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Crystals
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Crystallography of Enzymes
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Crystallography of Enzymes

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

Deadline for manuscript submissions: 30 September 2025 | Viewed by 10634

Special Issue Editors

Dr. Bo Liang

E-Mail Website
Guest Editor
Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, USA
Interests: biochemistry; structural biology; RNA biology; virology; neurobiology; cancer biology
Dr. Houfu Guo

E-Mail Website
Guest Editor
Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY, USA
Interests: protein biochemistry; structural biology; collagen; post-translational modifications; cancer biology; matrix biology

Special Issue Information

Dear Colleagues,

Enzymes are proteins or RNA molecules that act as biological catalysts for accelerating biochemical reactions by lowering their activation energies. Enzymes catalyze more than 5,000 known types of biochemical reactions; however, how enzymes carry out such diverse functions is still not fully understood. Since enzymes' unique three-dimensional (3D) structural architectures allow them to act on substrates and convert them to products, determining enzymes' structure is critical in elucidating their diverse functions. Just as Francis Crick, one of the greatest biologists, once said: "If you want to understand the function, study structure." Currently, X-ray crystallography remains the favored technique for determining enzyme structures. X-ray crystallography has been widely utilized to elucidate the atomic details of catalytic mechanisms and conformational changes in enzymes, such as active site binding to substrates or inhibitors. Such structural insights inform biology and biomedicine. Although many enzyme structures have been determined in the past several decades, more remain to be elucidated. Thus, we welcome structural biologists and biochemists to provide their views and perspectives on the crystallography of interesting and novel enzymes.

Dr. Bo Liang
Dr. Houfu Guo
Guest Editors

Manuscript Submission Information

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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.

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Keywords

  • enzyme
  • structure
  • function
  • X-ray crystallography
  • crystal
  • crystallization
  • catalytic mechanism
  • antagonist

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Published Papers (8 papers)

