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Crystallographic Studies of Enzymes (Volume II)

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A special issue of Crystals (ISSN 2073-4352). This special issue belongs to the section "Biomolecular Crystals".

Deadline for manuscript submissions: closed (31 March 2022) | Viewed by 27723

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Prof. Dr. Kyeong Kyu Kim

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Department of Molecular Cell Biology, School of Medicine, Sungkyunkwan University, Suwon 16419, Republic of Korea
Interests: enzymes; structures; esterase; deubiquitinase; noncanonical DNA
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Prof. Dr. T. Doohun Kim

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Department of Chemistry, College of Natural Science, Sookmyung Women's University, Seoul 04310, Korea
Interests: protein-ligand interactions; enzyme structures; assay development; immobilization of enzymes
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Dear Colleagues,

Enzymes play a major role in the control of key biological processes, including metabolism and signaling, by accelerating chemical processes. Therefore, examining their structures and reaction mechanisms is essential for understanding not only the biological processes at a molecule level but also their application in various fields, such as protein engineering and drug development. Indeed, enzymes such as protein kinases or proteases can be considered major drug targets for many diseases. Although cryoEM and NMR provide useful structural information, X-ray crystallography is the best because it elucidates the atomic structure of enzymes, which can be used as a frame for structure-based protein engineering or drug development. In these aspects, enzyme crystallography can be considered a door leading to a new world.

In this Special Issue, we intend to collect research manuscripts on enzyme crystallography. However, since the goal of this issue is to provide rich resources on enzymes regarding their structural and functional aspects, we also encourage manuscript submissions of studies on the structure, function, and application of enzymes, which would provide complementary information for enzyme crystallography.

Prof. Dr. Kyeong Kyu Kim
Prof. Dr. T. Doohun Kim
Guest Editors

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Keywords

crystallization methods and developments
crystallographic determination of enzyme structures
enzyme engineering based on crystal structures
computational and modeling studies of enzymes
structure-based applications of enzymes

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Crystallographic Studies of Enzymes in Crystals (13 articles)

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Editorial

Jump to: Research

2 pages, 162 KiB

Open AccessEditorial

Crystallographic Studies of Enzymes (Volume II)

by T. Doohun Kim and Kyeong Kyu Kim

Crystals 2022, 12(10), 1402; https://doi.org/10.3390/cryst12101402 - 4 Oct 2022

Viewed by 1160

Abstract

Enzymes play a major role in the control of key biological processes by accelerating chemical reactions [...] Full article

(This article belongs to the Special Issue Crystallographic Studies of Enzymes (Volume II))

Research

Jump to: Editorial

9 pages, 1985 KiB

Open AccessArticle

Identification, Characterization, and Preliminary X-ray Diffraction Analysis of a Novel Esterase (ScEst) from Staphylococcus chromogenes

by Jisub Hwang, Sangeun Jeon, Min Ju Lee, Wanki Yoo, Juwon Chang, Kyeong Kyu Kim, Jun Hyuck Lee, Hackwon Do and T. Doohun Kim

Crystals 2022, 12(4), 546; https://doi.org/10.3390/cryst12040546 - 13 Apr 2022

Cited by 2 | Viewed by 2043

Abstract

Ester prodrugs can develop novel antibiotics and have potential therapeutic applications against multiple drug-resistant bacteria. The antimicrobial activity of these prodrugs is activated after being cleaved by the esterases produced by the pathogen. Here, novel esterase ScEst originating from Staphylococcus chromogenes NCTC10530, which causes dairy cow mastitis, was identified, characterized, and analyzed using X-ray crystallography. The gene encoding ScEst was cloned into the pVFT1S vector and overexpressed in E. coli. The recombinant ScEst protein was obtained by affinity and size-exclusion purification. ScEst showed substrate preference for the short chain length of acyl derivatives. It was crystallized in an optimized solution composed of 0.25 M ammonium citrate tribasic (pH 7.0) and 20% PEG 3350 at 296 K. A total of 360 X-ray diffraction images were collected at a 1.66 Å resolution. ScEst crystal belongs to the space group of P2₁2₁2₁ with the unit cell parameters of a = 50.23 Å, b = 68.69 Å, c = 71.15 Å, and α = β = γ = 90°. Structure refinement after molecular replacement is under progress. Further biochemical studies will elucidate the hydrolysis mechanism of ScEst. Overall, this study is the first to report the functional characterization of an esterase from Staphylococcus chromogenes, which is potentially useful in elaborating its hydrolysis mechanism. Full article

