scholarly journals Aspartate or arginine? Validated redox state X-ray structures elucidate mechanistic subtleties of FeIV = O formation in bacterial dye-decolorizing peroxidases

2021 ◽  
Vol 26 (7) ◽  
pp. 743-761
Author(s):  
Marina Lučić ◽  
Michael T. Wilson ◽  
Dimitri A. Svistunenko ◽  
Robin L. Owen ◽  
Michael A. Hough ◽  
...  
Keyword(s):  
Redox State ◽  
Kinetic Studies ◽  
Site Directed Mutagenesis ◽  
X Rays ◽  
Mechanistic Studies ◽  
X Ray ◽  
X Ray Crystallography ◽  
Structural Framework ◽  
Solvent Isotope ◽  
Heme Peroxidases

AbstractStructure determination of proteins and enzymes by X-ray crystallography remains the most widely used approach to complement functional and mechanistic studies. Capturing the structures of intact redox states in metalloenzymes is critical for assigning the chemistry carried out by the metal in the catalytic cycle. Unfortunately, X-rays interact with protein crystals to generate solvated photoelectrons that can reduce redox active metals and hence change the coordination geometry and the coupled protein structure. Approaches to mitigate such site-specific radiation damage continue to be developed, but nevertheless application of such approaches to metalloenzymes in combination with mechanistic studies are often overlooked. In this review, we summarize our recent structural and kinetic studies on a set of three heme peroxidases found in the bacterium Streptomyces lividans that each belong to the dye decolourizing peroxidase (DyP) superfamily. Kinetically, each of these DyPs has a distinct reactivity with hydrogen peroxide. Through a combination of low dose synchrotron X-ray crystallography and zero dose serial femtosecond X-ray crystallography using an X-ray free electron laser (XFEL), high-resolution structures with unambiguous redox state assignment of the ferric and ferryl (FeIV = O) heme species have been obtained. Experiments using stopped-flow kinetics, solvent-isotope exchange and site-directed mutagenesis with this set of redox state validated DyP structures have provided the first comprehensive kinetic and structural framework for how DyPs can modulate their distal heme pocket Asp/Arg dyad to use either the Asp or the Arg to facilitate proton transfer and rate enhancement of peroxide heterolysis. Graphic abstract

scholarly journals Probing the Role of Asp-120(81) of Metallo-β-lactamase (IMP-1) by Site-directed Mutagenesis, Kinetic Studies, and X-ray Crystallography

2005 ◽  
Vol 280 (21) ◽  
pp. 20824-20832 ◽  
Author(s):  
Yoshihiro Yamaguchi ◽  
Takahiro Kuroki ◽  
Hisami Yasuzawa ◽  
Toshihiro Higashi ◽  
Wanchun Jin ◽  
...  
Keyword(s):  
Kinetic Studies ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
X Ray ◽  
X Ray Crystallography

scholarly journals A hybrid approach reveals the allosteric regulation of GTP cyclohydrolase I

2020 ◽  
Vol 117 (50) ◽  
pp. 31838-31849
Author(s):  
Rebecca Ebenhoch ◽  
Simone Prinz ◽  
Susann Kaltwasser ◽  
Deryck J. Mills ◽  
Robert Meinecke ◽  
...  
Keyword(s):  
Crystal Packing ◽  
Substrate Binding ◽  
Regulatory Protein ◽  
Allosteric Regulation ◽  
Hybrid Approach ◽  
Site Directed Mutagenesis ◽  
Gtp Cyclohydrolase I ◽  
Gtp Cyclohydrolase ◽  
X Ray ◽  
X Ray Crystallography

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). GFRP binds to GCH1 to form inhibited or activated complexes dependent on availability of cofactor ligands, BH4 and phenylalanine, respectively. We determined high-resolution structures of human GCH1−GFRP complexes by cryoelectron microscopy (cryo-EM). Cryo-EM revealed structural flexibility of specific and relevant surface lining loops, which previously was not detected by X-ray crystallography due to crystal packing effects. Further, we studied allosteric regulation of isolated GCH1 by X-ray crystallography. Using the combined structural information, we are able to obtain a comprehensive picture of the mechanism of allosteric regulation. Local rearrangements in the allosteric pocket upon BH4 binding result in drastic changes in the quaternary structure of the enzyme, leading to a more compact, tense form of the inhibited protein, and translocate to the active site, leading to an open, more flexible structure of its surroundings. Inhibition of the enzymatic activity is not a result of hindrance of substrate binding, but rather a consequence of accelerated substrate binding kinetics as shown by saturation transfer difference NMR (STD-NMR) and site-directed mutagenesis. We propose a dissociation rate controlled mechanism of allosteric, noncompetitive inhibition.

