A GH13 α-glucosidase from Weissella cibaria uncommonly acts on short-chain maltooligosaccharides

2021 ◽  
Vol 77 (8) ◽  
Author(s):  
Karan Wangpaiboon ◽  
Pasunee Laohawuttichai ◽  
Sun-Yong Kim ◽  
Tomoyuki Mori ◽  
Santhana Nakapong ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Domain ◽  
Hydrolytic Activity ◽  
Size Exclusion ◽  
Short Chain ◽  
Glycoside Hydrolase Family ◽  
Enzyme Active Site ◽  
Weissella Cibaria ◽  
Terminal Domain

α-Glucosidase (EC 3.2.1.20) is a carbohydrate-hydrolyzing enzyme which generally cleaves α-1,4-glycosidic bonds of oligosaccharides and starch from the nonreducing ends. In this study, the novel α-glucosidase from Weissella cibaria BBK-1 (WcAG) was biochemically and structurally characterized. WcAG belongs to glycoside hydrolase family 13 (GH13) and to the neopullanase subfamily. It exhibits distinct hydrolytic activity towards the α-1,4 linkages of short-chain oligosaccharides from the reducing end. The enzyme prefers to hydrolyse maltotriose and acarbose, while it cannot hydrolyse cyclic oligosaccharides and polysaccharides. In addition, WcAG can cleave pullulan hydrolysates and strongly exhibits transglycosylation activity in the presence of maltose. Size-exclusion chromatography and X-ray crystal structures revealed that WcAG forms a homodimer in which the N-terminal domain of one monomer is orientated in proximity to the catalytic domain of another, creating the substrate-binding groove. Crystal structures of WcAG in complexes with maltose, maltotriose and acarbose revealed a remarkable enzyme active site with accessible +2, +1 and −1 subsites, along with an Arg–Glu gate (Arg176–Glu296) in front of the active site. The −2 and −3 subsites were blocked by Met119 and Asn120 from the N-terminal domain of a different subunit, resulting in an extremely restricted substrate preference.

Crystal structures of the GH6 Orpinomyces sp. Y102 CelC7 enzyme with exo and endo activity and its complex with cellobiose

2019 ◽  
Vol 75 (12) ◽  
pp. 1138-1147
Author(s):  
Hsiao-Chuan Huang ◽  
Liu-Hong Qi ◽  
Yo-Chia Chen ◽  
Li-Chu Tsai
Keyword(s):  
Enzymatic Hydrolysis ◽  
Crystal Structures ◽  
Active Site ◽  
Binding Sites ◽  
Glycoside Hydrolase ◽  
Catalytic Domain ◽  
Glycoside Hydrolase Family ◽  
Substrate Binding Sites ◽  
Key Residues ◽  
Hydrolase Family

The catalytic domain (residues 128–449) of the Orpinomyces sp. Y102 CelC7 enzyme (Orp CelC7) exhibits cellobiohydrolase and cellotriohydrolase activities. Crystal structures of Orp CelC7 and its cellobiose-bound complex have been solved at resolutions of 1.80 and 2.78 Å, respectively. Cellobiose occupies subsites +1 and +2 within the active site of Orp CelC7 and forms hydrogen bonds to two key residues: Asp248 and Asp409. Furthermore, its substrate-binding sites have both tunnel-like and open-cleft conformations, suggesting that the glycoside hydrolase family 6 (GH6) Orp CelC7 enzyme may perform enzymatic hydrolysis in the same way as endoglucanases and cellobiohydrolases. LC-MS/MS analysis revealed cellobiose (major) and cellotriose (minor) to be the respective products of endo and exo activity of the GH6 Orp CelC7.

scholarly journals Crystal structures of DNA polymerase I capture novel intermediates in the DNA synthesis pathway

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Nicholas Chim ◽  
Lynnette N Jackson ◽  
Anh M Trinh ◽  
John C Chaput
Keyword(s):  
High Resolution ◽  
Dna Synthesis ◽  
Crystal Structures ◽  
Dna Polymerase ◽  
Active Site ◽  
Dna Polymerase I ◽  
Dynamic Nature ◽  
Enzyme Active Site ◽  
Polymerase I ◽  
In Cells

High resolution crystal structures of DNA polymerase intermediates are needed to study the mechanism of DNA synthesis in cells. Here we report five crystal structures of DNA polymerase I that capture new conformations for the polymerase translocation and nucleotide pre-insertion steps in the DNA synthesis pathway. We suggest that these new structures, along with previously solved structures, highlight the dynamic nature of the finger subdomain in the enzyme active site.

scholarly journals The third chitinase gene (chiC) of Serratia marcescens 2170 and the relationship of its product to other bacterial chitinases