(This article belongs to the Special Issue Crystallographic Studies of Enzymes (Volume II))

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9 pages, 7228 KiB

Open AccessCommunication

Identification, Characterization, and Preliminary X-ray Diffraction Analysis of a Single Stranded DNA Binding Protein (LjSSB) from Psychrophilic Lacinutrix jangbogonensis PAMC 27137

by Woong Choi, Jonghyeon Son, Aekyung Park, Hongshi Jin, Seung Chul Shin, Jun Hyuck Lee, T. Doohun Kim and Han-Woo Kim

Crystals 2022, 12(4), 538; https://doi.org/10.3390/cryst12040538 - 11 Apr 2022

Cited by 1 | Viewed by 1991

Abstract

Single-stranded DNA-binding proteins (SSBs) are essential for DNA metabolism, including repair and replication, in all organisms. SSBs have potential applications in molecular biology and in analytical methods. In this study, for the first time, we purified, structurally characterized, and analyzed psychrophilic SSB (LjSSB) from Lacinutrix jangbogonensis PAMC 27137 isolated from the Antarctic region. LjSSB has a relatively short amino acid sequence, consisting of 111 residues, with a molecular mass of 12.6 kDa. LjSSB protein was overexpressed in Escherichia coli BL21 (DE3) and analyzed for binding affinity using 20- and 35-mer deoxythymidine oligonucleotides (dT). In addition, the crystal structure of LjSSB at a resolution 2.6 Å was obtained. The LjSSB protein crystal belongs to the space group C222 with the unit cell parameters of a = 106.58 Å, b = 234.14 Å, c = 66.14 Å. The crystal structure was solved using molecular replacement, and subsequent iterative structure refinements and model building are currently under progress. Further, the complete structural information of LjSSB will provide a novel strategy for protein engineering and for the application on molecular biological techniques. Full article

(This article belongs to the Special Issue Crystallographic Studies of Enzymes (Volume II))

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13 pages, 4703 KiB

Open AccessArticle

New Insight into the Effects of Various Parameters on the Crystallization of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RuBisCO) from Alcaligenes eutrophus

by Hui-Woog Choe and Yong Ju Kim

Crystals 2022, 12(2), 196; https://doi.org/10.3390/cryst12020196 - 28 Jan 2022

Cited by 2 | Viewed by 2541

Abstract

Crystallization remains a bottleneck for determining the three-dimensional X-ray structure of proteins. Many parameters influence the complexity of protein crystallization. Therefore, it is not easy to systematically examine all of these parameters individually during crystallization because of a limited quantity of purified protein. We studied several factors that influence crystallization including protein concentration, pH, temperature, age, volume of crystallization, inhibitors, metal ions, seeding, and precipitating agents on RuBisCO samples from Alcaligenes eutrophus which are not only freshly purified, but are also dissolved both individually and in combination from microcrystals and precipitated droplets of recycled RuBisCO. Single-, twin-, and/or microcrystals are dependent upon the concentration of RuBisCO by both RuBisCO samples. The morphology, either orthorhombic- or monoclinic-space group, depends upon pH. Furthermore, ammonium sulfate((NH₄)₂SO₄) concentration at 20 °C (22% saturated) and/or at 4 °C (28% saturated) affected the crystallization of RuBisCO differently from one another. Finally, the age of RuBisCO also affected more uniformity and forming sharp edge during crystallization. Unexpected surprising monoclinic RuBisCO crystals were grown from dissolved microcrystals and precipitated droplets recycled RuBisCO samples. This quaternary RuBisCO single crystal, which contained Mg²⁺ and HCO₃ for an activated ternary complex and is inhibited with a transition substrate analogue, CABP (2-carboxyarabinitol-1,5-bisphosphate)⁻, diffracts better than 2.2 Å. It is different from Hansen S. et al. reported RuBisCO crystals which were grown ab initio in absence of Mg²⁺, HCO₃⁻ and CABP, a structure which was determined at 2.7 Å resolution. Full article

(This article belongs to the Special Issue Crystallographic Studies of Enzymes (Volume II))

