Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 13 de 13
Filter
Add more filters











Publication year range
1.
Angew Chem Int Ed Engl ; 62(4): e202211937, 2023 01 23.
Article in English | MEDLINE | ID: mdl-36308301

ABSTRACT

UDP-glucuronic acid (UDP-GlcA) 4-epimerase illustrates an important problem regarding enzyme catalysis: balancing conformational flexibility with precise positioning. The enzyme coordinates the C4-oxidation of the substrate by NAD+ and rotation of a decarboxylation-prone ß-keto acid intermediate in the active site, enabling stereoinverting reduction of the keto group by NADH. We reveal the elusive rotational landscape of the 4-keto intermediate. Distortion of the sugar ring into boat conformations induces torsional mobility in the enzyme's binding pocket. The rotational endpoints show that the 4-keto sugar has an undistorted 4 C1 chair conformation. The equatorially placed carboxylate group disfavors decarboxylation of the 4-keto sugar. Epimerase variants lead to decarboxylation upon removal of the binding interactions with the carboxylate group in the opposite rotational isomer of the substrate. Substitutions R185A/D convert the epimerase into UDP-xylose synthases that decarboxylate UDP-GlcA in stereospecific, configuration-retaining reactions.


Subject(s)
Racemases and Epimerases , Uridine Diphosphate Glucuronic Acid , Uridine Diphosphate Glucuronic Acid/metabolism , Decarboxylation , Rotation , Glucuronic Acid , Racemases and Epimerases/metabolism , Ketoses , NAD/chemistry
3.
ACS Catal ; 12(8): 4737-4743, 2022 Apr 15.
Article in English | MEDLINE | ID: mdl-35465242

ABSTRACT

Bifidobacterium bifidum lacto-N-biosidase (LnbB) is a critical enzyme for the degradation of human milk oligosaccharides in the gut microbiota of breast-fed infants. Guided by recent crystal structures, we unveil its molecular mechanism of catalysis using QM/MM metadynamics. We show that the oligosaccharide substrate follows 1 S 3/1,4 B → [4 E]‡ → 4 C 1/4 H 5 and 4 C 1/4 H 5 → [4 E/4 H 5]‡ → 1,4 B conformational itineraries for the two successive reaction steps, with reaction free energy barriers in agreement with experiments. The simulations also identify a critical histidine (His263) that switches between two orientations to modulate the pK a of the acid/base residue, facilitating catalysis. The reaction intermediate of LnbB is best depicted as an oxazolinium ion, with a minor population of neutral oxazoline. The present study sheds light on the processing of oligosaccharides of the early life microbiota and will be useful for the engineering of LnbB and similar glycosidases for biocatalysis.

4.
J Phys Chem B ; 126(4): 802-812, 2022 02 03.
Article in English | MEDLINE | ID: mdl-35073079

ABSTRACT

Glycoside hydrolases and glycosyltransferases are the main classes of enzymes that synthesize and degrade carbohydrates, molecules essential to life that are a challenge for classical chemistry. As such, considerable efforts have been made to engineer these enzymes and make them pliable to human needs, ranging from directed evolution to rational design, including mechanism engineering. Such endeavors fall short and are unreported in numerous cases, while even success is a necessary but not sufficient proof that the chemical rationale behind the design is correct. Here we review some of the recent work in CAZyme mechanism engineering, showing that computational simulations are instrumental to rationalize experimental data, providing mechanistic insight into how native and engineered CAZymes catalyze chemical reactions. We illustrate this with two recent studies in which (i) a glycoside hydrolase is converted into a glycoside phosphorylase and (ii) substrate specificity of a glycosyltransferase is engineered toward forming O-, N-, or S-glycosidic bonds.


