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A special issue of Molecules (ISSN 1420-3049). This special issue belongs to the section "Bioorganic Chemistry".

Deadline for manuscript submissions: closed (15 September 2021) | Viewed by 32474

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Prof. Dr. Stefan Janecek

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Laboratory of Protein Evolution, Institute of Molecular Biology, Slovak Academy of Sciences, SK-84551 Bratislava, Slovakia
Interests: amylolytic enzymes; starch/glycogen-binding domains; glycoside hydrolases; in-silico protein structure analysis; protein bioinformatics
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Special Issue Information

Dear Colleagues,

The area of carbohydrate-active enzymes belongs to one of the most enormously developing research fields that, in the last decades, has attracted continuously increasing attention from the scientific community. Since the beginning of the 1990s, the field has been tightly connected with the sequence-based classification of glycoside hydrolases (GHs) into GH families, established by Bernard Henrissat in 1991, and later involving other enzyme activities that, in addition to catalyzing the hydrolysis of glycoside linkages, can also catalyze the formation and/or modification of a variety of carbohydrates. Therefore, the carbohydrate-active enzymes are currently best recognized as being classified in the CAZy database (http://www.cazy.org/) covering, in addition to GH families, the families of glycosyltransferases (GTs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), and auxiliary activities (AAs); also covered is the segment devoted to their non-catalytic domains of the so-called carbohydrate-binding module (CBM) families. The scope of “CAZymes” thus includes a very wide space spanning from basic research to many practical applications, such as modern biotechnologies and top approaches in medicine.

For this Special Issue, both original full-length papers and insightful review articles on any aspects of CAZymes are equally welcome and considered.

Prof. Dr. Stefan Janecek
Guest Editor

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Keywords

new primary and tertiary structures
new enzyme specificities
new CAZy families
new practical applications
protein engineering and rational design
evolutionary questions

Published Papers (11 papers)

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Research

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24 pages, 2582 KiB

Open AccessArticle

Modulating Glycoside Hydrolase Activity between Hydrolysis and Transfer Reactions Using an Evolutionary Approach

by Rodrigo A. Arreola-Barroso, Alexey Llopiz, Leticia Olvera and Gloria Saab-Rincón

Molecules 2021, 26(21), 6586; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26216586 - 30 Oct 2021

Cited by 5 | Viewed by 2560

Abstract

The proteins within the CAZy glycoside hydrolase family GH13 catalyze the hydrolysis of polysaccharides such as glycogen and starch. Many of these enzymes also perform transglycosylation in various degrees, ranging from secondary to predominant reactions. Identifying structural determinants associated with GH13 family reaction specificity is key to modifying and designing enzymes with increased specificity towards individual reactions for further applications in industrial, chemical, or biomedical fields. This work proposes a computational approach for decoding the determinant structural composition defining the reaction specificity. This method is based on the conservation of coevolving residues in spatial contacts associated with reaction specificity. To evaluate the algorithm, mutants of α-amylase (TmAmyA) and glucanotransferase (TmGTase) from Thermotoga maritima were constructed to modify the reaction specificity. The K98P/D99A/H222Q variant from TmAmyA doubled the transglycosydation/hydrolysis (T/H) ratio while the M279N variant from TmGTase increased the hydrolysis/transglycosidation ratio five-fold. Molecular dynamic simulations of the variants indicated changes in flexibility that can account for the modified T/H ratio. An essential contribution of the presented computational approach is its capacity to identify residues outside of the active center that affect the reaction specificity. Full article

(This article belongs to the Special Issue Recent Advances in Carbohydrate-Active Enzymes)

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15 pages, 2644 KiB

Open AccessArticle

Discovery of a Kojibiose Hydrolase by Analysis of Specificity-Determining Correlated Positions in Glycoside Hydrolase Family 65

by Emma De Beul, Alana Jongbloet, Jorick Franceus and Tom Desmet

Molecules 2021, 26(20), 6321; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26206321 - 19 Oct 2021

