Crystallography and Its Impact on Carbonic Anhydrase Research

X-ray and neutron crystallography are powerful techniques utilized to study the structures of biomolecules. Visualization of enzymes in complex with substrate/product and the capture of intermediate states can be related to activity to facilitate understanding of the catalytic mechanism. Subsequent analysis of small molecule binding within the enzyme active site provides insight into mechanisms of inhibition, supporting the design of novel inhibitors using a structure-guided approach. The first X-ray crystal structures were determined for small, ubiquitous enzymes such as carbonic anhydrase (CA). CAs are a family of zinc metalloenzymes that catalyze the hydration of CO2, producing HCO3- and a proton. The CA structure and ping-pong mechanism have been extensively studied and are well understood. Though the function of CA plays an important role in a variety of physiological functions, CA has also been associated with diseases such as glaucoma, edema, epilepsy, obesity, and cancer and is therefore recognized as a drug target. In this review, a brief history of crystallography and its impact on CA research is discussed.

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Copper–oxygen Dynamics in Tyrosinase

10.26434/chemrxiv.9255251 ◽

2019 ◽

Author(s):

Nobutaka Fujieda ◽

Sachiko Yanagisawa ◽

Minoru Kubo ◽

Genji Kurisu ◽

Shinobu Itoh

Keyword(s):

Catalytic Mechanism ◽

Substrate Binding ◽

Active Form ◽

Copper Ion ◽

Atomic Resolution ◽

Oxygen Species ◽

X Ray ◽

Oxygen Dynamics ◽

Insight Into ◽

Copper Centre

To unveil the activation of dioxygen on the copper centre (Cu2O2core) of tyrosinase, we performed X-ray crystallograpy with active-form tyrosinase at near atomic resolution. This study provided a novel insight into the catalytic mechanism of the tyrosinase, including the rearrangement of copper-oxygen species as well as the intramolecular migration of copper ion induced by substrate-binding.

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SEM-based mineralogical mapping at the submicrometer-scale of modern Sardinian stromatolites

10.5194/egusphere-egu21-7169 ◽

2021 ◽

Author(s):

Juliette Debrie ◽

Dimitri Prêt ◽

Karim Benzerara ◽

Jean Paul Saint Martin

Keyword(s):

Mineralogical Composition ◽

Treatment Method ◽

Heterogeneous Distribution ◽

X Ray ◽

Mineral Phases ◽

Saint Martin ◽

History Of ◽

Phase Recognition ◽

Mineral Mixtures ◽

Insight Into

Stromatolites, i.e. macroscopically laminated carbonate rocks formed by diverse microbial communities, are particularly emblematic geobiological materials since they are the oldest evidence of life-mineral interactions, dated up to 3.5 Gyrs ago. &#160;They are found throughout the history of the Earth and have received strong attention because they provide precious information about microbial paleobiodiversity and paleoenvironments. However, while this information is interpreted based on our knowledge about modern analogs, the latter remains very incomplete. Here, we studied recently discovered modern stromatolites from Mari Ermi1, a coastal pond in Western Sardinia, that seasonally experience severe evaporation and broad salinity variations. For this purpose, we explored the mineralogical composition of these unique sedimentary archives and its spatial variations in order to gain better insight into how mineral phases record the conditions and processes of their formation. We investigated the heterogeneous distribution of minerals using quantitative X-ray chemical maps provided by energy dispersive x-ray spectrometry analyses coupled with scanning electron microscopy (SEM-EDXS). Hyperspectral maps were analyzed using an innovative data treatment method 2 allowing phase recognition within the complex mineral mixtures and solid solutions encountered. This method provided quantitative data on&#160;spatial distribution, modal content and associated calculated unit formulas for each identified mineral and phase with a hundred nanometer resolution. Based on these results, we will discuss the origin of the laminations in the stromatolites.Reference:1. Saint Martin, J.-P. & Saint Martin, S. Geo-Eco-Marina 21, 35&#8211;53 (2015a).2. Pr&#234;t, D. et al. American Mineralogist 95, 1379&#8211;1388 (2010).

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d-Alanine–d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.012936 ◽

2020 ◽

Vol 295 (23) ◽

pp. 7894-7904

Author(s):

Jordan L. Pederick ◽

Andrew P. Thompson ◽

Stephen G. Bell ◽

John B. Bruning

Keyword(s):