Subject(s)
Glycoside Hydrolases , Glycosyltransferases , Carbohydrates/chemistry , Computer Simulation , Glycoside Hydrolases/chemistry , Glycosyltransferases/metabolism , Humans , Substrate Specificity
5.
Angew Chem Int Ed Engl ; 60(11): 5754-5758, 2021 03 08.
Article in English | MEDLINE | ID: mdl-33528085

ABSTRACT

The recent discovery of zinc-dependent retaining glycoside hydrolases (GHs), with active sites built around a Zn(Cys)3 (Glu) coordination complex, has presented unresolved mechanistic questions. In particular, the proposed mechanism, depending on a Zn-coordinated cysteine nucleophile and passing through a thioglycosyl enzyme intermediate, remains controversial. This is primarily due to the expected stability of the intermediate C-S bond. To facilitate the study of this atypical mechanism, we report the synthesis of a cyclophellitol-derived ß-l-arabinofuranosidase inhibitor, hypothesised to react with the catalytic nucleophile to form a non-hydrolysable adduct analogous to the mechanistic covalent intermediate. This ß-l-arabinofuranosidase inhibitor reacts exclusively with the proposed cysteine thiol catalytic nucleophiles of representatives of GH families 127 and 146. X-ray crystal structures determined for the resulting adducts enable MD and QM/MM simulations, which provide insight into the mechanism of thioglycosyl enzyme intermediate breakdown. Leveraging the unique chemistry of cyclophellitol derivatives, the structures and simulations presented here support the assignment of a zinc-coordinated cysteine as the catalytic nucleophile and illuminate the finely tuned energetics of this remarkable metalloenzyme clan.


Subject(s)
Cyclohexanols/metabolism , Cysteine/metabolism , Enzyme Inhibitors/metabolism , Glycoside Hydrolases/metabolism , Biocatalysis , Crystallography, X-Ray , Cyclohexanols/chemistry , Cyclohexanols/pharmacology , Cysteine/chemistry , Density Functional Theory , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Glycoside Hydrolases/antagonists & inhibitors , Glycoside Hydrolases/chemistry , Molecular Dynamics Simulation , Molecular Structure
6.
Nat Commun ; 12(1): 367, 2021 01 14.
Article in English | MEDLINE | ID: mdl-33446650

ABSTRACT

Xylanolytic enzymes from glycoside hydrolase family 43 (GH43) are involved in the breakdown of hemicellulose, the second most abundant carbohydrate in plants. Here, we kinetically and mechanistically describe the non-reducing-end xylose-releasing exo-oligoxylanase activity and report the crystal structure of a native GH43 Michaelis complex with its substrate prior to hydrolysis. Two distinct calcium-stabilized conformations of the active site xylosyl unit are found, suggesting two alternative catalytic routes. These results are confirmed by QM/MM simulations that unveil the complete hydrolysis mechanism and identify two possible reaction pathways, involving different transition state conformations for the cleavage of xylooligosaccharides. Such catalytic conformational promiscuity in glycosidases is related to the open architecture of the active site and thus might be extended to other exo-acting enzymes. These findings expand the current general model of catalytic mechanism of glycosidases, a main reaction in nature, and impact on our understanding about their interaction with substrates and inhibitors.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/metabolism , Xanthomonas/enzymology , Bacterial Proteins/genetics , Binding Sites , Catalysis , Catalytic Domain , Crystallography, X-Ray , Glycoside Hydrolases/genetics , Kinetics , Models, Molecular , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Xanthomonas/chemistry , Xanthomonas/genetics , Xylose/chemistry , Xylose/metabolism
7.
Arch Biochem Biophys ; 681: 108256, 2020 03 15.
Article in English | MEDLINE | ID: mdl-31923393

ABSTRACT

Aldehyde dehydrogenases catalyze the NAD(P)+-dependent oxidation of aldehydes to their corresponding carboxylic acids. The three-dimensional structures of the human ALDH1A enzymes were recently obtained, while a complete kinetic characterization of them, under the same experimental conditions, is lacking. We show that the three enzymes, ALDH1A1, ALDH1A2 and ALDH1A3, have similar topologies, although with decreasing volumes in their substrate-binding pockets. The activity with aliphatic and retinoid aldehydes was characterized side-by-side, using an improved HPLC-based method for retinaldehyde. Hexanal was the most efficient substrate. ALDH1A1 displayed lower Km values with hexanal, trans-2-hexenal and citral, compared to ALDH1A2 and ALDH1A3. ALDH1A2 was the best enzyme for the lipid peroxidation product, 4-hydroxy-2-nonenal, in terms of kcat/Km. The catalytic efficiency towards all-trans and 9-cis-retinaldehyde was in general lower than for alkanals and alkenals. ALDH1A2 and ALDH1A3 showed higher catalytic efficiency for all-trans-retinaldehyde. The lower specificity of ALDH1A3 for 9-cis-retinaldehyde against the all-trans- isomer might be related to the smaller volume of its substrate-binding pocket. Magnesium inhibited ALDH1A1 and ALDH1A2, while it activated ALDH1A3, which is consistent with cofactor dissociation being the rate-limiting step for ALDH1A1 and ALDH1A2, and deacylation for ALDH1A3, with hexanal as a substrate. We mutated both ALDH1A1 (L114P) and ALDH1A2 (N475G, A476V, L477V, N478S) to mimic their counterpart substrate-binding pockets. ALDH1A1 specificity for citral was traced to residue 114 and to residues 458 to 461. Regarding retinaldehyde, the mutants did not show significant differences with their respective wild-type forms, suggesting that the mutated residues are not critical for retinoid specificity.


