Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed additional experiments and proposed a five-state kinetic model ...for photosynthetic oxygen evolution, known as Kok's S-state clock or cycle
. The model comprises four (meta)stable intermediates (S
, S
, S
and S
) and one transient S
state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn
CaO
) cluster in the oxygen-evolving complex
. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone Q
at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallography and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temperature, we visualize all (meta)stable states of Kok's cycle as high-resolution structures (2.04-2.08 Å). In addition, we report structures of two transient states at 150 and 400 µs, revealing notable structural changes including the binding of one additional 'water', Ox, during the S
→S
state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the additional oxygen Ox in the S
state between Ca and Mn1 supports O-O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O
release. Thus, our results exclude peroxo-bond formation in the S
state, and the nucleophilic attack of W3 onto W2 is unlikely.
A recent paper in BMC Biology presents a general method for mix-and-inject serial crystallography, to facilitate the visualization of enzyme intermediates via time-resolved serial femtosecond ...crystallography (tr-SFX). They apply their method to resolve in near atomic detail the cleavage and inactivation of the antibiotic ceftriaxone by a β-lactamase enzyme from Mycobacterium tuberculosis. Their work demonstrates the general applicability of time-resolved crystallography, from which dynamic structures, at atomic resolution, can be obtained.See research article: https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-018-0524-5 .
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cyclic guanosine 3′,5′‐monophosphate (cGMP) is an intracellular signalling molecule involved in many sensory and developmental processes. Synthesis of cGMP from GTP is catalysed by guanylate cyclase ...(GC) in a reaction analogous to cAMP formation by adenylate cyclase (AC). Although detailed structural information is available on the catalytic region of nucleotidyl cyclases (NCs) in various states, these atomic models do not provide a sufficient explanation for the substrate selectivity between GC and AC family members. Detailed structural information on the GC domain in its active conformation is largely missing, and no crystal structure of a GTP‐bound wild‐type GC domain has been published to date. Here, we describe the crystal structure of the catalytic domain of rhodopsin–GC (RhGC) from Catenaria anguillulae in complex with GTP at 1.7 Å resolution. Our study reveals the organization of a eukaryotic GC domain in its active conformation. We observe that the binding mode of the substrate GTP is similar to that of AC–ATP interaction, although surprisingly not all of the interactions predicted to be responsible for base recognition are present. The structure provides insights into potential mechanisms of substrate discrimination and activity regulation that may be common to all class III purine NCs.
Database
Structural data are available in Protein Data Bank database under the accession number 6SIR.
Enzymes
EC4.6.1.2.
Synthesis of cGMP, an important intracellular signalling molecule, is catalysed by guanylate cyclases. Although detailed structural information is available, current data do not provide a sufficient explanation for the substrate selectivity between guanylate and adenylate cyclases. We present the crystal structure of the GTP‐bound catalytic domain of rhodopsin–GC from Catenaria anguillulae at 1.7 Å resolution. Our study provides insights into potential mechanisms of substrate discrimination and activity regulation.
Where is crystallography going? Grimes, Jonathan M.; Hall, David R.; Ashton, Alun W. ...
Acta crystallographica. Section D, Structural biology,
February 2018, Letnik:
74, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Macromolecular crystallography (MX) has been a motor for biology for over half a century and this continues apace. A series of revolutions, including the production of recombinant proteins and ...cryo‐crystallography, have meant that MX has repeatedly reinvented itself to dramatically increase its reach. Over the last 30 years synchrotron radiation has nucleated a succession of advances, ranging from detectors to optics and automation. These advances, in turn, open up opportunities. For instance, a further order of magnitude could perhaps be gained in signal to noise for general synchrotron experiments. In addition, X‐ray free‐electron lasers offer to capture fragments of reciprocal space without radiation damage, and open up the subpicosecond regime of protein dynamics and activity. But electrons have recently stolen the limelight: so is X‐ray crystallography in rude health, or will imaging methods, especially single‐particle electron microscopy, render it obsolete for the most interesting biology, whilst electron diffraction enables structure determination from even the smallest crystals? We will lay out some information to help you decide.
Macromolecular crystallography has provided results that underpin much biological discovery and there is still scope for further development; however, a revolution in electron imaging now means that it can also routinely provide detailed atomic‐level descriptions. This article attempts to tease out where crystallography is going and consider what its place might be in the new landscape.
Peptidoglycan recognition proteins (PGRPs) are ubiquitous among animals and play pivotal functions in insect immunity. Non-catalytic PGRPs are involved in the activation of immune pathways by binding ...to the peptidoglycan (PGN), whereas amidase PGRPs are capable of cleaving the PGN into non-immunogenic compounds. Drosophila PGRP-LB belongs to the amidase PGRPs and downregulates the immune deficiency (IMD) pathway by cleaving meso-2,6-diaminopimelic (meso-DAP or DAP)-type PGN. While the recognition process is well analyzed for the non-catalytic PGRPs, little is known about the enzymatic mechanism for the amidase PGRPs, despite their essential function in immune homeostasis. Here, we analyzed the specific activity of different isoforms of Drosophila PGRP-LB towards various PGN substrates to understand their specificity and role in Drosophila immunity. We show that these isoforms have similar activity towards the different compounds. To analyze the mechanism of the amidase activity, we performed site directed mutagenesis and solved the X-ray structures of wild-type Drosophila PGRP-LB and its mutants, with one of these structures presenting a protein complexed with the tracheal cytotoxin (TCT), a muropeptide derived from the PGN. Only the Y78F mutation abolished the PGN cleavage while other mutations reduced the activity solely. Together, our findings suggest the dynamic role of the residue Y78 in the amidase mechanism by nucleophilic attack through a water molecule to the carbonyl group of the amide function destabilized by Zn2+.