Cited by 5 | Viewed by 3220

Abstract

The Glycoside Hydrolase Family 65 (GH65) is an enzyme family of inverting α-glucoside phosphorylases and hydrolases that currently contains 10 characterized enzyme specificities. However, its sequence diversity has never been studied in detail. Here, an in-silico analysis of correlated mutations was performed, revealing specificity-determining positions that facilitate annotation of the family’s phylogenetic tree. By searching these positions for amino acid motifs that do not match those found in previously characterized enzymes from GH65, several clades that may harbor new functions could be identified. Three enzymes from across these regions were expressed in E. coli and their substrate profile was mapped. One of those enzymes, originating from the bacterium Mucilaginibacter mallensis, was found to hydrolyze kojibiose and α-1,2-oligoglucans with high specificity. We propose kojibiose glucohydrolase as the systematic name and kojibiose hydrolase or kojibiase as the short name for this new enzyme. This work illustrates a convenient strategy for mapping the natural diversity of enzyme families and smartly mining the ever-growing number of available sequences in the quest for novel specificities. Full article

(This article belongs to the Special Issue Recent Advances in Carbohydrate-Active Enzymes)

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20 pages, 3942 KiB

Open AccessArticle

Utility of Bioluminescent Homogeneous Nucleotide Detection Assays in Measuring Activities of Nucleotide-Sugar Dependent Glycosyltransferases and Studying Their Inhibitors

by Laurie Engel, Juliano Alves, Jacquelyn Hennek, Said A. Goueli and Hicham Zegzouti

Molecules 2021, 26(20), 6230; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26206230 - 15 Oct 2021

Cited by 4 | Viewed by 2970

Abstract

Traditional glycosyltransferase (GT) activity assays are not easily configured for rapid detection nor for high throughput screening because they rely on radioactive product isolation, the use of heterogeneous immunoassays or mass spectrometry. In a typical glycosyltransferase biochemical reaction, two products are generated, a glycosylated product and a nucleotide released from the sugar donor substrate. Therefore, an assay that detects the nucleotide could be universal to monitor the activity of diverse glycosyltransferases in vitro. Here we describe three homogeneous and bioluminescent glycosyltransferase activity assays based on UDP, GDP, CMP, and UMP detection. Each of these assays are performed in a one-step detection that relies on converting the nucleotide product to ATP, then to bioluminescence using firefly luciferase. These assays are highly sensitive, robust and resistant to chemical interference. Various applications of these assays are presented, including studies on the specificity of sugar transfer by diverse GTs and the characterization of acceptor substrate-dependent and independent nucleotide-sugar hydrolysis. Furthermore, their utility in screening for specific GT inhibitors and the study of their mode of action are described. We believe that the broad utility of these nucleotide assays will enable the investigation of a large number of GTs and may have a significant impact on diverse areas of Glycobiology research. Full article

(This article belongs to the Special Issue Recent Advances in Carbohydrate-Active Enzymes)

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16 pages, 1450 KiB

Open AccessArticle

Chitinase Chit62J4 Essential for Chitin Processing by Human Microbiome Bacterium Clostridium paraputrificum J4

by Jan Dohnálek, Jarmila Dušková, Galina Tishchenko, Petr Kolenko, Tereza Skálová, Petr Novák, Karla Fejfarová and Jiří Šimůnek

Molecules 2021, 26(19), 5978; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26195978 - 02 Oct 2021

Cited by 5 | Viewed by 2296

Abstract

Commensal bacterium Clostridium paraputrificum J4 produces several extracellular chitinolytic enzymes including a 62 kDa chitinase Chit62J4 active toward 4-nitrophenyl N,N′-diacetyl-β-d-chitobioside (pNGG). We characterized the crude enzyme from bacterial culture fluid, recombinant enzyme rChit62J4, and its catalytic domain rChit62J4cat. This major chitinase, securing nutrition of the bacterium in the human intestinal tract when supplied with chitin, has a pH optimum of 5.5 and processes pNGG with K_m = 0.24 mM and k_cat = 30.0 s⁻¹. Sequence comparison of the amino acid sequence of Chit62J4, determined during bacterial genome sequencing, characterizes the enzyme as a family 18 glycosyl hydrolase with a four-domain structure. The catalytic domain has the typical TIM barrel structure and the accessory domains—2x Fn3/Big3 and a carbohydrate binding module—that likely supports enzyme activity on chitin fibers. The catalytic domain is highly homologous to a single-domain chitinase of Bacillus cereus NCTU2. However, the catalytic profiles significantly differ between the two enzymes despite almost identical catalytic sites. The shift of pI and pH optimum of the commensal enzyme toward acidic values compared to the soil bacterium is the likely environmental adaptation that provides C. paraputrificum J4 a competitive advantage over other commensal bacteria. Full article

