scholarly journals Structural basis of chitin utilization by a GH20 β-N-acetylglucosaminidase from Vibrio campbellii strain ATCC BAA-1116

2021 ◽  
Vol 77 (5) ◽  
pp. 674-689
Author(s):  
Piyanat Meekrathok ◽  
Marco Bürger ◽  
Arthur T. Porfetye ◽  
Sawitree Kumsaoad ◽  
Anuwat Aunkham ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Domain ◽  
Sole Source ◽  
Structural Basis ◽  
Carbohydrate Binding ◽  
Strain Atcc ◽  
Dimeric Interface ◽  
Catalytic Role ◽  
Vibrio Campbellii

Vibrio species play a crucial role in maintaining the carbon and nitrogen balance between the oceans and the land through their ability to employ chitin as a sole source of energy. This study describes the structural basis for the action of the GH20 β-N-acetylglucosaminidase (VhGlcNAcase) in chitin metabolism by Vibrio campbellii (formerly V. harveyi) strain ATCC BAA-1116. Crystal structures of wild-type VhGlcNAcase in the absence and presence of the sugar ligand, and of the unliganded D437A mutant, were determined. VhGlcNAcase contains three distinct domains: an N-terminal carbohydrate-binding domain linked to a small α+β domain and a C-terminal (β/α)8 catalytic domain. The active site of VhGlcNAcase has a narrow, shallow pocket that is suitable for accommodating a small chitooligosaccharide. VhGlcNAcase is a monomeric enzyme of 74 kDa, but its crystal structures show two molecules of enzyme per asymmetric unit, in which Gln16 at the dimeric interface of the first molecule partially blocks the entrance to the active site of the neighboring molecule. The GlcNAc unit observed in subsite −1 makes exclusive hydrogen bonds to the conserved residues Arg274, Tyr530, Asp532 and Glu584, while Trp487, Trp546, Trp582 and Trp505 form a hydrophobic wall around the −1 GlcNAc. The catalytic mutants D437A/N and E438A/Q exhibited a drastic loss of GlcNAcase activity, confirming the catalytic role of the acidic pair (Asp437–Glu438).

scholarly journals Structure based protein engineering to confer selectivity of guanine deaminase

2014 ◽  
Vol 70 (a1) ◽  
pp. C437-C437
Author(s):  
Aruna Bitra ◽  
Ruchi Anand
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Catalytic Efficiency ◽  
Structural Basis ◽  
Active Site Residues ◽  
X Ray ◽  
Secondary Activity ◽  
Guanine Deaminase ◽  
Differential Selectivity

Guanine deaminases (GDs) are important enzymes involved in both purine metabolism and nucleotide anabolism pathways. Here we present the molecular and catalytic mechanism of NE0047 and use the information obtained to engineer specific enzyme activities. NE0047 from Nitrosomonas europaea was found to be a high fidelity guanine deaminase (catalytic efficiency of 1.2 × 105 M–1 s–1). However; it exhibited secondary activity towards the structurally non-analogous triazine based compound ammeline. The X-ray structure of NE0047 in the presence of the substrate analogue 8-azaguanine help establish that the enzyme exists as a biological dimer and both the proper closure of the C-terminal loop and cross talk via the dimeric interface is crucial for conferring catalytic activity. It was further ascertained that the highly conserved active site residues Glu79 and Glu143 facilitate the deamination reaction by serving as proton shuttles. Moreover, to understand the structural basis of dual substrate specificity, X-ray structures of NE0047 in complex with a series of nucleobase analogs, nucleosides and substrate ammeline were determined. The crystal structures demonstrated that any substitutions in the parent substrates results in the rearrangement of the ligand in a catalytically unfavorable orientation and also impede the closure of catalytically important loop, thereby abrogating activity. However, ammeline was able to adopt a catalytically favorable orientation which, also allowed for proper loop closure. Based on the above knowledge of the crystal structures and the catalytic mechanism, the active site was subsequently engineered to fine-tune NE0047 activity. The mutated versions of the enzyme were designed so that they can function either exclusively as a GD or serve as specific ammeline deaminases. For example, mutations in the active site E143D and N66A confer the enzyme to be an unambiguous GD with no secondary activity towards ammeline. On the other hand, the N66Q mutant of NE0047 only deaminates ammeline. Additionally, a series of crystal structures of the mutant versions were solved that shed light on the structural basis of this differential selectivity.