scholarly journals Optical control, selection and analysis of population dynamics in ultrafast protein X-ray crystallography

2019 ◽  
Vol 377 (2145) ◽  
pp. 20170474 ◽  
Author(s):  
Christopher D. M. Hutchison ◽  
Jasper J. van Thor
Keyword(s):  
Structural Dynamics ◽  
Time Domain ◽  
Optical Pumping ◽  
High Repetition Rate ◽  
X Rays ◽  
X Ray Diffraction ◽  
X Ray ◽  
Vibrational Coherence ◽  
X Ray Crystallography ◽  
Ultrafast Optical

Ultrafast pump-probe X-ray crystallography has now been established at X-ray free electron lasers that operate at hard X-ray energies. We discuss the performance and development of current applications in terms of the available data quality and sensitivity to detect and analyse structural dynamics. A discussion of technical capabilities expected at future high repetition rate applications as well as future non-collinear multi-pulse schemes focuses on the possibility to advance the technique to the practical application of the X-ray crystallographic equivalent of an impulse time-domain Raman measurement of vibrational coherence. Furthermore, we present calculations of the magnitude of population differences and distributions prepared with ultrafast optical pumping of single crystals in the typical serial femtosecond crystallography geometry, which are developed for the general uniaxial and biaxial cases. The results present opportunities for polarization resolved anisotropic X-ray diffraction analysis of photochemical populations for the ultrafast time domain. This article is part of the theme issue ‘Measurement of ultrafast electronic and structural dynamics with X-rays’.

scholarly journals Closing the gap between electron and X-ray crystallography

2015 ◽  
Vol 71 (6) ◽  
pp. 737-739 ◽  
Author(s):  
Enrico Mugnaioli
Keyword(s):  
Electron Diffraction ◽  
Structure Characterization ◽  
Diffraction Tomography ◽  
X Rays ◽  
Electron Crystallography ◽  
X Ray ◽  
X Ray Crystallography ◽  
Closing The Gap ◽  
Refinement Algorithm

The development of a proper refinement algorithm that takes into account dynamical scattering guarantees, for electron crystallography, results approaching X-rays in terms of precision, accuracy and reliability. The combination of such dynamical refinement and electron diffraction tomography establishes a complete pathway for the structure characterization of single sub-micrometric crystals.

Crystal structure analysis of molecular dynamics using synchrotron X-rays

CrystEngComm ◽  
2015 ◽  
Vol 17 (46) ◽  
pp. 8786-8795 ◽  
Author(s):  
Manabu Hoshino ◽  
Shin-ichi Adachi ◽  
Shin-ya Koshihara
Keyword(s):  
Crystal Structure ◽  
Molecular Dynamics ◽  
Structure Analysis ◽  
Crystal Structure Analysis ◽  
X Rays ◽  
X Ray ◽  
X Ray Crystallography

X-ray crystallography using synchrotron X-rays enables observation of molecular dynamics in a crystal.

scholarly journals Expansion of Substrate Specificity and Catalytic Mechanism of Azoreductase by X-ray Crystallography and Site-directed Mutagenesis

2008 ◽  
Vol 283 (20) ◽  
pp. 13889-13896 ◽  
Author(s):  
Kosuke Ito ◽  
Masayuki Nakanishi ◽  
Woo-Cheol Lee ◽  
Yuehua Zhi ◽  
Hiroshi Sasaki ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Catalytic Mechanism ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
X Ray ◽  
X Ray Crystallography

scholarly journals Structural analysis of the bright monomeric yellow-green fluorescent protein mNeonGreen obtained by directed evolution

2016 ◽  
Vol 72 (12) ◽  
pp. 1298-1307 ◽  
Author(s):  
Damien Clavel ◽  
Guillaume Gotthard ◽  
David von Stetten ◽  
Daniele De Sanctis ◽  
Hélène Pasquier ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Directed Evolution ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
X Rays ◽  
Visible Absorption ◽  
X Ray ◽  
X Ray Crystallography ◽  
Green Fluorescent