(This article belongs to the Special Issue Recent Advances in Carbohydrate-Active Enzymes)

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17 pages, 21119 KiB

Open AccessFeature PaperArticle

In Silico Analysis of Fungal and Chloride-Dependent α-Amylases within the Family GH13 with Identification of Possible Secondary Surface-Binding Sites

by Zuzana Janíčková and Štefan Janeček

Molecules 2021, 26(18), 5704; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26185704 - 21 Sep 2021

Cited by 4 | Viewed by 2088

Abstract

This study brings a detailed bioinformatics analysis of fungal and chloride-dependent α-amylases from the family GH13. Overall, 268 α-amylase sequences were retrieved from subfamilies GH13_1 (39 sequences), GH13_5 (35 sequences), GH13_15 (28 sequences), GH13_24 (23 sequences), GH13_32 (140 sequences) and GH13_42 (3 sequences). Eight conserved sequence regions (CSRs) characteristic for the family GH13 were identified in all sequences and respective sequence logos were analysed in an effort to identify unique sequence features of each subfamily. The main emphasis was given on the subfamily GH13_32 since it contains both fungal α-amylases and their bacterial chloride-activated counterparts. In addition to in silico analysis focused on eventual ability to bind the chloride anion, the property typical mainly for animal α-amylases from subfamilies GH13_15 and GH13_24, attention has been paid also to the potential presence of the so-called secondary surface-binding sites (SBSs) identified in complexed crystal structures of some particular α-amylases from the studied subfamilies. As template enzymes with already experimentally determined SBSs, the α-amylases from Aspergillus niger (GH13_1), Bacillus halmapalus, Bacillus paralicheniformis and Halothermothrix orenii (all from GH13_5) and Homo sapiens (saliva; GH13_24) were used. Evolutionary relationships between GH13 fungal and chloride-dependent α-amylases were demonstrated by two evolutionary trees—one based on the alignment of the segment of sequences spanning almost the entire catalytic TIM-barrel domain and the other one based on the alignment of eight extracted CSRs. Although both trees demonstrated similar results in terms of a closer evolutionary relatedness of subfamilies GH13_1 with GH13_42 including in a wider sense also the subfamily GH13_5 as well as for subfamilies GH13_32, GH13_15 and GH13_24, some subtle differences in clustering of particular α-amylases may nevertheless be observed. Full article

(This article belongs to the Special Issue Recent Advances in Carbohydrate-Active Enzymes)

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15 pages, 2523 KiB

Open AccessArticle

Enhanced Activity by Genetic Complementarity: Heterologous Secretion of Clostridial Cellulases by Bacillus licheniformis and Bacillus velezensis

by Alexander Arsov, Kaloyan Petrov and Penka Petrova

Molecules 2021, 26(18), 5625; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26185625 - 16 Sep 2021

Cited by 11 | Viewed by 2604

Abstract

To adapt to various ecological niches, the members of genus Bacillus display a wide spectrum of glycoside hydrolases (GH) responsible for the hydrolysis of cellulose and lignocellulose. Being abundant and renewable, cellulose-containing plant biomass may be applied as a substrate in second-generation biotechnologies for the production of platform chemicals. The present study aims to enhance the natural cellulase activity of two promising 2,3-butanediol (2,3-BD) producers, Bacillus licheniformis 24 and B. velezensis 5RB, by cloning and heterologous expression of cel8A and cel48S genes of Acetivibrio thermocellus. In B. licheniformis, the endocellulase Cel8A (GH8) was cloned to supplement the action of CelA (GH9), while in B. velezensis, the cellobiohydrolase Cel48S (GH48) successfully complemented the activity of endo-cellulase EglS (GH5). The expression of the natural and heterologous cellulase genes in both hosts was demonstrated by reverse-transcription PCR. The secretion of clostridial cellulases was additionally enhanced by enzyme fusion to the subtilisin-like signal peptide, reaching a significant increase in the cellulase activity of the cell-free supernatants. The results presented are the first to reveal the possibility of genetic complementation for enhancement of cellulase activity in bacilli, thus opening the prospect for genetic improvement of strains with an important biotechnological application. Full article

