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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 October 2019) | Viewed by 42082

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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 control of key biological processes, including metabolism and signalling, 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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Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Crystals is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

crystallography
enzymes
structure
function
drug target
protein engineering

Related Special Issue

Crystallographic Studies of Enzymes (Volume II) in Crystals (12 articles)

Published Papers (13 papers)

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Editorial

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3 pages, 160 KiB

Open AccessEditorial

Crystallographic Studies of Enzymes

by T. Doohun Kim and Kyeong Kyu Kim

Crystals 2020, 10(1), 6; https://0-doi-org.brum.beds.ac.uk/10.3390/cryst10010006 - 20 Dec 2019

Viewed by 1880

Abstract

Enzymes are biological catalysts, which work to accelerate chemical reactions at the molecular level in living organisms. They are major players in the control of biological processes such as replication, transcription, protein synthesis, metabolism, and signaling. Like inorganic catalysts, enzymes function by decreasing the activation energy of chemical reactions, thereby enhancing the rate of the reactions. Enzymes are widely used for chemical, food, pharmaceutical, medicinal, analytical, clinical, forensic, and environmental applications. Therefore, studies on their structure, mechanism, and function, using a wide range of experimental and computational methods, are necessary to understand better enzymes in biological processes. For this special issue, “Crystallographic Studies of Enzymes", we have collected research papers on enzymes with structural aspects and functional aspects; here we briefly discuss the contents of such research papers as follows, with the aim of suggesting new directions of investigation in the fields of enzyme research, protein engineering, and drug development. Full article

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

Research

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13 pages, 5050 KiB

Open AccessArticle

Crystal Structure of Bacterial Cystathionine Γ-Lyase in The Cysteine Biosynthesis Pathway of Staphylococcus aureus

by Dukwon Lee, Soyeon Jeong, Jinsook Ahn, Nam-Chul Ha and Ae-Ran Kwon

Crystals 2019, 9(12), 656; https://0-doi-org.brum.beds.ac.uk/10.3390/cryst9120656 - 09 Dec 2019

Cited by 11 | Viewed by 4147

Abstract

Many enzymes require pyridoxal 5’-phosphate (PLP) as an essential cofactor and share active site residues in mediating diverse enzymatic reactions. Methionine can be converted into cysteine by cystathionine γ-lyases (CGLs) through a transsulfuration reaction dependent on PLP. In bacteria, MccB, also known as YhrB, exhibits CGL activity that cleaves the C–S bond of cystathionine at the γ position. In this study, we determined the crystal structure of MccB from Staphylococcus aureus in its apo- and PLP-bound forms. The structures of MccB exhibited similar molecular arrangements to those of MetC-mediating β-elimination with the same substrate and further illustrated PLP-induced structural changes in MccB. A structural comparison to MetC revealed a longer distance between the N-1 atom of the pyridine ring of PLP and the Oδ atom of the Asp residue, as well as a wider and more flexible active site environment in MccB. We also found a hydrogen bond network in Ser-water-Ser-Glu near the Schiff base nitrogen atom of the PLP molecule and propose the Ser-water-Ser-Glu motif as a general base for the γ-elimination process. Our study suggests the molecular mechanism for how homologous enzymes that use PLP as a cofactor catalyze different reactions with the same active site residues. Full article

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

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9 pages, 1847 KiB

Open AccessArticle

Crystal Structures of the 43 kDa ATPase Domain of Xanthomonas Oryzae pv. Oryzae Topoisomerase IV ParE Subunit and its Complex with Novobiocin

by Ha Yun Jung and Yong-Seok Heo

Crystals 2019, 9(11), 577; https://0-doi-org.brum.beds.ac.uk/10.3390/cryst9110577 - 05 Nov 2019