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Research

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11 pages, 2623 KiB  
Article
Structural Analysis of PlyKp104, a Novel Phage Endoysin
by Jung-Min Choi
Crystals 2025, 15(5), 448; https://doi.org/10.3390/cryst15050448 - 9 May 2025
Viewed by 177
Abstract
Antibiotic resistance has emerged as a critical global public health challenge, prompting increased interest in non-antibiotic antimicrobial strategies such as bacteriophage-derived endolysins. Although endolysins possess strong lytic potential, their application to Gram-negative bacteria remains limited due to the outer membrane barrier. PlyKp104 is [...] Read more.
Antibiotic resistance has emerged as a critical global public health challenge, prompting increased interest in non-antibiotic antimicrobial strategies such as bacteriophage-derived endolysins. Although endolysins possess strong lytic potential, their application to Gram-negative bacteria remains limited due to the outer membrane barrier. PlyKp104 is a recently identified phage-derived endolysin that exhibits lytic activity against Gram-negative bacteria without the aid of membrane permeabilizers. In this study, the crystal structure of PlyKp104 was determined at a resolution of 1.85 Å. PlyKp104 consists solely of a catalytic SLT domain, and structure-based analysis revealed a putative active site and key structural features associated with substrate binding. Comparative analysis with homologous structures suggested that PlyKp104 belongs to lytic transglycosylase family 1. B-factor analysis and hydrophobic interaction mapping indicated that the domain exhibits high structural stability, supported by conserved hydrophobic residues clustered in motifs I and II. During structure determination, an unidentified electron density was consistently observed near a neutral, hydrophobic surface region. Its shape and environment suggest the presence of a lipid-like molecule, implying a potential lipid-binding site. These findings provide structural insight into PlyKp104 and contribute to the understanding of endolysin mechanisms against Gram-negative bacteria, with implications for future protein engineering efforts. Full article
(This article belongs to the Special Issue Crystallography of Enzymes)
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16 pages, 7322 KiB  
Article
Structural Study of Thermostable Ginsenoside β-Glucosidase from Caldicellulosiruptor bescii
by Jung-Min Choi
Crystals 2025, 15(5), 447; https://doi.org/10.3390/cryst15050447 - 9 May 2025
Viewed by 244
Abstract
Protopanaxadiol-type ginsenosides, the major bioactive components of Panax ginseng, exhibit diverse pharmacological activities, but suffer from low oral bioavailability due to poor water solubility and membrane permeability. Enzymatic deglycosylation has emerged as an effective strategy to enhance their therapeutic potential; however, most [...] Read more.
Protopanaxadiol-type ginsenosides, the major bioactive components of Panax ginseng, exhibit diverse pharmacological activities, but suffer from low oral bioavailability due to poor water solubility and membrane permeability. Enzymatic deglycosylation has emerged as an effective strategy to enhance their therapeutic potential; however, most glucosidases lack sufficient thermostability for industrial applications. A β-glucosidase from the thermophilic bacterium Caldicellulosiruptor bescii (CbBGL) has demonstrated efficient conversion of major ginsenosides into compound K at elevated temperatures. In this study, the high-resolution crystal structure of CbBGL was determined at 1.9 Å. Structural analysis revealed that CbBGL adopts a classical (α/β)8 TIM barrel fold and functions as a homodimer. Comparative studies with other glucosidases highlighted structural features contributing to its thermostability, including moderate B-factor distribution and a limited hydrogen bond network. Docking analyses revealed a narrow, inverted conical substrate-binding cleft, which imposes specific binding orientations and underlies the enzyme’s stepwise deglycosylation mechanism. These insights provide a structural basis for CbBGL’s thermal resilience and substrate specificity, offering a valuable platform for the rational engineering of glucosidases in ginsenoside bioconversion processes. Full article
(This article belongs to the Special Issue Crystallography of Enzymes)
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14 pages, 12903 KiB  
Article
Biochemical and Structural Characterization of Glyoxylate Reductase/Hydroxypyruvate Reductase from Bacillus subtilis
by Thang Quyet Nguyen, Thai Huu Duong, Jin Kuk Yang and Wonchull Kang
Crystals 2025, 15(4), 298; https://doi.org/10.3390/cryst15040298 - 25 Mar 2025
Viewed by 286
Abstract
D-2-hydroxyacid dehydrogenases (2HADHs) catalyze the reversible reaction of 2-ketocarboxylic acid to the corresponding (R)-2-hydroxycarboxylic acids using NAD(P)H cofactor. As the preference of the cofactor and substrate varies among homologs, biochemical characterization is required to understand this enzyme. Here, we analyzed the biochemical properties [...] Read more.
D-2-hydroxyacid dehydrogenases (2HADHs) catalyze the reversible reaction of 2-ketocarboxylic acid to the corresponding (R)-2-hydroxycarboxylic acids using NAD(P)H cofactor. As the preference of the cofactor and substrate varies among homologs, biochemical characterization is required to understand this enzyme. Here, we analyzed the biochemical properties of Bacillus subtilis glyoxylate reductase/hydroxypyruvate reductase (BsGRHPR), which catalyzes the reduction of both glyoxylate (EC 1.1.1.26) and hydroxypyruvate (EC 1.1.1.81). Enzyme kinetics showed a preference for hydroxypyruvate over glyoxylate, with a seven-fold higher specificity constant. In addition, BsGRHPR displayed a strict preference for NADPH over NADH as a cofactor. The crystal structures of BsGRHPR in complex with formate were determined in the presence and absence of the cofactor at near-atomic resolution. Structural comparisons revealed conformational changes upon cofactor binding and key residues, such as Asp80, R157, R179, R239, Asp263, and Arg296. In addition, substrate-binding analysis highlighted conserved residues, including Val77, Gly78, His287, and S290. Our structures suggest that Glu137, His287, Ser290, and Arg296 serve as gatekeepers at the entrance of the tunnel. This comprehensive characterization of BsGRHPR elucidates its substrate specificity, cofactor preference, and catalytic mechanism, contributing to a broader understanding of GRHPR family enzymes, with potential implications for metabolic engineering applications. Full article
(This article belongs to the Special Issue Crystallography of Enzymes)
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17 pages, 4267 KiB  
Article
Crystallographic and NMR Study of Streptococcus pneumonia LCP Protein PsrSp Indicate the Importance of Dynamics in Four Long Loops for Ligand Specificity
by Tatyana Sandalova, Benedetta Maria Sala, Martin Moche, Hans-Gustaf Ljunggren, Evren Alici, Birgitta Henriques-Normark, Tatiana Agback, Dmitry Lesovoy, Peter Agback and Adnane Achour
Crystals 2024, 14(12), 1094; https://doi.org/10.3390/cryst14121094 - 19 Dec 2024
Viewed by 898
Abstract
The crystal structure of the extracellular region of the second pneumococcal LCP, a polyisoprenyl-teichoic acid-peptidoglycan teichoic acid transferase PsrSp, was determined and refined to 2.15 Å resolution. Despite the low sequence homology with other LCP proteins, the PsrSp maintains the [...] Read more.