Until recently, genes coding for homologues of the autofluorescent protein GFP had only been identified in marine organisms from the phyla Cnidaria and Arthropoda. New fluorescent-protein genes have now been found in the phylum Chordata, coding for particularly bright oligomeric fluorescent proteins such as the tetrameric yellow fluorescent proteinlanYFP fromBranchiostoma lanceolatum. A successful monomerization attempt led to the development of the bright yellow-green fluorescent protein mNeonGreen. The structures oflanYFP and mNeonGreen have been determined and compared in order to rationalize the directed evolution process leading from a bright, tetrameric to a still bright, monomeric fluorescent protein. An unusual discolouration of crystals of mNeonGreen was observed after X-ray data collection, which was investigated using a combination of X-ray crystallography and UV–visible absorption and Raman spectroscopies, revealing the effects of specific radiation damage in the chromophore cavity. It is shown that X-rays rapidly lead to the protonation of the phenolate O atom of the chromophore and to the loss of its planarity at the methylene bridge.

scholarly journals William Lawrence Bragg, 31 March 1890 - 1 July 1971

1979 ◽  
Vol 25 ◽  
pp. 74-143 ◽  
Keyword(s):  
Living Cell ◽  
Inorganic Compounds ◽  
X Rays ◽  
X Ray Diffraction ◽  
X Ray ◽  
X Ray Crystallography ◽  
Intensive Work ◽  
Unique Part

Walking along the Backs in Cambridge one day in the autumn of 1912 William Lawrence Bragg had an idea that led immediately to a dramatic advance in physics and has since transformed chemistry, mineralogy, metallurgy and, most recently, biology. He realized that the observations of X-ray diffraction by a crystal, which had been reported by von Laue and his associates earlier in that year, can be interpreted very simply as arising from reflexion of the X-rays by planes of atoms in the crystal and hence that the X-ray observations provide evidence from which the arrangement of atoms in the crystal may be determined. A few weeks of intensive work on simple inorganic compounds were enough to demonstrate the correctness of these ideas but the development of the method, at first in association with his father and later as the leader or guiding influence of a host of workers, was the labour of a lifetime. When he died on 1 July 1971, X-ray crystallography had revealed the arrangement of atoms in matter of all kinds from the simplest of salts to the macromolecules of the living cell. The story of his life is very largely the story of that achievement and the circumstances that led to his unique part in it.

X-ray analysis

1999 ◽  
Author(s):  
L. Sawyer ◽  
M. A. Turner
Keyword(s):  
Structure Determination ◽  
Protein Crystallography ◽  
General Description ◽  
X Rays ◽  
Actual Structure ◽  
X Ray ◽  
Preliminary Characterization ◽  
X Ray Crystallography ◽  
Full Structure ◽  
Unit Cell Dimensions

This chapter covers the preliminary characterization of the crystals in order to determine if they are suitable for a full structure determination. Probably more frustrating than failure to produce crystals at all, is the growth of beautiful crystals which do not diffract, which have very large unit cell dimensions, or which decay very rapidly in the X-ray beam, though this last problem has been largely overcome by freezing the sample. It is impossible in one brief chapter to give more than a flavour of what the X-ray crystallographic technique entails and it is assumed that the protein chemist growing the crystals will have contact with a protein crystallographer, who will carry out the actual structure determination and in whose laboratory state-of-the-art facilities exist. However, preliminary characterization can often be carried out with little more than the equipment which is widely available in Chemistry and Physics Departments and so the crystal grower remote from a protein crystallography laboratory can monitor the success of their experiments. The reader should refer to the first edition for protocols useful for photographic characterization but such techniques are seldom used nowadays. It must be remembered, in any case, that X-rays are dangerous and the inexperienced should not try to X-ray protein crystals without help. It is necessary to provide an overview of X-ray crystallography, to put the preliminary characterization in context. For a general description of the technique the reader should refer to Glusker et al. (1) or Stout and Jensen (2). For protein crystallography in particular, the books by McRee (3) and Drenth (4) describe many of the advances since the seminal work of Blundell and Johnson (5). Amongst many excellent introductory articles, those by Bragg (6), published years ago, and Glusker (7) are particularly recommended. The scattering or diffraction of X-rays is an interference phenomenon and the interference between the X-rays scattered from the atoms in the structure produces significant changes in the observed diffraction in different directions. This variation in intensity with direction arises because the path differences taken by the scattered X-ray beams are of the same magnitude as the separation of the atoms in the molecule.

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