Subject(s)
Aldehyde Dehydrogenase 1 Family/metabolism , Aldehyde Oxidoreductases/metabolism , Neoplastic Stem Cells/metabolism , Retinal Dehydrogenase/metabolism , Tretinoin/metabolism , Humans , Magnesium/metabolism , Models, Molecular , Neoplastic Stem Cells/pathology , Retinaldehyde/metabolism , Substrate Specificity
8.
J Am Chem Soc ; 142(5): 2120-2124, 2020 02 05.
Article in English | MEDLINE | ID: mdl-31917561

ABSTRACT

Glycoside hydrolases and phosphorylases are two major classes of enzymes responsible for the cleavage of glycosidic bonds. Here we show that two GH84 O-GlcNAcase enzymes can be converted to efficient phosphorylases by a single point mutation. Noteworthy, the mutated enzymes are over 10-fold more active than naturally occurring glucosaminide phosphorylases. We rationalize this novel transformation using molecular dynamics and QM/MM metadynamics methods, showing that the mutation changes the electrostatic potential at the active site and reduces the energy barrier for phosphorolysis by 10 kcal·mol-1. In addition, the simulations unambiguously reveal the nature of the intermediate as a glucose oxazolinium ion, clarifying the debate on the nature of such a reaction intermediate in glycoside hydrolases operating via substrate-assisted catalysis.


Subject(s)
Glycoside Hydrolases/metabolism , Phosphorylases/metabolism , Point Mutation , Catalytic Domain , Glycoside Hydrolases/genetics
9.
Curr Opin Chem Biol ; 53: 183-191, 2019 12.
Article in English | MEDLINE | ID: mdl-31731209

ABSTRACT

Modeling catalysis in carbohydrate-active enzymes is a daunting challenge because of the high flexibility and diversity of both enzymes and carbohydrates. Glycoside hydrolases (GHs) are an illustrative example, where conformational changes and subtle interactions have been shown to be critical for catalysis. GHs have pivotal roles in industry (e.g. biofuel or detergent production) and biomedicine (e.g. targets for cancer and diabetes), and thus, a huge effort is devoted to unveil their molecular mechanisms. Besides experimental techniques, computational methods have served to provide an in-depth understanding of GH mechanisms, capturing complex reaction coordinates and the conformational itineraries that substrates follow during the whole catalytic pathway, providing a framework that ultimately may assist the engineering of these enzymes and the design of new inhibitors.


Subject(s)
Biocatalysis , Glycoside Hydrolases/metabolism , Models, Molecular , Glycoside Hydrolases/chemistry , Protein Conformation
10.
Front Chem ; 7: 269, 2019.
Article in English | MEDLINE | ID: mdl-31114783

ABSTRACT

By means of QM(DFT)/MM metadynamics we have unraveled the hydrolytic reaction mechanism of Neisseria polysaccharea amylosucrase (NpAS), a member of GH13 family. Our results provide an atomistic picture of the active site reorganization along the catalytic double-displacement reaction, clarifying whether the glycosyl-enzyme reaction intermediate features an α-glucosyl unit in an undistorted 4 C 1 conformation, as inferred from structural studies, or a distorted 1 S 3-like conformation, as expected from mechanistic analysis of glycoside hydrolases (GHs). We show that, even though the first step of the reaction (glycosylation) results in a 4 C 1 conformation, the α-glucosyl unit undergoes an easy conformational change toward a distorted conformation as the active site preorganizes for the forthcoming reaction step (deglycosylation), in which an acceptor molecule, i.e., a water molecule for the hydrolytic reaction, performs a nucleophilic attack on the anomeric carbon. The two conformations (4 C 1 ad E 3) can be viewed as two different states of the glycosyl-enzyme intermediate (GEI), but only the E 3 state is preactivated for catalysis. These results are consistent with the general conformational itinerary observed for α-glucosidases.