Herein, the structure resulting from in situ turnover in a chemically challenging quaternary ammonium oxidative demethylation reaction was captured via crystallographic analysis and analyzed via ...single-crystal spectroscopy. Crystal structures were determined for the Rieske-type monooxygenase, stachydrine demethylase, in the unliganded state (at 1.6 Å resolution) and in the product complex (at 2.2 Å resolution). The ligand complex was obtained from enzyme aerobically cocrystallized with the substrate stachydrine (N,N-dimethylproline). The ligand electron density in the complex was interpreted as proline, generated within the active site at 100 K by the absorption of X-ray photon energy and two consecutive demethylation cycles. The oxidation state of the Rieske iron–sulfur cluster was characterized by UV–visible spectroscopy throughout X-ray data collection in conjunction with resonance Raman spectra collected before and after diffraction data. Shifts in the absorption band wavelength and intensity as a function of absorbed X-ray dose demonstrated that the Rieske center was reduced by solvated electrons generated by X-ray photons; the kinetics of the reduction process differed dramatically for the liganded complex compared to unliganded demethylase, which may correspond to the observed turnover in the crystal.
To take full advantage of advanced data collection techniques and high beam flux at next‐generation macromolecular crystallography beamlines, rapid and reliable methods will be needed to mount and ...align many samples per second. One approach is to use an acoustic ejector to eject crystal‐containing droplets onto a solid X‐ray transparent surface, which can then be positioned and rotated for data collection. Proof‐of‐concept experiments were conducted at the National Synchrotron Light Source on thermolysin crystals acoustically ejected onto a polyimide `conveyor belt'. Small wedges of data were collected on each crystal, and a complete dataset was assembled from a well diffracting subset of these crystals. Future developments and implementation will focus on achieving ejection and translation of single droplets at a rate of over one hundred per second.
The proton transfer reaction between the substrate nitroethane and Asp-402 catalyzed by nitroalkane oxidase and the uncatalyzed process in water have been investigated using a path-integral ...free-energy perturbation method. Although the dominating effect in rate acceleration by the enzyme is the lowering of the quasiclassical free energy barrier, nuclear quantum effects also contribute to catalysis in nitroalkane oxidase. in particular, the overall nuclear quantum effects have greater contributions to lowering the classical barrier in the enzyme, and there is a larger difference in quantum effects between proton and deuteron transfer for the enzymatic reaction than that in water. Both experiment and computation show that primary KIEs are enhanced in the enzyme, and the computed Swain-Schaad exponent for the enzymatic reaction is exacerbated relative to that in the absence of the enzyme. In addition, the computed tunneling transmission coefficient is approximately three times greater for the enzyme reaction than the uncatalyzed reaction, and the origin of the difference may be attributed to a narrowing effect in the effective potentials for tunneling in the enzyme than that in aqueous solution.
Redox reactions are central to biochemistry and are both controlled by and induce protein structural changes. Here, we describe structural rearrangements and crosstalk within the
Bacillus cereus
...ribonucleotide reductase R2b–NrdI complex, a di-metal carboxylate-flavoprotein system, as part of the mechanism generating the essential catalytic free radical of the enzyme. Femtosecond crystallography at an X-ray free electron laser was utilized to obtain structures at room temperature in defined redox states without suffering photoreduction. Together with density functional theory calculations, we show that the flavin is under steric strain in the R2b–NrdI protein complex, likely tuning its redox properties to promote superoxide generation. Moreover, a binding site in close vicinity to the expected flavin O
2
interaction site is observed to be controlled by the redox state of the flavin and linked to the channel proposed to funnel the produced superoxide species from NrdI to the di-manganese site in protein R2b. These specific features are coupled to further structural changes around the R2b–NrdI interaction surface. The mechanistic implications for the control of reactive oxygen species and radical generation in protein R2b are discussed.
Choline oxidase catalyzes the oxidation of choline to glycine betaine, a compatible solute that accumulates in pathogenic bacteria and plants so they can withstand osmotic and temperature stresses. ...The crystal structure of choline oxidase was determined and refined to a resolution of 1.86 Å with data collected at 100 K using synchrotron X-ray radiation. The structure reveals a covalent linkage between His99 Nε2 and FAD C8M atoms, and a 123 Å3 solvent-excluded cavity adjacent to the re face of the flavin. A hypothetical model for choline docked into the cavity suggests that several aromatic residues and Glu312 may orient the cationic substrate for efficient catalysis. The role of the negative charge on Glu312 was investigated by engineering variant enzymes in which Glu312 was replaced with alanine, glutamine, or aspartate. The Glu312Ala enzyme was inactive. The Glu312Gln enzyme exhibited a K d value for choline at least 500 times larger than that of the wild-type enzyme. The Glu312Asp enzyme had a k cat/K O 2 value similar to that of the wild-type enzyme but k cat and k cat/K m values that were 230 and 35 times lower, respectively, than in the wild-type enzyme. These data are consistent with the spatial location of the negative charge on residue 312 being important for the oxidation of the alcohol substrate. Solvent viscosity and substrate kinetic isotope effects suggest the presence of an internal equilibrium in the Glu312Asp enzyme prior to the hydride transfer reaction. Altogether, the crystallographic and mechanistic data suggest that Glu312 is important for binding and positioning of the substrate in the active site of choline oxidase.
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