(This article belongs to the Special Issue Recent Advances in Carbohydrate-Active Enzymes)

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15 pages, 2073 KiB

Open AccessArticle

Glucosylation of (±)-Menthol by Uridine-Diphosphate-Sugar Dependent Glucosyltransferases from Plants

by Elisabeth Kurze, Victoria Ruß, Nadia Syam, Isabelle Effenberger, Rafal Jonczyk, Jieren Liao, Chuankui Song, Thomas Hoffmann and Wilfried Schwab

Molecules 2021, 26(18), 5511; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26185511 - 10 Sep 2021

Cited by 4 | Viewed by 2561

Abstract

Menthol is a cyclic monoterpene alcohol of the essential oils of plants of the genus Mentha, which is in demand by various industries due to its diverse sensorial and physiological properties. However, its poor water solubility and its toxic effect limit possible applications. Glycosylation offers a solution as the binding of a sugar residue to small molecules increases their water solubility and stability, renders aroma components odorless and modifies bioactivity. In order to identify plant enzymes that catalyze this reaction, a glycosyltransferase library containing 57 uridine diphosphate sugar-dependent enzymes (UGTs) was screened with (±)-menthol. The identity of the products was confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. Five enzymes were able to form (±)-menthyl-β-d-glucopyranoside in whole-cell biotransformations: UGT93Y1, UGT93Y2, UGT85K11, UGT72B27 and UGT73B24. In vitro enzyme activity assays revealed highest catalytic activity for UGT93Y1 (7.6 nkat/mg) from Camellia sinensis towards menthol and its isomeric forms. Although UGT93Y2 shares 70% sequence identity with UGT93Y1, it was less efficient. Of the five enzymes, UGT93Y1 stood out because of its high in vivo and in vitro biotransformation rate. The identification of novel menthol glycosyltransferases from the tea plant opens new perspectives for the biotechnological production of menthyl glucoside. Full article

(This article belongs to the Special Issue Recent Advances in Carbohydrate-Active Enzymes)

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Review

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15 pages, 2855 KiB

Open AccessReview

Production of Large-Ring Cyclodextrins by Amylomaltases

by Kuakarun Krusong, Abbas Ismail, Karan Wangpaiboon and Piamsook Pongsawasdi

Molecules 2022, 27(4), 1446; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules27041446 - 21 Feb 2022

Cited by 7 | Viewed by 2159

Abstract

Amylomaltase is a well-known glucan transferase that can produce large ring cyclodextrins (LR-CDs) or so-called cycloamyloses via cyclization reaction. Amylomaltases have been found in several microorganisms and their optimum temperatures are generally around 60–70 °C for thermostable amylomaltases and 30–45 °C for the enzymes from mesophilic bacteria and plants. The optimum pHs for mesophilic amylomaltases are around pH 6.0–7.0, while the thermostable amylomaltases are generally active at more acidic conditions. Size of LR-CDs depends on the source of amylomaltases and the reaction conditions including pH, temperature, incubation time, and substrate. For example, in the case of amylomaltase from Corynebacterium glutamicum, LR-CD productions at alkaline pH or at a long incubation time favored products with a low degree of polymerization. In this review, we explore the synthesis of LR-CDs by amylomaltases, structural information of amylomaltases, as well as current applications of LR-CDs and amylomaltases. Full article

(This article belongs to the Special Issue Recent Advances in Carbohydrate-Active Enzymes)

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13 pages, 1905 KiB

Open AccessReview

The Ectodomains of rBAT and 4F2hc Are Fake or Orphan α-Glucosidases

by Joana Fort, Adrià Nicolàs-Aragó and Manuel Palacín

Molecules 2021, 26(20), 6231; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26206231 - 15 Oct 2021

