Enzymatic C4‐Epimerization of UDP‐Glucuronic Acid: Precisely Steered Rotation of a Transient 4‐Keto Intermediate for an Inverted Reaction without Decarboxylation

Borg, Annika J. E.; Esquivias, Oriol; Coines, Joan; Rovira, Carme; Nidetzky, Bernd

doi:10.1002/anie.202211937

Cited by 3 publications

(12 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It contributes to holding the C5 carboxylate in an equatorial orientation (Fig. 1b) which disfavors decarboxylation of the chemically labile 4-keto-intermediate on stereo-electronic grounds 51 .…”

Section: Resultsmentioning

confidence: 99%

“…Considering the controversial views, we figured that fresh evidence from the study of a different but relevantly similar (alcohol dehydrogenase-like) enzyme would be important to gain an advanced perspective. UDP-glucuronic acid 4-epimerase (UGAepi) [48][49][50][51] seemed to be an excellent candidate to analyze the functional interconnection between active-site preorganization, conformational sampling and energetics of the catalytic reaction.…”

mentioning

confidence: 99%

“…1a). Each hydride transfer is facilitated by a concerted proton transfer 48,50,51 . The transiently formed UDP-4-keto-hexuronic acid is rotated in the active site so that keto-group reduction can happen from both faces of the carbonyl.…”

mentioning

confidence: 99%

“…1) 50 . Repositioning of the 4-keto-hexuronic acid moiety involves extensive sampling of sugar ring pucker, yet the active-site configuration is almost unchanged in the process 51 . Biochemical study of the Bacillus cereus UGAepi establishes that C-H activation for substrate oxidation is rate-limiting for the overall C4 epimerization of UDP-GlcA 48 .…”

mentioning

confidence: 99%

“…The essential feature that the experimental k cat reflects a single chemical step of the enzymatic mechanism has enabled us here to apply temperature dependence studies and isotope effect measurements to probe the energetics and conformational characteristics of UGAepi activity 51 . Based on thermodynamic activation parameters, we find that approach from the ensemble-averaged GS to the TSRC involves restricting the configurational freedom of enzyme-substrate complex in considerable degree (ÀTΔS z ≤ 44 kJ/mol at 25 °C).…”

mentioning

confidence: 99%

See 4 more Smart Citations

Interplay of structural preorganization and conformational sampling in UDP-glucuronic acid 4-epimerase catalysis

Rapp,

Borg,

Nidetzky

2024

Nat Commun

Self Cite

View full text Add to dashboard Cite

Understanding enzyme catalysis as connected to protein motions is a major challenge. Here, based on temperature kinetic studies combined with isotope effect measurements, we obtain energetic description of C-H activation in NAD-dependent UDP-glucuronic acid C4 epimerase. Approach from the ensemble-averaged ground state (GS) to the transition state-like reactive conformation (TSRC) involves, alongside uptake of heat ($${\Delta {{{{{\rm{H}}}}}}}^{{{\ddagger}} }$$ Δ H ‡ = 54 kJ mol−1), significant loss in entropy ($$-T{\Delta {{{{{\rm{S}}}}}}}^{{{\ddagger}} }$$ − T Δ S ‡ = 20 kJ mol−1; 298 K) and negative activation heat capacity ($${\Delta {{{{{\rm{C}}}}}}}_{{{{{{\rm{p}}}}}}}^{{{\ddagger}} }$$ Δ C p ‡ = −0.64 kJ mol−1 K−1). Thermodynamic changes suggest the requirement for restricting configurational freedom at the GS to populate the TSRC. Enzyme variants affecting the electrostatic GS preorganization reveal active-site interactions important for precise TSRC sampling and H-transfer. Collectively, our study captures thermodynamic effects associated with TSRC sampling and establishes rigid positioning for C-H activation in an enzyme active site that requires conformational flexibility in fulfillment of its natural epimerase function.

show abstract

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Interplay of structural preorganization and conformational sampling in UDP-glucuronic acid 4-epimerase catalysis

Rapp,

Borg,

Nidetzky

2024

Nat Commun

Self Cite

View full text Add to dashboard Cite

show abstract

Enzymatic C4‐Epimerization of UDP‐Glucuronic Acid: Precisely Steered Rotation of a Transient 4‐Keto Intermediate for an Inverted Reaction without Decarboxylation

Borg

Esquivias

Coines³

et al. 2022

Angew Chem Int Ed

View full text Add to dashboard Cite

UDP-glucuronic acid (UDP-GlcA) 4-epimerase illustrates an important problem regarding enzyme catalysis: balancing conformational flexibility with precise positioning. The enzyme coordinates the C4oxidation of the substrate by NAD + and rotation of a decarboxylation-prone β-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 C 1 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.

show abstract

Molecular Mechanism of Double-Displacement Retaining β-Kdo Glycosyltransferase WbbB

Rao,

Zhu,

Liu

et al. 2024

J. Phys. Chem. B

View full text Add to dashboard Cite

Glycosyltransferases (GTs) are pivotal enzymes involved in glycosidic bond synthesis, which can lead to either retention or inversion of the glycosyl moiety's anomeric configuration. However, the catalytic mechanism for retaining GTs remains a subject of controversy. In this study, we employ MD and QM/MM metadynamics to investigate the double-displacement catalytic mechanism of the retaining β-Kdo transferase WbbB. Our findings demonstrate that the nucleophile Asp232 initiates the reaction by attacking the sugar ring containing a carboxylate at the anomeric position, forming a covalent adduct. Subsequently, the adduct undergoes a rotational rearrangement, ensuring proper orientation of the anomeric carbon for the acceptor substrate. In the second step, Glu158 acts as the catalytic base to abstract the proton of the acceptor substrate to complete the transglycosylation reaction. Notably, His265 does not function as the anticipated catalytic acid; instead, it stabilizes the phosphate group through H-bonding interactions. Our simulations support the double-displacement mechanism implicated from the crystallographic studies of WbbB. This mechanism deviates from the common S N i-type and retaining glycoside hydrolase mechanisms, thereby expanding our understanding of GT catalytic mechanisms.

show abstract

Enzymatic C4‐Epimerization of UDP‐Glucuronic Acid: Precisely Steered Rotation of a Transient 4‐Keto Intermediate for an Inverted Reaction without Decarboxylation

Cited by 3 publications

References 43 publications

Interplay of structural preorganization and conformational sampling in UDP-glucuronic acid 4-epimerase catalysis

Interplay of structural preorganization and conformational sampling in UDP-glucuronic acid 4-epimerase catalysis

Enzymatic C4‐Epimerization of UDP‐Glucuronic Acid: Precisely Steered Rotation of a Transient 4‐Keto Intermediate for an Inverted Reaction without Decarboxylation

Molecular Mechanism of Double-Displacement Retaining β-Kdo Glycosyltransferase WbbB

Contact Info

Product

Resources

About