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6 pages, 869 KiB

Open AccessCommunication

Secondary Structure and X-ray Crystallographic Analysis of the Glideosome-Associated Connector (GAC) from Toxoplasma gondii

by Amit Kumar, Xu Zhang, Oscar Vadas, Fisentzos A. Stylianou, Nicolas Dos Santos Pacheco, Sarah L. Rouse, Marc L. Morgan, Dominique Soldati-Favre and Steve Matthews

Crystals 2022, 12(1), 110; https://doi.org/10.3390/cryst12010110 - 15 Jan 2022

Cited by 2 | Viewed by 2701

Abstract

A model for parasitic motility has been proposed in which parasite filamentous actin (F-actin) is attached to surface adhesins by a large component of the glideosome, known as the glideosome-associated connector protein (GAC). This large 286 kDa protein interacts at the cytoplasmic face of the plasma membrane with the phosphatidic acid-enriched inner leaflet and cytosolic tails of surface adhesins to connect them to the parasite actomyosin system. GAC is observed initially to the conoid at the apical pole and re-localised with the glideosome to the basal pole in gliding parasite. GAC presumably functions in force transmission to surface adhesins in the plasma membrane and not in force generation. Proper connection between F-actin and the adhesins is as important for motility and invasion as motor operation itself. This notion highlights the need for new structural information on GAC interactions, which has eluded the field since its discovery. We have obtained crystals that diffracted to 2.6–2.9 Å for full-length GAC from Toxoplasma gondii in native and selenomethionine-labelled forms. These crystals belong to space group P2₁2₁2₁; cell dimensions are roughly a = 119 Å, b = 123 Å, c = 221 Å, α = 90°, β = 90° and γ = 90° with 1 molecule per asymmetric unit, suggesting a more compact conformation than previously proposed Full article

(This article belongs to the Special Issue Crystallographic Studies of Enzymes (Volume II))

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8 pages, 1346 KiB

Open AccessEditor’s ChoiceCommunication

Sequence Analysis and Preliminary X-ray Crystallographic Analysis of an Acetylesterase (LgEstI) from Lactococcus garvieae

by Hackwon Do, Ying Wang, Chang Woo Lee, Wanki Yoo, Sangeun Jeon, Jisub Hwang, Min Ju Lee, Kyeong Kyu Kim, Han-Woo Kim, Jun Hyuck Lee and T. Doohun Kim

Crystals 2022, 12(1), 46; https://doi.org/10.3390/cryst12010046 - 29 Dec 2021

Cited by 3 | Viewed by 2016

Abstract

A gene encoding LgEstI was cloned from a bacterial fish pathogen, Lactococcus garvieae. Sequence and bioinformatic analysis revealed that LgEstI is close to the acetyl esterase family and had maximum similarity to a hydrolase (UniProt: Q5UQ83) from Acanthamoeba polyphaga mimivirus (APMV). Here, we present the results of LgEstI overexpression and purification, and its preliminary X-ray crystallographic analysis. The wild-type LgEstI protein was overexpressed in Escherichia coli, and its enzymatic activity was tested using p-nitrophenyl of varying lengths. LgEstI protein exhibited higher esterase activity toward p-nitrophenyl acetate. To better understand the mechanism underlying LgEstI activity and subject it to protein engineering, we determined the high-resolution crystal structure of LgEstI. First, the wild-type LgEstI protein was crystallized in 0.1 M Tris-HCl buffer (pH 7.1), 0.2 M calcium acetate hydrate, and 19% (w/v) PEG 3000, and the native X-ray diffraction dataset was collected up to 2.0 Å resolution. The crystal structure was successfully determined using a molecular replacement method, and structure refinement and model building are underway. The upcoming complete structural information of LgEstI may elucidate the substrate-binding mechanism and provide novel strategies for subjecting LgEstI to protein engineering. Full article

(This article belongs to the Special Issue Crystallographic Studies of Enzymes (Volume II))