Cited by 9 | Viewed by 2159

Abstract

It is known that 4F2hc and rBAT are the heavy subunits of the heteromeric amino acid transporters (HATs). These heavy subunits are N-glycosylated proteins, with an N-terminal domain, one transmembrane domain and a bulky extracellular domain (ectodomain) that belongs to the α-amylase family. The heavy subunits are covalently linked to a light subunit from the SLC7 family, which is responsible for the amino acid transport activity, forming a heterodimer. The functions of 4F2hc and rBAT are related mainly to the stability and trafficking of the HATs in the plasma membrane of vertebrates, where they exert the transport activity. Moreover, 4F2hc is a modulator of integrin signaling, has a role in cell fusion and it is overexpressed in some types of cancers. On the other hand, some mutations in rBAT are found to cause the malfunctioning of the b^0,+ transport system, leading to cystinuria. The ectodomains of 4F2hc and rBAT share both sequence and structure homology with α-amylase family members. Very recently, cryo-EM has revealed the structure of several HATs, including the ectodomains of rBAT and 4F2hc. Here, we analyze available data on the ectodomains of rBAT and 4Fhc and their relationship with the α-amylase family. The physiological relevance of this relationship remains largely unknown. Full article

(This article belongs to the Special Issue Recent Advances in Carbohydrate-Active Enzymes)

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27 pages, 51924 KiB

Open AccessReview

Cellular and Molecular Engineering of Glycan Sialylation in Heterologous Systems

by Ryoma Hombu, Sriram Neelamegham and Sheldon Park

Molecules 2021, 26(19), 5950; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26195950 - 30 Sep 2021

Cited by 4 | Viewed by 4290

Abstract

Glycans have been shown to play a key role in many biological processes, such as signal transduction, immunogenicity, and disease progression. Among the various glycosylation modifications found on cell surfaces and in biomolecules, sialylation is especially important, because sialic acids are typically found at the terminus of glycans and have unique negatively charged moieties associated with cellular and molecular interactions. Sialic acids are also crucial for glycosylated biopharmaceutics, where they promote stability and activity. In this regard, heterogenous sialylation may produce variability in efficacy and limit therapeutic applications. Homogenous sialylation may be achieved through cellular and molecular engineering, both of which have gained traction in recent years. In this paper, we describe the engineering of intracellular glycosylation pathways through targeted disruption and the introduction of carbohydrate active enzyme genes. The focus of this review is on sialic acid-related genes and efforts to achieve homogenous, humanlike sialylation in model hosts. We also discuss the molecular engineering of sialyltransferases and their application in chemoenzymatic sialylation and sialic acid visualization on cell surfaces. The integration of these complementary engineering strategies will be useful for glycoscience to explore the biological significance of sialic acids on cell surfaces as well as the future development of advanced biopharmaceuticals. Full article

(This article belongs to the Special Issue Recent Advances in Carbohydrate-Active Enzymes)

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23 pages, 6519 KiB

Open AccessReview

Structural Insights in Mammalian Sialyltransferases and Fucosyltransferases: We Have Come a Long Way, but It Is Still a Long Way Down

by Ravneet Kaur Grewal, Abdul Rajjak Shaikh, Suresh Gorle, Manjeet Kaur, Paula Alexendra Videira, Luigi Cavallo and Mohit Chawla

Molecules 2021, 26(17), 5203; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26175203 - 27 Aug 2021

Cited by 14 | Viewed by 3692

Abstract

Mammalian cell surfaces are modified with complex arrays of glycans that play major roles in health and disease. Abnormal glycosylation is a hallmark of cancer; terminal sialic acid and fucose in particular have high levels in tumor cells, with positive implications for malignancy. Increased sialylation and fucosylation are due to the upregulation of a set of sialyltransferases (STs) and fucosyltransferases (FUTs), which are potential drug targets in cancer. In the past, several advances in glycostructural biology have been made with the determination of crystal structures of several important STs and FUTs in mammals. Additionally, how the independent evolution of STs and FUTs occurred with a limited set of global folds and the diverse modular ability of catalytic domains toward substrates has been elucidated. This review highlights advances in the understanding of the structural architecture, substrate binding interactions, and catalysis of STs and FUTs in mammals. While this general understanding is emerging, use of this information to design inhibitors of STs and FUTs will be helpful in providing further insights into their role in the manifestation of cancer and developing targeted therapeutics in cancer. Full article

(This article belongs to the Special Issue Recent Advances in Carbohydrate-Active Enzymes)

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