scholarly journals Substrate-modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR

2013 ◽  
Vol 288 (23) ◽  
pp. 17008-17018 ◽  
Author(s):  
D. Fernando Estrada ◽  
Jennifer S. Laurence ◽  
Emily E. Scott
Keyword(s):  
Cytochrome P450 ◽  
Binding Site ◽  
Active Site ◽  
Catalytic Domain ◽  
Cytochrome B5 ◽  
Structural Basis ◽  
Reaction Conditions ◽  
Reversible Binding ◽  
Important Interaction

The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction that is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b5 on the two CYP17A1-mediated reactions and, thus, communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17α-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site.

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.

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.

Multitasking in the gut: the X-ray structure of the multidomain BbgIII from Bifidobacterium bifidum offers possible explanations for its alternative functions

2021 ◽  
Vol 77 (12) ◽  
Author(s):  
Olga V. Moroz ◽  
Elena Blagova ◽  
Andrey A. Lebedev ◽  
Filomeno Sánchez Rodríguez ◽  
Daniel J. Rigden ◽  
...  
Keyword(s):  
Active Site ◽  
Model Building ◽  
Catalytic Domain ◽  
Bifidobacterium Bifidum ◽  
Host Cells ◽  
Intact Protein ◽  
Lactose Hydrolysis ◽  
Carbohydrate Binding ◽  
X Ray ◽  
Carbohydrate Binding Modules

β-Galactosidases catalyse the hydrolysis of lactose into galactose and glucose; as an alternative reaction, some β-galactosidases also catalyse the formation of galactooligosaccharides by transglycosylation. Both reactions have industrial importance: lactose hydrolysis is used to produce lactose-free milk, while galactooligosaccharides have been shown to act as prebiotics. For some multi-domain β-galactosidases, the hydrolysis/transglycosylation ratio can be modified by the truncation of carbohydrate-binding modules. Here, an analysis of BbgIII, a multidomain β-galactosidase from Bifidobacterium bifidum, is presented. The X-ray structure has been determined of an intact protein corresponding to a gene construct of eight domains. The use of evolutionary covariance-based predictions made sequence docking in low-resolution areas of the model spectacularly easy, confirming the relevance of this rapidly developing deep-learning-based technique for model building. The structure revealed two alternative orientations of the CBM32 carbohydrate-binding module relative to the GH2 catalytic domain in the six crystallographically independent chains. In one orientation the CBM32 domain covers the entrance to the active site of the enzyme, while in the other orientation the active site is open, suggesting a possible mechanism for switching between the two activities of the enzyme, namely lactose hydrolysis and transgalactosylation. The location of the carbohydrate-binding site of the CBM32 domain on the opposite site of the module to where it comes into contact with the catalytic GH2 domain is consistent with its involvement in adherence to host cells. The role of the CBM32 domain in switching between hydrolysis and transglycosylation modes offers protein-engineering opportunities for selective β-galactosidase modification for industrial purposes in the future.

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.

Crystal structures of the starch-binding domain from Rhizopus oryzae glucoamylase reveal a polysaccharide-binding path

2008 ◽  
Vol 416 (1) ◽  
pp. 27-36 ◽  
Author(s):  
Jung-Yu Tung ◽  
Margaret Dah-Tsyr Chang ◽  
Wei-I Chou ◽  
Yen-Yi Liu ◽  
Yi-Hung Yeh ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Hydrophobic Interaction ◽  
Binding Sites ◽  
Catalytic Domain ◽  
Rhizopus Oryzae ◽  
Binding Domain ◽  
Scatchard Analysis ◽  
Carbohydrate Binding ◽  
Binding Isotherm ◽  
Long Chain