Cited by 3 | Viewed by 2449

Abstract

Topoisomerase IV, one of the best-established antibacterial targets, is an enzyme crucial for chromosome segregation and cell division by catalyzing changes in DNA topology through breaking and rejoining DNA. This enzyme functions as a heterotetramer consisting of two ParC and two ParE subunits. Aminocoumarin class inhibitors target the ParE subunit, while widely used quinolones target the ParC subunit. Here, we determined the crystal structure of the ParE 43 kDa ATPase domain from Xanthomonas oryzae pv. oryzae. Size exclusion chromatography showed that the ParE ATPase domain exists as a monomer in solution, while it dimerizes when ATP is added. Structural comparison with the structure of Escherichia coli ParE in complex with an ATP analogue showed large conformational change of the subdomains within the protein. We also determined the structure of the ParE ATPase domain in complex with novobiocin, a natural product aminocoumarin class inhibitor, revealing its binding mode and the structural change within the ATP-binding site induced by novobiocin binding. These results could provide a basis for the design of more potent topoisomerase IV inhibitors with improved antibacterial activity. Full article

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

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12 pages, 3589 KiB

Open AccessArticle

Crystal Structure of IlvC, a Ketol-Acid Reductoisomerase, from Streptococcus Pneumoniae

by Gyuhee Kim, Donghyuk Shin, Sumin Lee, Jaesook Yun and Sangho Lee

Crystals 2019, 9(11), 551; https://0-doi-org.brum.beds.ac.uk/10.3390/cryst9110551 - 24 Oct 2019

Cited by 4 | Viewed by 3031

Abstract

Biosynthesis of branched-chain amino acids (BCAAs), including isoleucine, leucine and valine, is required for survival and virulence of a bacterial pathogen such as Streptococcus pneumoniae. IlvC, a ketol-acid reductoisomerase (E.C. 1.1.1.86) with NADP(H) and Mg²⁺ as cofactors from the pathogenic Streptococcus pneumoniae (SpIlvC), catalyzes the second step in the BCAA biosynthetic pathway. To elucidate the structural basis for the IlvC-mediated reaction, we determined the crystal structure of SpIlvC at 1.69 Å resolution. The crystal structure of SpIlvC contains an asymmetric dimer in which one subunit is in apo-form and the other in NADP(H) and Mg²⁺-bound form. Crystallographic analysis combined with an activity assay and small-angle X-ray scattering suggested that SpIlvC retains dimeric arrangement in solution and that D83 in the NADP(H) binding site and E195 in the Mg²⁺ binding site are the most critical in the catalytic activity of SpIlvC. Crystal structures of SpIlvC mutants (R49E, D83G, D191G and E195S) revealed local conformational changes only in the NADP(H) binding site. Taken together, our results establish the molecular mechanism for understanding functions of SpIlvC in pneumococcal growth and virulence. Full article

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

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8 pages, 1814 KiB

Open AccessArticle

Crystal Structures of Putative Flavin Dependent Monooxygenase from Alicyclobacillus Acidocaldarius

by Hyunjin Moon, Sungwook Shin and Jungwoo Choe

Crystals 2019, 9(11), 548; https://0-doi-org.brum.beds.ac.uk/10.3390/cryst9110548 - 23 Oct 2019

Cited by 1 | Viewed by 2049

Abstract

Flavin dependent monooxygenases catalyze various reactions to play a key role in biological processes, such as catabolism, detoxification, and biosynthesis. Group D flavin dependent monooxygenases are enzymes with an Acyl-CoA dehydrogenase (ACAD) fold and use Flavin adenine dinucleotide (FAD) or Flavin mononucleotide (FMN) as a cofactor. In this research, crystal structures of Alicyclobacillus acidocaldarius protein formerly annotated as an ACAD were determined in Apo and FAD bound state. Although our structure showed high structural similarity to other ACADs, close comparison of substrate binding pocket and phylogenetic analysis showed that this protein is more closely related to other bacterial group D flavin dependent monooxygenases, such as DszC (sulfoxidase) and DnmZ and Kijd3 (nitrososynthases). Full article