Binding Site ◽

Active Site ◽

Drug Target ◽

Catalytic Mechanism ◽

Thermus Thermophilus ◽

Monovalent Cation ◽

Antibiotic Drug ◽

Domains Of Life ◽

Alanine Dipeptide ◽

Insight Into

The ATP-grasp superfamily of enzymes shares an atypical nucleotide-binding site known as the ATP-grasp fold. These enzymes are involved in many biological pathways in all domains of life. One ATP-grasp enzyme, d-alanine–d-alanine ligase (Ddl), catalyzes ATP-dependent formation of the d-alanyl–d-alanine dipeptide essential for bacterial cell wall biosynthesis and is therefore an important antibiotic drug target. Ddl is activated by the monovalent cation (MVC) K+, but despite its clinical relevance and decades of research, how this activation occurs has not been elucidated. We demonstrate here that activating MVCs bind adjacent to the active site of Ddl from Thermus thermophilus and used a combined biochemical and structural approach to characterize MVC activation. We found that TtDdl is a type II MVC-activated enzyme, retaining activity in the absence of MVCs. However, the efficiency of TtDdl increased ∼20-fold in the presence of activating MVCs, and it was maximally activated by K+ and Rb+ ions. A strict dependence on ionic radius of the MVC was observed, with Li+ and Na+ providing little to no TtDdl activation. To understand the mechanism of MVC activation, we solved crystal structures of TtDdl representing distinct catalytic stages in complex with K+, Rb+, or Cs+. Comparison of these structures with apo TtDdl revealed no evident conformational change on MVC binding. Of note, the identified MVC binding site is structurally conserved within the ATP-grasp superfamily. We propose that MVCs activate Ddl by altering the charge distribution of its active site. These findings provide insight into the catalytic mechanism of ATP-grasp enzymes.

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Synchrotron Radiation Research and Analysis of the Particulate Matter in Deep Ice Cores: An Overview of the Technical Challenges

Condensed Matter ◽

10.3390/condmat4030061 ◽

2019 ◽

Vol 4 (3) ◽

pp. 61 ◽

Cited By ~ 1

Author(s):

Giannantonio Cibin ◽

Augusto Marcelli ◽

Valter Maggi ◽

Giovanni Baccolo ◽

Dariush Hampai ◽

...

Keyword(s):

Synchrotron Radiation ◽

Ice Core ◽

Ice Cores ◽

Atmospheric Conditions ◽

Airborne Dust ◽

X Ray ◽

History Of ◽

X Ray Absorption ◽

Radiation Research ◽

Insight Into

Airborne dust extracted from deep ice core perforations can provide chemical and mineralogical insight into the history of the climate and atmospheric conditions, with unrivalled temporal resolution, time span and richness of information. The availability of material for research and the natural complexity of the particulate, however, pose significant challenges to analytical methods. We present the developments undertaken to optimize the experimental techniques, materials and protocols for synchrotron radiation-based analysis, in particular for the acquisition of combined Synchrotron Radiation X-Ray Fluorescence and X-ray Absorption Spectroscopy data.

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Structural studies on dehydroshikimate dehydratase

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314095242 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C475-C475

Author(s):

James Peek ◽

Dinesh Christendat

Keyword(s):

Sole Carbon Source ◽

Catalytic Mechanism ◽

Substrate Recognition ◽

Structural Studies ◽

Sugar Phosphate ◽

X Ray ◽

Structural Insight ◽

Functional Relevance ◽

Insight Into

The soil bacterium, Pseudomonas putida, is capable of using the alicyclic compound quinate as a sole carbon source. During this process, quinate is converted to 3-dehydroshikimate, which subsequently undergoes a dehydration to form protocatechuate. The latter transformation is performed by the enzyme dehydroshikimate dehydratase (DSD). We have recombinantly produced DSD from P. putida and are currently performing x-ray crystallographic studies on the enzyme to gain structural insight into its catalytic mechanism and mode of substrate recognition. Initial crystals of DSD diffracted to 2.7 Ä resolution, but exhibited strong twinning. A redesigned construct has recently yielded crystals that diffract to similar resolution, but with a significantly reduced tendency toward twinning. Interestingly, sequence analysis of P. putida DSD reveals that the protein is in fact a fusion of two distinct domains: an N-terminal sugar phosphate isomerase-like domain associated with DSD activity, and a C-terminal hydroxyphenylpyruvate dioxygenase (HPPD)-like domain with unknown functional significance. Structural characterization of the protein may provide novel insight into the functional relevance of the unusual HPPD-like domain.

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New insight into catalytic mechanism of serine proteases from ultra-high resolution X-ray studies

Acta Crystallographica Section A Foundations of Crystallography ◽

10.1107/s0108767308088326 ◽

2008 ◽

Vol 64 (a1) ◽

pp. C365-C366

Author(s):

J. Raczynska ◽

M. Dauter ◽

T. Borchert ◽

W. Rypniewski

Keyword(s):

High Resolution ◽

Serine Proteases ◽

Catalytic Mechanism ◽

X Ray ◽

Insight Into

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Structural and mechanistic analysis of the arsenate respiratory reductase provides insight into environmental arsenic transformations

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1807984115 ◽

2018 ◽

Vol 115 (37) ◽

pp. E8614-E8623 ◽

Cited By ~ 26

Author(s):

Nathaniel R. Glasser ◽

Paul H. Oyala ◽

Thomas H. Osborne ◽

Joanne M. Santini ◽

Dianne K. Newman

Keyword(s):