11.
Phys Chem Chem Phys ; 21(16): 8457-8463, 2019 Apr 17.
Article in English | MEDLINE | ID: mdl-30951074

ABSTRACT

Deciphering the transport through outer-membrane porins is crucial to understand how anti-infectives enter Gram-negative bacteria and perform their function. Here we elucidated the transport mechanism of substrates through the Pseudomonads sugar-specific porin OprB by means of multiscale modeling. We used molecular dynamics simulations to quantify the energetics of transport and thus a diffusion model to quantify the macroscopic flux of molecules through OprB. Our results show that Trp171 and several glutamate residues in the constriction region are key for the transport of glucose, the preferred natural substrate, through OprB. The unveiled transport mechanism suggests that 2-acetamido-1,2-dideoxynojirimycin (DNJ-NAc), an anti-infective structurally similar to glucose, can enter the cell via OprB. We quantified its energetics and macroscopic flux through OprB providing a comparative analysis with the natural substrate. Thus this pore can be considered as a promising gateway for exploiting the Trojan Horse strategy in pathogenic bacteria.


Subject(s)
Bacterial Proteins/metabolism , Glucose/metabolism , Porins/metabolism , Pseudomonas Infections/microbiology , Pseudomonas putida/metabolism , Anti-Infective Agents/metabolism , Bacterial Proteins/chemistry , Biological Transport , Humans , Models, Molecular , Porins/chemistry , Protein Conformation , Pseudomonas putida/chemistry , Substrate Specificity
12.
Chemistry ; 24(72): 19258-19265, 2018 Dec 20.
Article in English | MEDLINE | ID: mdl-30276896

ABSTRACT

The enzymatic hydrolysis of chitin, one of the most abundant carbohydrates in nature, is achieved by chitinases, enzymes of increasing importance in biomedicine and industry. Unlike most retaining glycosidases, family GH18 chitinases follow a substrate-assisted mechanism in which the 2-acetamido group of one N-acetylglucosamine monomer, rather than a basic residue of the enzyme, reacts with the sugar anomeric carbon, forming an intermediate that has been described as an oxazolinium ion. Based on QM/MM metadynamics simulations on chitinase B from Serratia marcescens, we show that the reaction intermediate of GH18 chitinases features instead a neutral oxazoline in a 4 C1 /4 H5 conformation, with an oxazolinium ion being formed on the pathway towards the reaction products. The role of a well-defined hydrogen-bond network that operates around the N-acetyl group, orchestrating catalysis by protonation events, is discussed.


Subject(s)
Chitinases/chemistry , Acetylglucosamine/chemistry , Catalysis , Chitin/chemistry , Chitin/metabolism , Chitinases/metabolism , Hydrogen Bonding , Hydrogenation , Protein Conformation , Serratia marcescens/enzymology
13.
Chem Commun (Camb) ; 53(66): 9238-9241, 2017 Aug 25.
Article in English | MEDLINE | ID: mdl-28766587

ABSTRACT

The non-hydrolyzable S-linked azasugars, 1,6-α-mannosylthio- and 1,6-α-mannobiosylthioisofagomine, were synthesized and shown to bind with high affinity to a family 76 endo-1,6-α-mannanase from Bacillus circulans. X-ray crystallography showed an atypical interaction of the isofagomine nitrogen with the catalytic acid/base. Molecular dynamics simulations reveal that the atypical binding results from sulfur perturbing the most stable form away from the nucleophile interaction preferred for the O-linked congener.


Subject(s)
Aza Compounds/pharmacology , Carbohydrates/pharmacology , Enzyme Inhibitors/pharmacology , Mannosidases/antagonists & inhibitors , Aza Compounds/chemical synthesis , Aza Compounds/chemistry , Bacillus/enzymology , Carbohydrate Conformation , Carbohydrates/chemical synthesis , Carbohydrates/chemistry , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Mannosidases/metabolism , Models, Molecular
SELECTION OF CITATIONS
SEARCH DETAIL