1999 ◽  
Vol 343 (3) ◽  
pp. 587-596 ◽  
Author(s):  
Kazushi SUZUKI ◽  
Mayumi TAIYOJI ◽  
Noriko SUGAWARA ◽  
Naoki NIKAIDOU ◽  
Bernard HENRISSAT ◽  
...  
Keyword(s):  
Serratia Marcescens ◽  
Catalytic Domain ◽  
Signal Sequence ◽  
Early Stage ◽  
Hydrolytic Activity ◽  
Chitinase Gene ◽  
Glycoside Hydrolase Family ◽  
Chitin Binding Domain ◽  
The Third ◽  
Fn3 Domain

The third chitinase gene (chiC) of Serratia marcescens 2170, specifying chitinases C1 and C2, was identified. Chitinase C1 lacks a signal sequence and consists of a catalytic domain belonging to glycoside hydrolase family 18, a fibronectin type III-like domain (Fn3 domain) and a C-terminal chitin-binding domain (ChBD). Chitinase C2 corresponds to the catalytic domain of C1 and is probably generated by proteolytic removal of the Fn3 and ChBDs. The loss of the C-terminal portion reduced the hydrolytic activity towards powdered chitin and regenerated chitin, but not towards colloidal chitin and glycol chitin, illustrating the importance of the ChBD for the efficient hydrolysis of crystalline chitin. Phylogenetic analysis showed that bacterial family 18 chitinases can be clustered in three subfamilies which have diverged at an early stage of bacterial chitinase evolution. Ser. marcescens chitinase C1 is found in one subfamily, whereas chitinases A and B of the same bacterium belong to another subfamily. Chitinase C1 is the only Ser. marcescens chitinase that has an Fn3 domain. The presence of multiple, divergent, chitinases in a single chitinolytic bacterium is perhaps necessary for efficient synergistic degradation of chitin.

scholarly journals Biological and Structural Characterization of Trypanosoma cruzi Phosphodiesterase C and Implications for Design of Parasite Selective Inhibitors

2012 ◽  
Vol 287 (15) ◽  
pp. 11788-11797 ◽  
Author(s):  
Huanchen Wang ◽  
Stefan Kunz ◽  
Gong Chen ◽  
Thomas Seebeck ◽  
Yiqian Wan ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Crystal Structures ◽  
Active Site ◽  
Catalytic Domain ◽  
Trypanosoma Evansi ◽  
Active Site Residues ◽  
Dual Specificity ◽  
Pde Inhibitors ◽  
Starting Model

Trypanosoma cruzi phosphodiesterase C (TcrPDEC) is a potential new drug target for the treatment of Chagas disease but has not been well studied. This study reports the enzymatic properties of various kinetoplastid PDECs and the crystal structures of the unliganded TcrPDEC1 catalytic domain and its complex with an inhibitor. Mutations of PDEC during the course of evolution led to inactivation of PDEC in Trypanosoma brucei/Trypanosoma evansi/Trypanosoma congolense, whereas the enzyme is active in all other kinetoplastids. The TcrPDEC1 catalytic domain hydrolyzes both cAMP and cGMP with a Km of 23.8 μm and a kcat of 31 s−1 for cAMP and a Km of 99.1 μm and a kcat of 17 s−1 for cGMP, thus confirming its dual specificity. The crystal structures show that the N-terminal fragment wraps around the TcrPDEC catalytic domain and may thus regulate its enzymatic activity via direct interactions with the active site residues. A PDE5 selective inhibitor that has an IC50 of 230 nm for TcrPDEC1 binds to TcrPDEC1 in an orientation opposite to that of sildenafil. This observation, together with the screen of the inhibitory potency of human PDE inhibitors against TcrPDEC, implies that the scaffold of some human PDE inhibitors might be used as the starting model for design of parasite PDE inhibitors. The structural study also identified a unique parasite pocket that neighbors the active site and may thus be valuable for the design of parasite-specific inhibitors.

scholarly journals Identification of a ligand binding hot spot and structural motifs replicating aspects of tyrosyl-DNA phosphodiesterase I (TDP1) phosphoryl recognition by crystallographic fragment cocktail screening

2019 ◽  
Vol 47 (19) ◽  
pp. 10134-10150 ◽  
Author(s):  
George T Lountos ◽  
Xue Zhi Zhao ◽  
Evgeny Kiselev ◽  
Joseph E Tropea ◽  
Danielle Needle ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Small Molecule ◽  
Anticancer Agents ◽  
Active Site ◽  
Catalytic Domain ◽  
Hot Spot ◽  
Alkylating Agents ◽  
Fragment Screening ◽  
Rational Development ◽  
Biochemical Assays