Competitive Inhibitor ◽

Diffusion Limit ◽

X Ray ◽

X Ray Crystallography ◽

Arsenic Mobility ◽

Mechanistic Analysis ◽

Ping Pong ◽

Arsenate Respiration ◽

Natural Inhibitor ◽

Insight Into

Arsenate respiration by bacteria was discovered over two decades ago and is catalyzed by diverse organisms using the well-conserved Arr enzyme complex. Until now, the mechanisms underpinning this metabolism have been relatively opaque. Here, we report the structure of an Arr complex (solved by X-ray crystallography to 1.6-Å resolution), which was enabled by an improved Arr expression method in the genetically tractable arsenate respirerShewanellasp. ANA-3. We also obtained structures bound with the substrate arsenate (1.8 Å), the product arsenite (1.8 Å), and the natural inhibitor phosphate (1.7 Å). The structures reveal a conserved active-site motif that distinguishes Arr [(R/K)GRY] from the closely related arsenite respiratory oxidase (Arx) complex (XGRGWG). Arr activity assays using methyl viologen as the electron donor and arsenate as the electron acceptor display two-site ping-pong kinetics. A Mo(V) species was detected with EPR spectroscopy, which is typical for proteins with a pyranopterin guanine dinucleotide cofactor. Arr is an extraordinarily fast enzyme that approaches the diffusion limit (Km= 44.6 ± 1.6 μM,kcat= 9,810 ± 220 seconds−1), and phosphate is a competitive inhibitor of arsenate reduction (Ki= 325 ± 12 μM). These observations, combined with knowledge of typical sedimentary arsenate and phosphate concentrations and known rates of arsenate desorption from minerals in the presence of phosphate, suggest that (i) arsenate desorption limits microbiologically induced arsenate reductive mobilization and (ii) phosphate enhances arsenic mobility by stimulating arsenate desorption rather than by inhibiting it at the enzymatic level.

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An unprecedented insight into the catalytic mechanism of copper nitrite reductase from atomic-resolution and damage-free structures

Science Advances ◽

10.1126/sciadv.abd8523 ◽

2021 ◽

Vol 7 (1) ◽

pp. eabd8523

Author(s):

Samuel L. Rose ◽

Svetlana V. Antonyuk ◽

Daisuke Sasaki ◽

Keitaro Yamashita ◽

Kunio Hirata ◽

...

Keyword(s):

Bound States ◽

Catalytic Mechanism ◽

Catalytic Efficiency ◽

Atomic Resolution ◽

Reaction Intermediates ◽

X Ray ◽

Nitrite Reductases ◽

Radiation Induced ◽

Insight Into ◽

Shed Light

Copper-containing nitrite reductases (CuNiRs), encoded by nirK gene, are found in all kingdoms of life with only 5% of CuNiR denitrifiers having two or more copies of nirK. Recently, we have identified two copies of nirK genes in several α-proteobacteria of the order Rhizobiales including Bradyrhizobium sp. ORS 375, encoding a four-domain heme-CuNiR and the usual two-domain CuNiR (Br2DNiR). Compared with two of the best-studied two-domain CuNiRs represented by the blue (AxNiR) and green (AcNiR) subclasses, Br2DNiR, a blue CuNiR, shows a substantially lower catalytic efficiency despite a sequence identity of ~70%. Advanced synchrotron radiation and x-ray free-electron laser are used to obtain the most accurate (atomic resolution with unrestrained SHELX refinement) and damage-free (free from radiation-induced chemistry) structures, in as-isolated, substrate-bound, and product-bound states. This combination has shed light on the protonation states of essential catalytic residues, additional reaction intermediates, and how catalytic efficiency is modulated.

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The structure of a family GH25 lysozyme fromAspergillus fumigatus

Acta Crystallographica Section F Structural Biology and Crystallization Communications ◽

10.1107/s1744309110025601 ◽

2010 ◽

Vol 66 (9) ◽

pp. 973-977 ◽

Cited By ~ 8

Author(s):

Justyna E. Korczynska ◽

Steffen Danielsen ◽

Ulrika Schagerlöf ◽

Johan P. Turkenburg ◽

Gideon J. Davies ◽

...

Keyword(s):

Active Site ◽

Glycoside Hydrolase ◽

Catalytic Mechanism ◽

Medical Application ◽

Glycoside Hydrolase Family ◽

X Ray ◽

Fungal Structure ◽

The Family ◽

Hydrolase Family ◽

Insight Into

Lysins are important biomolecules which cleave the bacterial cell-wall polymer peptidoglycan. They are finding increasing commercial and medical application. In order to gain an insight into the mechanism by which these enzymes operate, the X-ray structure of a CAZy family GH25 `lysozyme' fromAspergillus fumigatuswas determined. This is the first fungal structure from the family and reveals a modified α/β-barrel-like fold in which an eight-stranded β-barrel is flanked by three α-helices. The active site lies toward the bottom of a negatively charged pocket and its layout has much in common with other solved members of the GH25 and related GH families. A conserved active-site DXE motif may be implicated in catalysis, lending further weight to the argument that this glycoside hydrolase family operatesviaa `substrate-assisted' catalytic mechanism.

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