| 1. |
- Drago, Victoria N., et al.
(författare)
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Neutron diffraction from a microgravity-grown crystal reveals the active site hydrogens of the internal aldimine form of tryptophan synthase
- 2024
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Ingår i: Cell Reports Physical Science. - 2666-3864. ; 5:2
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Tidskriftsartikel (refereegranskat)abstract
- Pyridoxal 5′-phosphate (PLP), the biologically active form of vitamin B6, is an essential cofactor in many biosynthetic pathways. The emergence of PLP-dependent enzymes as drug targets and biocatalysts, such as tryptophan synthase (TS), has underlined the demand to understand PLP-dependent catalysis and reaction specificity. The ability of neutron diffraction to resolve the positions of hydrogen atoms makes it an ideal technique to understand how the electrostatic environment and selective protonation of PLP regulates PLP-dependent activities. Facilitated by microgravity crystallization of TS with the Toledo Crystallization Box, we report the 2.1 Å joint X-ray/neutron (XN) structure of TS with PLP in the internal aldimine form. Positions of hydrogens were directly determined in both the α- and β-active sites, including PLP cofactor. The joint XN structure thus provides insight into the selective protonation of the internal aldimine and the electrostatic environment of TS necessary to understand the overall catalytic mechanism.
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| 2. |
- Hong, Liang, et al.
(författare)
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Structure and Dynamics of a Compact State of a Multidomain Protein, the Mercuric Ion Reductase
- 2014
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 107:2, s. 393-400
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Tidskriftsartikel (refereegranskat)abstract
- The functional efficacy of colocalized, linked protein domains is dependent on linker flexibility and system compaction. However, the detailed characterization of these properties in aqueous solution presents an enduring challenge. Here, we employ a novel, to our knowledge, combination of complementary techniques, including small-angle neutron scattering, neutron spin-echo spectroscopy, and all-atom molecular dynamics and coarse-grained simulation, to identify and characterize in detail the structure and dynamics of a compact form of mercuric ion reductase (MerA), an enzyme central to bacterial mercury resistance. MerA possesses metallochaperone-like N-terminal domains (NmerA) tethered to its catalytic core domain by linkers. The NmerA domains are found to interact principally through electrostatic interactions with the core, leashed by the linkers so as to subdiffuse on the surface over an area close to the core C-terminal Hg(II)-binding cysteines. How this compact, dynamical arrangement may facilitate delivery of Hg(II) from NmerA to the core domain is discussed.
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| 3. |
- Lian, Peng, et al.
(författare)
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Mechanistic Investigation of Dimethylmercury Formation Mediated by a Sulfide Mineral Surface
- 2021
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Ingår i: Journal of Physical Chemistry A. - : American Chemical Society (ACS). - 1089-5639 .- 1520-5215. ; 125:24, s. 5397-5405
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Tidskriftsartikel (refereegranskat)abstract
- Mercury (Hg) pollution is a global environmental problem. The abiotic formation of dimethylmercury (DMeHg) from monomethylmercury (MMeHg) may account for a large portion of DMeHg in oceans. Previous experimental work has shown that abiotic formation of DMeHg from MMeHg can be facilitated by reduced sulfur groups on sulfide mineral surfaces. In that work, a mechanism was proposed in which neighboring MMeHg moieties bound to sulfide sites on a mineral surface react through an SN2-type mechanism to form DMeHg and incorporate the remaining Hg atoms into the mineral surface. Here, we perform density functional theory calculations to explore the mechanisms of DMeHg formation on the 110 surface of a CdS(s) (hawleyite) nanoparticle. We show that coordination of MMeHg substituents to adjacent reduced sulfur groups protruding from the surface indeed facilitates DMeHg formation and that the reaction proceeds through direct transmethylation from one MMeHg substituent to another. Coordination of Hg by multiple S atoms provides a transition-state stabilization and activates a C–Hg bond for methyl transfer. In addition, solvation effects play an important role in the surface reconstruction of the nanoparticle and in decreasing the energetic barrier for DMeHg formation relative to the corresponding reaction in vacuo.
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| 4. |
- Wan, Qun, et al.
(författare)
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Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography.
- 2015
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 112:40, s. 12384-12389
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Tidskriftsartikel (refereegranskat)abstract
- Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.
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