GA (glucoamylase) hydrolyses starch and polysaccharides to β-D-glucose. RoGA (Rhizopus oryzae GA) consists of two functional domains, an N-terminal SBD (starch-binding domain) and a C-terminal catalytic domain, which are connected by an O-glycosylated linker. In the present study, the crystal structures of the SBD from RoGA (RoGACBM21) and the complexes with β-cyclodextrin (SBD–βCD) and maltoheptaose (SBD–G7) were determined. Two carbohydrate binding sites, I (Trp47) and II (Tyr32), were resolved and their binding was co-operative. Besides the hydrophobic interaction, two unique polyN loops comprising consecutive asparagine residues also participate in the sugar binding. A conformational change in Tyr32 was observed between unliganded and liganded SBDs. To elucidate the mechanism of polysaccharide binding, a number of mutants were constructed and characterized by a quantitative binding isotherm and Scatchard analysis. A possible binding path for long-chain polysaccharides in RoGACBM21 was proposed.

scholarly journals Machine learning reveals sequence-function relationships in family 7 glycoside hydrolases

2020 ◽  
Author(s):  
Japheth E. Gado ◽  
Brent E. Harrison ◽  
Mats Sandgren ◽  
Jerry Ståhlberg ◽  
Gregg T. Beckham ◽  
...  
Keyword(s):  
Machine Learning ◽  
Active Site ◽  
Catalytic Domain ◽  
Cellulose Degradation ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Functional Variation ◽  
Functional Relationships ◽  
Experimental Findings ◽  
And Function

AbstractFamily 7 glycoside hydrolases (GH7) are among the principal enzymes for cellulose degradation in nature and industrially. These important enzymes are often bimodular, comprised of a catalytic domain attached to a carbohydrate binding module (CBM) via a flexible linker, and exhibit a long active site that binds cello-oligomers of up to ten glucosyl moieties. GH7 cellulases consist of two major subtypes: cellobiohydrolases (CBH) and endoglucanases (EG). Despite the critical biological and industrial importance of GH7 enzymes, there remain gaps in our understanding of how GH7 sequence and structure relate to function. Here, we employed machine learning to gain insights into relationships between sequence, structure, and function across the GH7 family. Machine-learning models, using the number of residues in the active-site loops as features, were able discriminate GH7 CBHs and EGs with up to 99% accuracy. The lengths of the A4, B2, B3, and B4 loops were strongly correlated with functional subtype across the GH7 family. Position-specific classification rules were derived such that specific amino acids at 42 different sequence positions predicted the functional subtype with accuracies greater than 87%. A random forest model trained on residues at 19 positions in the catalytic domain predicted the presence of a CBM with 89.5% accuracy. We propose these positions play vital roles in the functional variation of GH7 cellulases. Taken together, our results complement numerous experimental findings and present functional relationships that can be applied when prospecting GH7 cellulases from nature, for sequence annotation, and to understand or manipulate function.

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.

 L-allo-Threonine aldolase with an H128Y/S292R mutation fromAeromonas jandaeiDK-39 reveals the structural basis of changes in substrate stereoselectivity

2014 ◽  
Vol 70 (6) ◽  
pp. 1695-1703 ◽  
Author(s):  
Hui-Min Qin ◽  
Fabiana Lica Imai ◽  
Takuya Miyakawa ◽  
Michihiko Kataoka ◽  
Nahoko Kitamura ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Active Site ◽  
Fold Increase ◽  
Saturation Mutagenesis ◽  
Structural Basis ◽  
Tyrosine Residue ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Threonine Aldolase

L-allo-Threonine aldolase (LATA), a pyridoxal-5′-phosphate-dependent enzyme fromAeromonas jandaeiDK-39, stereospecifically catalyzes the reversible interconversion of L-allo-threonine to glycine and acetaldehyde. Here, the crystal structures of LATA and its mutant LATA_H128Y/S292R were determined at 2.59 and 2.50 Å resolution, respectively. Their structures implied that conformational changes in the loop consisting of residues Ala123–Pro131, where His128 moved 4.2 Å outwards from the active site on mutation to a tyrosine residue, regulate the substrate specificity for L-allo-threonineversusL-threonine. Saturation mutagenesis of His128 led to diverse stereoselectivity towards L-allo-threonine and L-threonine. Moreover, the H128Y mutant showed the highest activity towards the two substrates, with an 8.4-fold increase towards L-threonine and a 2.0-fold increase towards L-allo-threonine compared with the wild-type enzyme. The crystal structures of LATA and its mutant LATA_H128Y/S292R reported here will provide further insights into the regulation of the stereoselectivity of threonine aldolases targeted for the catalysis of L-allo-threonine/L-threonine synthesis.

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