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

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12 pages, 14151 KiB

Open AccessArticle

Characterization and Structural Determination of Cold-Adapted Monodehydroascorbate Reductase, MDHAR, from the Antarctic Hairgrass Deschampsia Antarctica

by Ae Kyung Park, Il-Sup Kim, Hackwon Do, Hyun Kim, Woong Choi, Seung-Woo Jo, Seung Chul Shin, Jun Hyuck Lee, Ho-Sung Yoon and Han-Woo Kim

Crystals 2019, 9(10), 537; https://0-doi-org.brum.beds.ac.uk/10.3390/cryst9100537 - 18 Oct 2019

Cited by 6 | Viewed by 2773

Abstract

Ascorbic acid (AsA) is an abundant component of plants and acts as a strong and active antioxidant. In order to maintain the antioxidative capacity of AsA, the rapid regeneration of AsA is regulated by dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR). To understand how MDHAR functions under extreme temperature conditions, this study characterized its biochemical properties and determined the crystal structure of MDHAR from the Antarctic hairgrass Deschampsia antarctica (DaMDHAR) at 2.2 Å resolution. This allowed for a structural comparison with the mesophilic MDHAR from Oryza sativa L. japonica (OsMDHAR). In the functional analysis, yeast cells expressing DaMDHAR were tolerant to freezing and thawing cycles. It is possible that the expression of DaMDHAR in yeast enhanced the tolerance for ROS-induced abiotic stress. Full article

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

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10 pages, 1845 KiB

Open AccessArticle

Crystal Structure of the Active Site Mutant Form of Soluble Fumarate Reductase, Osm1

by Chang Min Kim, Sunghark Kwon, Kyung Ho Jung, Hye Lin Chun, Hyun Ji Ha and Hyun Ho Park

Crystals 2019, 9(10), 504; https://0-doi-org.brum.beds.ac.uk/10.3390/cryst9100504 - 27 Sep 2019

Cited by 2 | Viewed by 2551

Abstract

Soluble fumarate reductase is essential for survival under anaerobic conditions. This enzyme can maintain the redox balance in the cell by catalyzing the reduction of fumarate to succinate. Although the overall reaction mechanism of soluble fumarate reductase in yeast, Osm1, has been proposed by a previous structural study, the details of the underlying mechanism are not completely elucidated. The present study provides the structural information regarding the active site mutant form of Osm1 (R326A), thus, revealing that R326A mutation does not affect the substrate binding. Structural alterations of the residues surrounding the active site, and the missing 2nd flavin adenine dinucleotide (FAD) in the previously defined 2nd FAD binding site, were observed as characteristic features of the Osm1 R326A crystal structure. Based on these findings, we provided a clue that can explain the loss of activity of Osm1 R326A. Full article

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

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13 pages, 3419 KiB

Open AccessArticle

Comparison of Candida Albicans Fatty Acid Amide Hydrolase Structure with Homologous Amidase Signature Family Enzymes

by Cho-Ah Min, Ji-Sook Yun, Eun Hwa Choi, Ui Wook Hwang, Dong-Hyung Cho, Je-Hyun Yoon and Jeong Ho Chang

Crystals 2019, 9(9), 472; https://0-doi-org.brum.beds.ac.uk/10.3390/cryst9090472 - 10 Sep 2019

Cited by 3 | Viewed by 3066

Abstract

Fatty acid amide hydrolase (FAAH) is a well-characterized member of the amidase signature (AS) family of serine hydrolases. The membrane-bound FAAH protein is responsible for the catabolism of neuromodulatory fatty acid amides, including anandamide and oleamide, that regulate a wide range of mammalian behaviors, including pain perception, inflammation, sleep, and cognitive/emotional state. To date, limited crystal structures of FAAH and non-mammalian AS family proteins have been determined and used for structure-based inhibitor design. In order to provide broader structural information, the crystal structure of FAAH from the pathogenic fungus Candida albicans was determined at a resolution of 2.2 Å. A structural comparison with a brown rat Rattus norvegicus FAAH as well as with other bacterial AS family members, MAE2 and PAM, showed overall similarities but there were several discriminative regions found: the transmembrane domain and the hydrophobic cap of the brown rat FAAH were completely absent in the fungal FAAH structure. Along with these results, a phylogenetic analysis of 19 species within the AS family showed that fungal FAAHs diverged from a common ancestor before the separation of eukarya and prokarya. Taken together, this study provides insights into developing more potent inhibitors of FAAH as well as expanding our knowledge of the relationships between AS family members. Full article