The crystal structure of the extracellular region of the second pneumococcal LCP, a polyisoprenyl-teichoic acid-peptidoglycan teichoic acid transferase PsrSp, was determined and refined to 2.15 Å resolution. Despite the low sequence homology with other LCP proteins, the PsrSp maintains the fold of the LCP domain, and the positions of the residues suggested to participate in the transferase function are conserved. The tunnel found in the PsrSp between the central β-sheet and three α-helices is wide enough to accommodate polyisoprenyl-teichoic acid. Comparison of the crystallographic temperature factors of LCP from distinct bacteria demonstrated that the four long loops located close to the teichoic acid and peptidoglycan binding sites have different relative mobilities. To compare the dynamics of the PsrSp in crystalline state and in solution, NMR spectra were recorded, and 88% of the residues were assigned in the 1H-15N TROSY HSQC spectra. Perfect accordance in the secondary structure of the crystal structure of PsrSp with NMR data demonstrated correct assignment. Moreover, the relative mobility of the essential loops estimated from the crystallographic B-factor is in good agreement with order parameter S2, predicted from chemical shift. We hypothesize that the dynamics of these loops are important for the substrate promiscuity of LCP proteins. Full article
(This article belongs to the Special Issue Crystallography of Enzymes)
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14 pages, 4767 KiB  
Article
Structural Flexibility of the Monomeric Red Fluorescent Protein DsRed
by Ki Hyun Nam
Crystals 2024, 14(1), 62; https://doi.org/10.3390/cryst14010062 - 2 Jan 2024
Cited by 3 | Viewed by 2096
Abstract
The monomeric red fluorescent protein DsRed (mDsRed) is widely used as an optical probe for multicolor applications in flow cytometry or fluorescence microscopy. Understanding the structure and dynamics of mDsRed provides fundamental information for its practical applications. The mDsRed crystal structure has been [...] Read more.
The monomeric red fluorescent protein DsRed (mDsRed) is widely used as an optical probe for multicolor applications in flow cytometry or fluorescence microscopy. Understanding the structure and dynamics of mDsRed provides fundamental information for its practical applications. The mDsRed crystal structure has been reported, but the structural dynamics have not been fully elucidated. Herein, the crystal structure of mDsRed was determined at 2.9 Å resolution, and the molecular flexibility was analyzed. mDsRed contains a solvent-accessible hole between the β7-strand and β9-α10 loop, which is connected to the chromophore. A partial disorder was present in the electron density map of the tyrosine-ring group of the mDsRed chromophore, indicating a flexible conformation of the chromophore. The refined mDsRed chromophore displayed a cis-conformation with a nonplanar configuration between the tyrosine and imidazoline rings of the chromophore. Temperature factor analysis indicated that the β-barrel fold of mDsRed is rigid, while the loops at the top and bottom of the β-barrel are relatively flexible. The β-barrel surface of mDsRed was closer to the native conformation compared with the previously reported Zn-bound state of mDsRed. These structural findings extend our understanding of the molecular flexibility of mDsRed. Full article
(This article belongs to the Special Issue Crystallography of Enzymes)
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13 pages, 7480 KiB  
Article
Crystal Structure of Aspartate Semialdehyde Dehydrogenase from Porphyromonas gingivalis
by Jisub Hwang, Hackwon Do, Youn-Soo Shim and Jun Hyuck Lee
Crystals 2023, 13(8), 1274; https://doi.org/10.3390/cryst13081274 - 18 Aug 2023
Viewed by 1577
Abstract
Aspartate semialdehyde dehydrogenase (ASADH) catalyzes the biosynthesis of several essential amino acids, including lysine, methionine, and threonine, and bacterial cell components. Thus, ASADH is a crucial target for developing new antimicrobial agents that can potentially disrupt the biosynthesis of essential amino acids, thereby [...] Read more.
Aspartate semialdehyde dehydrogenase (ASADH) catalyzes the biosynthesis of several essential amino acids, including lysine, methionine, and threonine, and bacterial cell components. Thus, ASADH is a crucial target for developing new antimicrobial agents that can potentially disrupt the biosynthesis of essential amino acids, thereby inhibiting the growth of pathogens. Herein, the crystal structures of ASADH obtained from Porphyromonas gingivalis (PgASADH, UniProtKB code A0A1R4DY25) were determined in apo- and adenosine-2′-5′-diphosphate (2′,5′-ADP)-bound complex forms at a resolution of 1.73 Å. The apo- and 2′,5′-ADP-complexed crystals of PgASADH belonged to the space groups of I212121 and C2221, respectively. Analytical size-exclusion chromatography showed a stable PgASADH dimer in a solution. Clustering analysis and structural comparison studies performed on PgASADH and previously known ASADHs revealed that ASADHs, including PgASADH, can be classified into three types depending on sequential and structural differences at the α-helical subdomain region. These findings provide valuable insights into developing structure-based species-specific new antibacterial drugs against the oral pathogen P. gingivalis. Full article
(This article belongs to the Special Issue Crystallography of Enzymes)
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11 pages, 4004 KiB  
Article
Crystal Structure and Functional Characterization of an S-Formylglutathione Hydrolase (BuSFGH) from Burkholderiaceae sp.
by Jisub Hwang, Hackwon Do, Youn-Soo Shim and Jun Hyuck Lee
Crystals 2023, 13(4), 621; https://doi.org/10.3390/cryst13040621 - 4 Apr 2023
Viewed by 1805
Abstract
S-formylglutathione hydrolases (SFGHs) catalyze the hydrolysis of S-formylglutathione to formate and glutathione using the conserved serine hydrolase catalytic triad residues (Ser-His-Asp). SFGHs have broad substrate specificity, including, for example, ester bond-containing substrates. Here, we report the crystal structure of Burkholderiaceae sp. SFGH ( [...] Read more.
S-formylglutathione hydrolases (SFGHs) catalyze the hydrolysis of S-formylglutathione to formate and glutathione using the conserved serine hydrolase catalytic triad residues (Ser-His-Asp). SFGHs have broad substrate specificity, including, for example, ester bond-containing substrates. Here, we report the crystal structure of Burkholderiaceae sp. SFGH (BuSFGH) at 1.73 Å resolution. Structural analysis showed that the overall structure of BuSFGH has a typical α/β hydrolase fold, with a central β-sheet surrounded by α-helices. Analytical ultracentrifugation analysis showed that BuSFGH formed a stable dimer in solution. The enzyme activity assay indicated that BuSFGH has a high preference for short-chain p-nitrophenyl esters, such as p-nitrophenyl acetate. The activity of BuSFGH toward p-nitrophenyl acetate was five times higher than that of p-nitrophenyl butylate. Molecular modeling studies on the p-nitrophenyl acetate-bound BuSFGH structure indicate that Gly52, Leu53, Trp96, His147, Ser148, Trp182, Phe228, and His259 residues may be crucial for substrate binding. Collectively, these results are useful for understanding the substrate-binding mechanism and substrate specificity of BuSFGH. They can also provide useful insights for designing modified BuSFGHs with different substrate specificities. Full article
(This article belongs to the Special Issue Crystallography of Enzymes)
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Review

