Catalytic mechanism of α-phosphate attack in dUTPase is revealed by X-ray crystallographic snapshots of distinct intermediates, 31P-NMR spectroscopy and reaction path modelling

Barábas, O.; Németh, Viktória; Bodor, Andrea; Perczel, András; Rosta, Edina; Kele, Zoltán; Zagyva, Imre; Szabadka, Zoltan; Grolmusz, Vince; Wilmanns, Matthias; Vértessy, Beáta G.

doi:10.1093/nar/gkt756

Cited by 17 publications

(24 citation statements)

References 89 publications

(119 reference statements)

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“…3 A and C). The reversible movement of catalytic side chains has been observed before for other enzymes (43,44). In addition, we observe that the noncatalytic substrate-binding residue R380 also alternates between two different conformations throughout the catalytic cycle ( Fig.…”

Section: Discussionsupporting

confidence: 82%

Structural snapshots illustrate the catalytic cycle of β-galactocerebrosidase, the defective enzyme in Krabbe disease

Hill

Graham

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et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Glycosphingolipids are ubiquitous components of mammalian cell membranes, and defects in their catabolism by lysosomal enzymes cause a diverse array of diseases. Deficiencies in the enzyme β-galactocerebrosidase (GALC) cause Krabbe disease, a devastating genetic disorder characterized by widespread demyelination and rapid, fatal neurodegeneration. Here, we present a series of highresolution crystal structures that illustrate key steps in the catalytic cycle of GALC. We have captured a snapshot of the short-lived enzyme-substrate complex illustrating how wild-type GALC binds a bona fide substrate. We have extensively characterized the enzyme kinetics of GALC with this substrate and shown that the enzyme is active in crystallo by determining the structure of the enzyme-product complex following extended soaking of the crystals with this same substrate. We have also determined the structure of a covalent intermediate that, together with the enzymesubstrate and enzyme-product complexes, reveals conformational changes accompanying the catalytic steps and provides key mechanistic insights, laying the foundation for future design of pharmacological chaperones.β-galactosylceramidase | lysosomal storage disease | glycosyl hydrolase | pharmacological chaperone therapy T he recycling and degradation of eukaryotic membrane components occurs in the lysosome and is essential for cellular maintenance. The molecular mechanisms of lysosomal lipid degradation are primarily informed by the study of a class of human diseases, sphingolipidoses, which are caused by inherited defects in glycosphingolipid catabolism. Krabbe disease is a devastating neurodegenerative disorder that is caused by deficiencies in the lysosomal enzyme β-galactocerebrosidase (GALC) (enzyme commission 3.2.1.46). It is essential for the catabolism of galactosphingolipids, including the principal lipid component of myelin, β-D-galactocerebroside ( Fig. 1A) (1). GALC function has also been implicated in cancer cell metabolism, primary open-angle glaucoma and the maintenance of a hematopoietic stem cell niche (2-4).GALC catalyzes the hydrolysis of β-D-galactocerebroside to β-D-galactose and ceramide, as well as the breakdown of psychosine to β-D-galactose and sphingosine. In both cases, removal of the galactosyl moiety is thought to occur via a retaining twostep glycosidic bond hydrolysis reaction (5, 6). Our recent structure of murine GALC identified two active site glutamate residues geometrically consistent with this mechanism (7). In the first step, the carboxylate group of E258 is hypothesized to perform a nucleophilic attack at ring position C 1 , forming an enzymesubstrate intermediate, releasing the first product (ceramide or sphingosine) as the leaving group. In the second step, E182 is thought to act as a general acid/base to deprotonate a water molecule, which then attacks the ring, releasing the enzyme and the second product (galactose).Defects in GALC lead to the accumulation of cytotoxic metabolites that elicit complex, and still only partially under...

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Section: Discussionsupporting

confidence: 82%

Structural snapshots illustrate the catalytic cycle of β-galactocerebrosidase, the defective enzyme in Krabbe disease

Hill

Graham

Read

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…We recently identified a one-step associative A N D N catalytic mechanism for the hydrolysis at the α-phosphate, involving also a coupled proton transfer step from the nucleophilic water to the catalytic Asp83 residue. 33 Here we show that essentially the same mechanism takes place in all three dUTPase systems investigated. The coupled proton transfer and the phosphate cleavage occur in a single step that has a loose dissociative transition state (TS) structure.…”

Section: Introductionsupporting

confidence: 61%

“…This mechanism is also consistent with the experimentally observed pH change that is used to directly follow the reaction kinetics, 56 and with the lack of observed intermediates for the phosphate hydrolysis. 33 It is found in E. coli dUTPase that two protons transfer to the reaction medium in a concerted mode, immediately following the rate limiting step. 56 These proton transfer events were also found dependent on two ionizable groups in the protein substrate complex with pK a values of 5.8 and 10.3.…”

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confidence: 99%

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

et al. 2015

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Most enzymes present a catalytic mechanism where one or more proton transfer events occur coupled tightly together with the enzymatic chemical reaction. We show here that inactivating mutations decouple this proton transfer step from the phosphate cleavage reaction in dUTPase. Homotrimeric dUTPase enzymes catalyze the hydrolysis of dUTP to dUMP and pyrophosphate, using largely similar structural and functional groups as most AAA+ enzymes. dUTPases typically use a single Mg 2+ ion as a cofactor in the active site that is formed by direct protein-protein contacts including all three protomers. Here we focus on the C-terminal arm structural motif, which has sequence and functional similarities to P-loop motifs, and is required for catalysis. In this work, we have studied the functional roles of the C-terminal arm in ligand binding and catalysis by using QM/MM (quantum mechanics/molecular mechanics) calculations in conjunction with site-directed mutagenesis experiments. We also present a new method to assess the metal ion coordination symmetry during the catalytic reaction. Using this new implementation, we identified that the coordination symmetry follows a consistent pattern in the three systems studied, reaching the most symmetrical state near the transition states. We found that the phosphate cleavage proceeds with a concerted bimolecular (A N D N ) mechanism with a loose dissociative transition state, and that it is coupled with a proton transfer step involving a unanimously conserved Asp residue. We show that the main mechanistic effect of the lack of the C-terminal arm is to decouple the phosphate cleavage from the subsequent proton transfer step, resulting in a highbarrier altered reaction pathway.

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“…We tested the Stl inhibition on the wild type enzyme (mtDUT WT ) and on the quasi wild type variant (mtDUT H145W ) containing a tryptophan substitution at the active site [20,44]. The active site architecture of dUTPase is well-known from the literature [45][46][47][48]. The mutated position is conserved for aromatic residues.…”

Section: Stl Inhibits the Enzymatic Activity Of M Tuberculosis Dutpamentioning

confidence: 99%

Cross-species inhibition of dUTPase via the Staphylococcal Stl protein perturbs dNTP pool and colony formation in Mycobacterium

et al. 2015

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Catalytic mechanism of α-phosphate attack in dUTPase is revealed by X-ray crystallographic snapshots of distinct intermediates, 31P-NMR spectroscopy and reaction path modelling

Cited by 17 publications

References 89 publications

Structural snapshots illustrate the catalytic cycle of β-galactocerebrosidase, the defective enzyme in Krabbe disease

Structural snapshots illustrate the catalytic cycle of β-galactocerebrosidase, the defective enzyme in Krabbe disease

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

Cross-species inhibition of dUTPase via the Staphylococcal Stl protein perturbs dNTP pool and colony formation in Mycobacterium

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