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

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13 pages, 2237 KiB

Open AccessArticle

Crystal Structure of NADPH-Dependent Methylglyoxal Reductase Gre2 from Candida Albicans

by Giang Thu Nguyen, Shinae Kim, Hyeonseok Jin, Dong-Hyung Cho, Hang-Suk Chun, Woo-Keun Kim and Jeong Ho Chang

Crystals 2019, 9(9), 471; https://0-doi-org.brum.beds.ac.uk/10.3390/cryst9090471 - 10 Sep 2019

Cited by 3 | Viewed by 3384

Abstract

Gre2 is a key enzyme in the methylglyoxal detoxification pathway; it uses NADPH or NADH as an electron donor to reduce the cytotoxic methylglyoxal to lactaldehyde. This enzyme is a member of the short-chain dehydrogenase/reductase (SDR) superfamily whose members catalyze this type of reaction with a broad range of substrates. To elucidate the structural features, we determined the crystal structures of the NADPH-dependent methylglyoxal reductase Gre2 from Candida albicans (CaGre2) for both the apo-form and NADPH-complexed form at resolutions of 2.8 and 3.02 Å, respectively. The CaGre2 structure is composed of two distinct domains: the N-terminal cofactor-binding domain and the C-terminal substrate-binding domain. Extensive comparison of CaGre2 with its homologous structures reveals conformational changes in α12 and β3′ of the NADPH-complex forms. This study may provide insights into the structural and functional variation of SDR family proteins. Full article

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

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8 pages, 1802 KiB

Open AccessCommunication

Triglycine-Based Approach for Identifying the Substrate Recognition Site of an Enzyme

by Ki Hyun Nam

Crystals 2019, 9(9), 444; https://0-doi-org.brum.beds.ac.uk/10.3390/cryst9090444 - 26 Aug 2019

Cited by 3 | Viewed by 3516

Abstract

Various peptides or non-structural amino acids are recognized by their specific target proteins, and perform a biological role in various pathways in vivo. Understanding the interactions between target protein and peptides (or non-structural amino acids) provides key information on the molecular interactions, which can be potentially translated to the development of novel drugs. However, it is experimentally challenging to determine the crystal structure of protein–peptide complexes. To obtain structural information on the substrate recognition of the peptide-recognizing enzyme, X-ray crystallographic studies were performed using triglycine (Gly-Gly-Gly) as the main-chain of the peptide. The crystal structure of Parengyodontium album Proteinase K in complex with triglcyine was determined at a 1.4 Å resolution. Two different bound conformations of triglycine were observed at the substrate recognition site. The triglycine backbone forms stable interactions with β5-α4 and α5-β6 loops of the main-chain. One of the triglycine-binding conformations was identical to the binding mode of a peptide-based inhibitor from a previously reported crystal structure of Proteinase K. Triglycine has potential application in X-ray crystallography in order to identify the substrate recognition sites in the peptide binding enzymes. Full article

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

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11 pages, 2406 KiB

Open AccessArticle

Structural Analyses of Helicobacter Pylori FolC Conducting Glutamation in Folate Metabolism

by Joon Sung Park, Hyoun Sook Kim, Sang Ho Park, Mi Seul Park, Sung-Min Kang, Hyun-Jung Kim and Byung Woo Han

Crystals 2019, 9(8), 429; https://0-doi-org.brum.beds.ac.uk/10.3390/cryst9080429 - 19 Aug 2019