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8 pages, 2187 KiB

Open AccessCommunication

Structural and Biochemical Studies of Bacillus subtilis MobB

by Dajeong Kim, Sarah Choi, Hyunjin Kim and Jungwoo Choe

Crystals 2021, 11(10), 1262; https://doi.org/10.3390/cryst11101262 - 18 Oct 2021

Cited by 1 | Viewed by 1874

Abstract

The biosynthesis of molybdenum cofactor for redox enzymes is carried out by multiple enzymes in bacteria including MobA and MobB. MobA is known to catalyze the attachment of GMP to molybdopterin to form molybdopterin guanine dinucleotide. MobB is a GTP binding protein that enhances the activity of MobA by forming the MobA:MobB complex. However, the mechanism of activity enhancement by MobB is not well understood. The structure of Bacillus subtilis MobB was determined to 2.4 Å resolution and it showed an elongated homodimer with an extended β-sheet. Bound sulfate ions were observed in the Walker A motifs, indicating a possible phosphate-binding site for GTP molecules. The binding assay showed that the affinity between B. subtilis MobA and MobB increased in the presence of GTP, suggesting a possible role of MobB as an enhancer of MobA activity. Full article

(This article belongs to the Special Issue Crystallographic Studies of Enzymes (Volume II))

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19 pages, 5508 KiB

Open AccessArticle

The Depth-Dependent Mechanical Behavior of Anisotropic Native and Cross-Linked HheG Enzyme Crystals

by Marta Kubiak, Marcel Staar, Ingo Kampen, Anett Schallmey and Carsten Schilde

Crystals 2021, 11(7), 718; https://doi.org/10.3390/cryst11070718 - 22 Jun 2021

Cited by 6 | Viewed by 2189

Abstract

Enzymes are able to catalyze various specific reactions under mild conditions and can, therefore, be applied in industrial processes. To ensure process profitability, the enzymes must be reusable while ensuring their enzymatic activity. To improve the processability and immobilization of the biocatalyst, the enzymes can be, e.g., crystallized, and the resulting crystals can be cross-linked. These mechanically stable and catalytically active particles are called CLECs (cross-linked enzyme crystals). In this study, the influence of cross-linking on the mechanical and catalytic properties of the halohydrin dehalogenase (HheG) crystals was investigated using the nanoindentation technique. Considering the viscoelastic behavior of protein crystals, a mechanical investigation was performed at different indentation rates. In addition to the hardness, for the first time, depth-dependent fractions of elastic and plastic deformation energies were determined for enzyme crystals. The results showed that the hardness of HheG enzyme crystals are indentation-rate-insensitive and decrease with increases in penetration depth. Our investigation of the fraction of plastic deformation energy indicated anisotropic crystal behavior and higher irreversible deformation for prismatic crystal faces. Due to cross-linking, the fraction of elastic energy of anisotropic crystal faces increased from 8% for basal faces to 68% for prismatic crystal faces. This study demonstrates that mechanically enhanced CLECs have good catalytic activity and are, therefore, suitable for industrial use. Full article

(This article belongs to the Special Issue Crystallographic Studies of Enzymes (Volume II))

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16 pages, 4924 KiB

Open AccessArticle

Crystal Structure of Nitrilase-Like Protein Nit2 from Kluyveromyces lactis

by Chaewon Jin, Hyeonseok Jin, Byung-Cheon Jeong, Dong-Hyung Cho, Hang-Suk Chun, Woo-Keun Kim and Jeong Ho Chang

Crystals 2021, 11(5), 499; https://doi.org/10.3390/cryst11050499 - 1 May 2021

Cited by 1 | Viewed by 2850

Abstract

The nitrilase superfamily, including 13 branches, plays various biological functions in signaling molecule synthesis, vitamin metabolism, small-molecule detoxification, and posttranslational modifications. Most of the mammals and yeasts have Nit1 and Nit2 proteins, which belong to the nitrilase-like (Nit) branch of the nitrilase superfamily. Recent studies have suggested that Nit1 is a metabolite repair enzyme, whereas Nit2 shows ω-amidase activity. In addition, Nit1 and Nit2 are suggested as putative tumor suppressors through different ways in mammals. Yeast Nit2 (yNit2) is a homolog of mouse Nit1 based on similarity in sequence. To understand its specific structural features, we determined the crystal structure of Nit2 from Kluyveromyces lactis (KlNit2) at 2.2 Å resolution and compared it with the structure of yeast-, worm-, and mouse-derived Nit2 proteins. Based on our structural analysis, we identified five distinguishable structural features from 28 structural homologs. This study might potentially provide insights into the structural relationships of a broad spectrum of nitrilases. Full article

(This article belongs to the Special Issue Crystallographic Studies of Enzymes (Volume II))

