Deciphering the enzymatic mechanism of sugar ring contraction in UDP-apiose biosynthesis

Savino, Simone; Borg, Annika J. E.; Dennig, Alexander; Pfeiffer, Martin; Giorgi, Francesca De; Weber, Hansjörg; Dubey, Kshatresh Dutta; Rovira, Carme; Mattevi, Andrea; Nidetzky, Bernd

doi:10.1038/s41929-019-0382-8

Cited by 23 publications

(32 citation statements)

References 36 publications

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“…The crevice between the two domains encloses the active site (27)(28)(29). As for other SDR family enzymes, two protein chains are arranged to form a tight homo-dimer (11,30,31) where each subunit (37 kDa molecular weight) interacts with two adjacent α-helices generating an extensively intermolecular hydrophobic core of four α-helices (14,16) (Fig. 1).…”

Section: Overall Structurementioning

confidence: 99%

“…SDRs are characterized by a strictly conserved catalytic triad, Ser/Thr-Tyr-Lys. The tyrosine residue functions as the base that abstracts a proton from the substrate 4'-OH group and promotes C4' oxidation by NAD + (12)(13)(14)(15)(16)(17)(18). The generated 4-keto-hexose-uronic acid intermediate is unstable and converted into different products depending on the enzyme (Scheme 1).…”

Section: Introductionmentioning

confidence: 99%

“…As demonstrated by recent work, these enzymes fine tune the rates of the individual redox steps to prevent the accumulation of the 4-keto-hexoseuronic acid, minimizing the undesired decarboxylation and/or release of this reactive intermediate (23). While the catalytic mechanisms of UDP-xylose synthase and UDP-apiose/xylose synthase have been thoroughly elucidated (13,16,24), the epimerization reaction by UDP-glucuronic acid 4epimerase remains partly unexplored. In this work, we carried out a comprehensive structural analysis of UDP-glucuronic acid 4-epimerase from Bacillus cereus (BcUGAepi) by solving six high-resolution crystal structures that elucidate different steps along the reaction.…”

Section: Introductionmentioning

confidence: 99%

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Crystallographic snapshots of UDP-glucuronic acid 4-epimerase ligand binding, rotation, and reduction

Iacovino

Savino

Borg

et al. 2020

Journal of Biological Chemistry

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UDP-glucuronic acid is converted to UDP-galacturonic acid en route to a variety of sugar-containing metabolites. This reaction is performed by a NAD+-dependent epimerase belonging to the short-chain dehydrogenase/reductase family. We present several high-resolution crystal structures of the UDP-glucuronic acid epimerase from Bacillus cereus. The geometry of the substrate-NAD+ interactions is finely arranged to promote hydride transfer. The exquisite complementarity between glucuronic acid and its binding site is highlighted by the observation that the unligated cavity is occupied by a cluster of ordered waters whose positions overlap the polar groups of the sugar substrate. Co-crystallization experiments led to a structure where substrate- and product-bound enzymes coexist within the same crystal. This equilibrium structure reveals the basis for a “swing & flip” rotation of the pro-chiral 4-keto-hexose-uronic acid intermediate that results from glucuronic acid oxidation, placing the C4’ atom in position for receiving a hydride ion on the opposite side of the sugar ring. The product-bound active site is almost identical to that of the substrate-bound structure and satisfies all hydrogen-bonding requirements of the ligand. The structure of the apo-enzyme together with kinetic isotope effect and mutagenesis experiments further outlines a few flexible loops that exist in discrete conformations, imparting structural malleability required for ligand rotation while avoiding leakage of the catalytic intermediate and/or side-reactions. These data highlight the double nature of the enzymatic mechanism: the active site features a high degree of precision in substrate recognition combined with the flexibility required for intermediate rotation.

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Section: Overall Structurementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Crystallographic snapshots of UDP-glucuronic acid 4-epimerase ligand binding, rotation, and reduction

Iacovino

Savino

Borg

et al. 2020

Journal of Biological Chemistry

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“…All belong to the extended subclass of SDRs that are distinct from the canonical SDR type in having NAD + coenzyme tightly bound to their structure [11–13]. Common feature of their mechanism appears to be a transient UDP‐4‐keto‐hexose‐uronic acid intermediate which is formed by proton abstraction from C4‐OH together with hydride transfer to enzyme‐NAD + [13–19]. At this stage, the catalytic paths of the individual enzymes diverge (Fig.…”