Cited by 1 | Viewed by 3142

Abstract

FolC plays important roles in the folate metabolism of cells by attaching l-Glu to dihydropteroate (DHP) and folate, which are known activities of dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS), respectively. Here, we determined the crystal structure of Helicobacter pylori FolC (HpFolC) at 1.95 Å resolution using the single-wavelength anomalous diffraction method. HpFolC has globular N- and C-terminal domains connected by a single loop, and a binding site for ATP is located between the two domains. Apo-HpFolC was crystallized in the presence of citrate in a crystallization solution, which was held in the ATP-binding site. Structural motifs such as the P-loop and Ω-loop of HpFolC for binding of ATP and two magnesium ions are well conserved in spite of the low overall sequence similarity to other FolC/FPGSs. The Ω-loop would also recognize a folate molecule, and the DHP-binding loop of HpFolC is expected to exhibit a unique recognition mode on DHP, compared with other FolCs. Because human FolC is known to only exhibit FPGS activity, the DHFS activity of bacterial FolC is an attractive target for the eradication of pathogenic bacteria. Consequently, our structural analyses of HpFolC provide a valuable foundation for a universal antibacterial strategy against H. pylori as well as other pathogenic bacteria. Full article

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

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7 pages, 2043 KiB

Open AccessArticle

Crystal Structure of Chaperonin GroEL from Xanthomonas oryzae pv. oryzae

by Huyen-Thi Tran, Jongha Lee, Hyunjae Park, Jeong-Gu Kim, Seunghwan Kim, Yeh-Jin Ahn and Lin-Woo Kang

Crystals 2019, 9(8), 399; https://0-doi-org.brum.beds.ac.uk/10.3390/cryst9080399 - 02 Aug 2019

Cited by 1 | Viewed by 3274

Abstract

Xanthomonas oryzae pv. oryzae (Xoo) is a plant pathogen that causes bacterial blight of rice, with outbreaks occurring in most rice-growing countries. Thus far, there is no effective pesticide against bacterial blight. Chaperones in bacterial pathogens are important for the stabilization and delivery of effectors into host cells to cause disease. In bacteria, GroEL/GroES complex mediates protein folding and protects proteins against misfolding and aggregation caused by environmental stress. We determined the crystal structure of GroEL from Xanthomonas oryzae pv. oryzae (XoGroEL) at 3.2 Å resolution, which showed the open form of two conserved homoheptameric rings stacked back-to-back. In the open form structure, the apical domain of XoGroEL had a higher B factor than the intermediate and equatorial domains, indicating that the apical domain had a flexible conformation before the binding of substrate unfolded protein and ATP. The XoGroEL structure will be helpful in understanding the function and catalytic mechanism of bacterial chaperonin GroELs. Full article

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

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Review

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27 pages, 6140 KiB

Open AccessReview

Carboxylic Ester Hydrolases in Bacteria: Active Site, Structure, Function and Application

by Changsuk Oh, T. Doohun Kim and Kyeong Kyu Kim

Crystals 2019, 9(11), 597; https://0-doi-org.brum.beds.ac.uk/10.3390/cryst9110597 - 14 Nov 2019

Cited by 23 | Viewed by 5954

Abstract

Carboxylic ester hydrolases (CEHs), which catalyze the hydrolysis of carboxylic esters to produce alcohol and acid, are identified in three domains of life. In the Protein Data Bank (PDB), 136 crystal structures of bacterial CEHs (424 PDB codes) from 52 genera and metagenome have been reported. In this review, we categorize these structures based on catalytic machinery, structure and substrate specificity to provide a comprehensive understanding of the bacterial CEHs. CEHs use Ser, Asp or water as a nucleophile to drive diverse catalytic machinery. The α/β/α sandwich architecture is most frequently found in CEHs, but 3-solenoid, β-barrel, up-down bundle, α/β/β/α 4-layer sandwich, 6 or 7 propeller and α/β barrel architectures are also found in these CEHs. Most are substrate-specific to various esters with types of head group and lengths of the acyl chain, but some CEHs exhibit peptidase or lactamase activities. CEHs are widely used in industrial applications, and are the objects of research in structure- or mutation-based protein engineering. Structural studies of CEHs are still necessary for understanding their biological roles, identifying their structure-based functions and structure-based engineering and their potential industrial applications. Full article

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

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