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10 pages, 2686 KiB

Open AccessArticle

Crystal Structure of an Active Site Mutant Form of IRG1 from Bacillus subtilis

by Hyun Ho Park

Crystals 2021, 11(4), 350; https://doi.org/10.3390/cryst11040350 - 29 Mar 2021

Cited by 1 | Viewed by 2120

Abstract

Immune-responsive gene1 (IRG1), an enzyme that is overexpressed during immune reactions, catalyzes the production of itaconate from cis-aconitate. Itaconate is a multifunctional immuno-metabolite that displays antibacterial and antiviral activities. The recent resolution of its structure has enabled the mechanism underlying IRG1 function to be speculated on. However, the precise mechanism underlying the enzymatic reaction of IRG1 remains vague owing to the absence of information regarding the structure of the IRG1/substrate or the product complex. In this study, we determined the high-resolution structure of the active site mutant form of IRG1 from Bacillus subtilis (bsIRG1_H102A). Structural analysis detected unidentified electron densities around the active site. Structural comparison with the wildtype revealed that H102 was critical for the precise location of the side chain of residues around active site of IRG1. Finally, the activity of bsIRG1 was extremely low compared with that of mammalian IRG1. The current structural study will expectedly help understand the working mechanism of IRG1. Full article

(This article belongs to the Special Issue Crystallographic Studies of Enzymes (Volume II))

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7 pages, 2086 KiB

Open AccessCommunication

Purification and Crystallographic Analysis of a Novel Cold-Active Esterase (HaEst1) from Halocynthiibacter arcticus

by Sangeun Jeon, Jisub Hwang, Wanki Yoo, Joo Won Chang, Hackwon Do, Han-Woo Kim, Kyeong Kyu Kim, Jun Hyuck Lee and T. Doohun Kim

Crystals 2021, 11(2), 170; https://doi.org/10.3390/cryst11020170 - 8 Feb 2021

Cited by 2 | Viewed by 2072

Abstract

This report deals with the purification, characterization, and a preliminary crystallographic study of a novel cold-active esterase (HaEst1) from Halocynthiibacter arcticus. Primary sequence analysis reveals that HaEst1 has a catalytic serine in G-x-S-x-G motif. The recombinant HaEst1 was cloned, expressed, and purified. SDS-PAGE and zymographic analysis were carried out to characterize the properties of HaEst1. A single crystal of HaEst1 was obtained in a solution containing 10% (w/v) PEG 8000/8% ethylene glycol, 0.1 M Hepes-NaOH, pH 7.5. Diffraction data were collected to 2.10 Å resolution with P2₁ space group. The final R_merge and R_p.i.m values were 7.6% and 3.5% for 50–2.10 Å resolution. The unit cell parameters were a = 35.69 Å, b = 91.21 Å, c = 79.15 Å, and β = 96.9°. Full article

(This article belongs to the Special Issue Crystallographic Studies of Enzymes (Volume II))

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22 pages, 3778 KiB

Open AccessArticle

Molecular Dynamics Investigation of Phenolic Oxidative Coupling Protein Hyp-1 Derived from Hypericum perforatum

by Joanna Smietanska, Tomasz Kozik, Radoslaw Strzalka, Ireneusz Buganski and Janusz Wolny

Crystals 2021, 11(1), 43; https://doi.org/10.3390/cryst11010043 - 6 Jan 2021

Cited by 1 | Viewed by 2919

Abstract

Molecular dynamics (MD) simulations provide a physics-based approach to understanding protein structure and dynamics. Here, we used this intriguing tool to validate the experimental structural model of Hyp-1, a pathogenesis-related class 10 (PR-10) protein from the medicinal herb Hypericum perforatum, with potential application in various pharmaceutical therapies. A nanosecond MD simulation using the all-atom optimized potentials for liquid simulations (OPLS–AA) force field was performed to reveal that experimental atomic displacement parameters (ADPs) underestimate their values calculated from the simulation. The average structure factors obtained from the simulation confirmed to some extent the relatively high compliance of experimental and simulated Hyp-1 models. We found, however, many outliers between the experimental and simulated side-chain conformations within the Hyp-1 model, which prompted us to propose more reasonable energetically preferred rotameric forms. Therefore, we confirmed that MD simulation may be applicable for the verification of refined, experimental models and the explanation of their structural intricacies. Full article

(This article belongs to the Special Issue Crystallographic Studies of Enzymes (Volume II))

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