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14 pages, 14944 KiB  
Review
Structural Aspects of the ROS1 Kinase Domain and Oncogenic Mutations
by Juliana F. Vilachã, Tsjerk A. Wassenaar and Siewert J. Marrink
Crystals 2024, 14(2), 106; https://doi.org/10.3390/cryst14020106 - 23 Jan 2024
Cited by 2 | Viewed by 2616
Abstract
Protein kinases function as pivotal regulators in biological events, governing essential cellular processes through the transfer of phosphate groups from ATP molecules to substrates. Dysregulation of kinase activity is frequently associated with cancer, ocasionally arising from chromosomal translocation events that relocate genes encoding [...] Read more.
Protein kinases function as pivotal regulators in biological events, governing essential cellular processes through the transfer of phosphate groups from ATP molecules to substrates. Dysregulation of kinase activity is frequently associated with cancer, ocasionally arising from chromosomal translocation events that relocate genes encoding kinases. Fusion proteins resulting from such events, particularly those involving the proto-oncogene tyrosine-protein kinase ROS (ROS1), manifest as constitutively active kinases, emphasizing their role in oncogenesis. Notably, the chromosomal reallocation of the ros1 gene leads to fusion of proteins with the ROS1 kinase domain, implicated in various cancer types. Despite their prevalence, targeted inhibition of these fusion proteins relies on repurposed kinase inhibitors. This review comprehensively surveys experimentally determined ROS1 structures, emphasizing the pivotal role of X-ray crystallography in providing high-quality insights. We delve into the intricate interactions between ROS1 and kinase inhibitors, shedding light on the structural basis for inhibition. Additionally, we explore point mutations identified in patients, employing molecular modeling to elucidate their structural impact on the ROS1 kinase domain. By integrating structural insights with in vitro and in silico data, this review advances our understanding of ROS1 kinase in cancer, offering potential avenues for targeted therapeutic strategies. Full article
(This article belongs to the Special Issue Crystallography of Enzymes)
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