Abstract Tyrosyl DNA-phosphodiesterase I (TDP1) repairs type IB topoisomerase (TOP1) cleavage complexes generated by TOP1 inhibitors commonly used as anticancer agents. TDP1 also removes DNA 3′ end blocking lesions generated by chain-terminating nucleosides and alkylating agents, and base oxidation both in the nuclear and mitochondrial genomes. Combination therapy with TDP1 inhibitors is proposed to synergize with topoisomerase targeting drugs to enhance selectivity against cancer cells exhibiting deficiencies in parallel DNA repair pathways. A crystallographic fragment screening campaign against the catalytic domain of TDP1 was conducted to identify new lead compounds. Crystal structures revealed two fragments that bind to the TDP1 active site and exhibit inhibitory activity against TDP1. These fragments occupy a similar position in the TDP1 active site as seen in prior crystal structures of TDP1 with bound vanadate, a transition state mimic. Using structural insights into fragment binding, several fragment derivatives have been prepared and evaluated in biochemical assays. These results demonstrate that fragment-based methods can be a highly feasible approach toward the discovery of small-molecule chemical scaffolds to target TDP1, and for the first time, we provide co-crystal structures of small molecule inhibitors bound to TDP1, which could serve for the rational development of medicinal TDP1 inhibitors.

scholarly journals Redirecting substrate regioselectivity using engineered ΔN123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mounir Benkoulouche ◽  
Akli Ben Imeddourene ◽  
Louis-Antoine Barel ◽  
Guillaume Le Heiget ◽  
Sandra Pizzut ◽  
...  
Keyword(s):  
Active Site ◽  
Shigella Flexneri ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Active Enzymes ◽  
Enzyme Active Site ◽  
Molecular Determinants ◽  
Dynamics Simulations ◽  
Carbohydrate Components ◽  
Glycosylation Reaction ◽  
Hydrolase Family

AbstractThe (chemo-)enzymatic synthesis of oligosaccharides has been hampered by the lack of appropriate enzymatic tools with requisite regio- and stereo-specificities. Engineering of carbohydrate-active enzymes, in particular targeting the enzyme active site, has notably led to catalysts with altered regioselectivity of the glycosylation reaction thereby enabling to extend the repertoire of enzymes for carbohydrate synthesis. Using a collection of 22 mutants of ΔN123-GBD-CD2 branching sucrase, an enzyme from the Glycoside Hydrolase family 70, containing between one and three mutations in the active site, and a lightly protected chemically synthesized tetrasaccharide as an acceptor substrate, we showed that altered glycosylation product specificities could be achieved compared to the parental enzyme. Six mutants were selected for further characterization as they produce higher amounts of two favored pentasaccharides compared to the parental enzyme and/or new products. The produced pentasaccharides were shown to be of high interest as they are precursors of representative haptens of Shigella flexneri serotypes 3a, 4a and 4b. Furthermore, their synthesis was shown to be controlled by the mutations introduced in the active site, driving the glucosylation toward one extremity or the other of the tetrasaccharide acceptor. To identify the molecular determinants involved in the change of ΔN123-GBD-CD2 regioselectivity, extensive molecular dynamics simulations were carried out in combination with in-depth analyses of amino acid residue networks. Our findings help to understand the inter-relationships between the enzyme structure, conformational flexibility and activity. They also provide new insight to further engineer this class of enzymes for the synthesis of carbohydrate components of bacterial haptens.

scholarly journals How similar are enzyme active site geometries derived from quantum mechanical theozymes to crystal structures of enzyme-inhibitor complexes? Implications for enzyme design

2007 ◽  
Vol 16 (9) ◽  
pp. 1851-1866 ◽  
Author(s):  
Jason DeChancie ◽  
Fernando R. Clemente ◽  
Adam J.T. Smith ◽  
Hakan Gunaydin ◽  
Yi-Lei Zhao ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Enzyme Inhibitor ◽  
Quantum Mechanical ◽  
Enzyme Design ◽  
Enzyme Active Site

scholarly journals Functional Analysis of a Novel β-(1,3)-Glucanase from Corallococcus sp. Strain EGB Containing a Fascin-Like Module

2017 ◽  
Vol 83 (16) ◽  
Author(s):  
Jie Zhou ◽  
Zhoukun Li ◽  
Jiale Wu ◽  
Lifeng Li ◽  
Ding Li ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Functional Characterization ◽  
Hydrolytic Activity ◽  
Site Directed Mutagenesis ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Content Type ◽  
Binding Function ◽  
Catalytic Module

