fe cofactor. A MnIII(µ-O)(µ-OH)FeIII species is identified as the first EPR-active species and represents a common state between the two classes of redox-active Mn/Fe proteins. The species downstream from the MnIII(µ-O)(µ-OH)FeIII state exhibits unique EPR properties, including unprecedented spectral breadth and isotope-dependent g-tensors, which are attributed to a weakly coupled, hydrogen-bonded MnIII(µ-OH)FeIII species. This final intermediate precedes formation of the MnIII/FeIII resting state and is suggested to be relevant to understanding the endogenous reactivity of R2lox.


Assuntos
Manganês , Ribonucleotídeo Redutases , Espectroscopia de Ressonância de Spin Eletrônica , Elétrons , Ferro/química , Ligantes , Manganês/química , Ribonucleotídeo Redutases/química
2.
Biochemistry ; 54(18): 2817-27, 2015 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-25897619

RESUMO

Bactoprenyl diphosphate (BPP), a two-E eight-Z configuration C55 isoprenoid, serves as a critical anchor for the biosynthesis of complex glycans central to bacterial survival and pathogenesis. BPP is formed by the polymerase undecaprenyl pyrophosphate synthase (UppS), which catalyzes the elongation of a single farnesyl diphosphate (FPP) with eight Z-configuration isoprene units from eight isopentenyl diphosphates. In vitro analysis of UppS and other polyprenyl diphosphate synthases requires the addition of a surfactant such as Triton X-100 to stimulate the release of the hydrophobic product from the enzyme for effective and efficient turnover. Here using a fluorescent 2-nitrileanilinogeranyl diphosphate analogue of FPP, we have found that a wide range of surfactants can stimulate release of product from UppS and that the structure of the surfactant has a major impact on the lengths of products produced by the protein. Of particular importance, shorter chain surfactants promote the release of isoprenoids with four to six Z-configuration isoprene additions, while larger chain surfactants promote the formation of natural isoprenoid lengths (8Z) and larger. We have found that the product chain lengths can be readily controlled and coarsely tuned by adjusting surfactant identity, concentration, and reaction time. We have also found that binary mixtures of just two surfactants can be used to fine-tune isoprenoid lengths. The surfactant effects discovered do not appear to be significantly altered with an alternative isoprenoid substrate. However, the surfactant effects do appear to be dependent on differences in UppS between bacterial species. This work provides new insights into surfactant effects in enzymology and highlights how these effects can be leveraged for the chemoenzymatic synthesis of otherwise difficult to obtain glycan biosynthesis probes. This work also provides key reagents for the systematic analysis of structure-activity relationships between glycan biosynthesis enzymes and isoprenoid structure.


Assuntos
Alquil e Aril Transferases/química , Proteínas de Bactérias/química , Corantes Fluorescentes/síntese química , Tensoativos/química , Terpenos/síntese química , Bacteroides fragilis/enzimologia , Estereoisomerismo , Fatores de Tempo
3.
ACS Omega ; 8(17): 15790-15798, 2023 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-37151508

RESUMO

Complex poly- and oligosaccharides on the surface of bacteria provide a unique fingerprint to different strains of pathogenic and symbiotic microbes that could be exploited for therapeutics or sensors selective for specific glycans. To discover reagents that can selectively interact with specific bacterial glycans, a system for both the chemoenzymatic preparation and immobilization of these materials would be ideal. Bacterial glycans are typically synthesized in nature on the C55 polyisoprenoid bactoprenyl (or undecaprenyl) phosphate. However, this long-chain isoprenoid can be difficult to work with in vitro. Here, we describe the addition of a chemically functional benzylazide tag to polyisoprenoids. We have found that both the organic-soluble and water-soluble benzylazide isoprenoid can serve as a substrate for the well-characterized system responsible for Campylobacter jejuni N-linked heptasaccharide assembly. Using the organic-soluble analogue, we demonstrate the use of an N-acetyl-glucosamine epimerase that can be used to lower the cost of glycan assembly, and using the water-soluble analogue, we demonstrate the immobilization of the C. jejuni heptasaccharide on magnetic beads. These conjugated beads are then shown to interact with soybean agglutinin, a lectin known to interact with N-acetyl-galactosamine in the C. jejuni heptasaccharide. The methods provided could be used for a wide variety of applications including the discovery of new glycan-interacting partners.

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