Section: Introductionmentioning

confidence: 99%

Mechanistic characterization of UDP‐glucuronic acid 4‐epimerase

et al. 2020

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UDP-glucuronic acid (UDP-GlcA) is a central precursor in sugar nucleotide biosynthesis and common substrate for C4-epimerases and decarboxylases releasing UDP-galacturonic acid (UDP-GalA) and UDP-pentose products, respectively. Despite the different reactions catalyzed, the enzymes are believed to share mechanistic analogy rooted in their joint membership to the short-chain dehydrogenase/reductase (SDR) protein superfamily: Oxidation at the substrate C4 by enzyme-bound NAD + initiates the catalytic pathway. Here, we present mechanistic characterization of the C4-epimerization of UDP-GlcA, which in comparison with the corresponding decarboxylation has been largely unexplored. The UDP-GlcA 4-epimerase from Bacillus cereus functions as a homodimer and contains one NAD + /subunit (k cat = 0.25 AE 0.01 s −1). The epimerization of UDP-GlcA proceeds via hydride transfer from and to the substrate's C4 while retaining the enzyme-bound cofactor in its oxidized form (≥ 97%) at steady state and without trace of decarboxylation. The k cat for UDP-GlcA conversion shows a kinetic isotope effect of 2.0 (AE0.1) derived from substrate deuteration at C4. The proposed enzymatic mechanism involves a transient UDP-4-keto-hexose-uronic acid intermediate whose formation is rate-limiting overall, and is governed by a conformational step before hydride abstraction from UDP-GlcA. Precise positioning of the substrate in a kinetically slow binding step may be important for the epimerase to establish stereo-electronic constraints in which decarboxylation of the labile β-keto acid species is prevented effectively. Mutagenesis and pH studies implicate the conserved Tyr149 as the catalytic base for substrate oxidation and show its involvement in the substrate positioning step. Collectively, this study suggests that based on overall mechanistic analogy, stereo-electronic control may be a distinguishing feature of catalysis by SDR-type epimerases and decarboxylases active on UDP-GlcA.

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“…SDRs have been classified into seven families: classical, extended, atypical, intermediate, divergent, complex and unassigned, and the classical type is the most prominent (Persson and Kallberg, 2013;Gräff et al, 2019). SDRs show diverse substrate spectra, including steroids, alcohols, sugars, aromatic compounds, and xenobiotics, and for this reason, more and more SDRs have been extensively explored for industrial production (Persson and Kallberg, 2013;Luo et al, 2019;Savino et al, 2019;Shanati et al, 2019;Zhou et al, 2019;Su et al, 2020). SDRs play diverse roles in core metabolism and specific metabolism pathways such as steroidal metabolism, detoxification and drug resistance (Sonawane et al, 2018;Laskar and Younus, 2019).…”

Section: Introductionmentioning

confidence: 99%

Short-Chain Dehydrogenase NcmD Is Responsible for the C-10 Oxidation of Nocamycin F in Nocamycin Biosynthesis

Zhang

et al. 2020

Front. Microbiol.

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Nocamycins I and II, featured with a tetramic acid scaffold, were isolated from the broth of Saccharothrix syringae NRRL B-16468. The biosynthesis of nocamycin I require an intermediate bearing a hydroxyl group at the C-10 position. A short chain dehydrogenase/reductase NcmD was proposed to catalyze the conversion of the hydroxyl group to ketone at the C-10 position. By using the λ-RED recombination technology, we generated the NcmD deletion mutant strain S. syringae MoS-1005, which produced a new intermediate nocamycin F with a hydroxyl group at C-10 position. We then overexpressed NcmD in Escherichia coli BL21 (DE3), purified the His6-tagged protein NcmD to homogeneity and conducted in vitro enzymatic assays. NcmD showed preference to the cofactor NAD+, and it effectively catalyzed the conversion from nocamyin F to nocamycin G, harboring a ketone group at C-10 position. However, NcmD showed no catalytic activity toward nocamyin II. NcmD achieved maximum catalytic activity at 45°C and pH 8.5. The kinetics of NcmD toward nocamycin F was investigated at 45°C, pH 8.5 in the presence of 2 mM NAD+. The Km and kcat values were 131 ± 13 μM and 65 ± 5 min−1, respectively. In this study, we have characterized NcmD as a dehydrogenase, which is involved in forming the ketone group at the C-10 position of nocamycin F. The results provide new insights to the nocamycin biosynthetic pathway.

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Deciphering the enzymatic mechanism of sugar ring contraction in UDP-apiose biosynthesis

Cited by 23 publications

References 36 publications

Crystallographic snapshots of UDP-glucuronic acid 4-epimerase ligand binding, rotation, and reduction

Crystallographic snapshots of UDP-glucuronic acid 4-epimerase ligand binding, rotation, and reduction

Mechanistic characterization of UDP‐glucuronic acid 4‐epimerase

Short-Chain Dehydrogenase NcmD Is Responsible for the C-10 Oxidation of Nocamycin F in Nocamycin Biosynthesis

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