ABSTRACT A novel β-(1,3)-glucanase gene designated lamC, cloned from Corallococcus sp. strain EGB, contains a fascin-like module and a glycoside hydrolase family 16 (GH16) catalytic module. LamC displays broad hydrolytic activity toward various polysaccharides. Analysis of the hydrolytic products revealed that LamC is an exo-acting enzyme on β-(1,3)(1,3)- and β-(1,6)-linked glucan substrates and an endo-acting enzyme on β-(1,4)-linked glucan and xylan substrates. Site-directed mutagenesis of conserved catalytic Glu residues (E304A and E309A) demonstrated that these activities were derived from the same active site. Excision of the fascin-like module resulted in decreased activity toward β-(1,3)(1,3)-linked glucans. The carbohydrate-binding assay showed that the fascin-like module was a novel β-(1,3)-linked glucan-binding module. The functional characterization of the fascin-like module and catalytic module will help us better understand these enzymes and modules. IMPORTANCE In this report of a bacterial β-(1,3)(1,3)-glucanase containing a fascin-like module, we reveal the β-(1,3)(1,3)-glucan-binding function of the fascin-like module present in the N terminus of LamC. LamC displays exo-β-(1,3)/(1,6)-glucanase and endo-β-(1,4)-glucanase/xylanase activities with a single catalytic domain. Thus, LamC was identified as a novel member of the GH16 family.

scholarly journals Crystal Structure of the Salmonella enterica Serovar Typhimurium Virulence Factor SrfJ, a Glycoside Hydrolase Family Enzyme

2009 ◽  
Vol 191 (21) ◽  
pp. 6550-6554 ◽  
Author(s):  
Yeon-Gil Kim ◽  
Jin-Hong Kim ◽  
Kyung-Jin Kim
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Salmonella Enterica ◽  
Glycoside Hydrolase ◽  
Catalytic Domain ◽  
Membrane Lipids ◽  
Glycoside Hydrolase Family ◽  
Secretion Systems ◽  
High Amino Acid ◽  
Serovar Typhimurium

ABSTRACT To cause infection, Salmonella enterica serovar Typhimurium uses type III secretion systems, which are encoded on two Salmonella pathogenicity islands, SPI-1 and SPI-2, the latter of which is thought to play a crucial role in bacterial proliferation in Salmonella-containing vacuoles (SCVs) after invading cells. S. Typhimurium SrfJ, located outside SPI-2, is also known to be involved in Salmonella pathogenicity and has high amino acid sequence homology with human lysosomal glucosylceramidase (GlcCerase). We present the first crystal structure of SrfJ at a resolution of 1.8 Å. The overall fold of SrfJ shares high structure similarities with that of human GlcCerase, comprising two distinctive domains: a (β/α)8-barrel catalytic domain and a β-sandwich domain. As in human GlcCerase, the pocket-shaped active site of SrfJ is located on the C-terminal side of the barrel, and two conserved glutamic acid residues are used for the enzyme catalysis. Moreover, a glycerol-bound form of SrfJ reveals that the glucose ring moiety of the substrate might similarly bind to the enzyme as to human GlcCerase, suggesting that SrfJ might function as a glycoside hydrolase. Although some structural differences are observed between SrfJ and human GlcCerase in the substrate entrance of the active site, we speculate that, based on the high structural similarities to human GlcCerase in the overall fold and the active-site environment, SrfJ might have a GlcCerase activity and use the activity to enhance Salmonella virulence by modifying SCV membrane lipids.

scholarly journals Structure of a thermostable serralysin fromSerratiasp. FS14 at 1.1 Å resolution

2016 ◽  
Vol 72 (1) ◽  
pp. 10-15 ◽  
Author(s):  
Dongxia Wu ◽  
Tinting Ran ◽  
Weiwu Wang ◽  
Dongqing Xu
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bonds ◽  
Active Site ◽  
Comparative Studies ◽  
Catalytic Domain ◽  
Molecular Surface ◽  
Terminal Domain ◽  
Catalytic Active Site ◽  
Active Site Cavity ◽  
Β Sheets

Serralysin is a well studied metalloprotease, and typical serralysins are not thermostable. The serralysin isolated fromSerratiasp. FS14 was found to be thermostable, and in order to reveal the mechanism responsible for its thermostability, the crystal structure of serralysin fromSerratiasp. FS14 was solved to a crystallographicRfactor of 0.1619 at 1.10 Å resolution. Similar to its homologues, it mainly consists of two domains: an N-terminal catalytic domain and a `parallel β-roll' C-terminal domain. Comparative studies show that the shape of the catalytic active-site cavity is more open owing to the 189–198 loop, with a short 310-helix protruding further from the molecular surface, and that the β-sheets comprising the `parallel β-roll' are longer than those in its homologues. The formation of hydrogen bonds from one of the nonconserved residues (Asn200) to Lys27 may contribute to the thermostability.

Sign in / Sign up

Export Citation Format

Share Document