Stereo-electronic control of reaction selectivity in short-chain dehydrogenases: Decarboxylation, epimerization, and dehydration

Sugar nucleotide–modifying enzymes of the short-chain dehydrogenase/reductase type use transient oxidation–reduction by a tightly bound nicotinamide cofactor as a common strategy of catalysis to promote a diverse set of reactions, including decarboxylation, singleor double-site epimerization, and dehydration. Although the basic mechanistic principles have been worked out decades ago, the finely tuned control of reactivity and selectivity in several of these enzymes remains enigmatic. Recent evidence on uridine 5'-diphosphate (UDP)glucuronic acid decarboxylases (UDP-xylose synthase, UDPapiose/UDP-xylose synthase) and UDP-glucuronic acid-4epimerase suggests that stereo-electronic constraints established at the enzyme’s active site control the selectivity, and the timing of the catalytic reaction steps, in the conversion of the common substrate toward different products. The mechanistic idea of stereo-electronic control is extended to epimerases and dehydratases that deprotonate the Ca of the transient ketohexose intermediate. The human guanosine 5'-diphosphate (GDP)-mannose 4,6-dehydratase was recently shown to use a minimal catalytic machinery, exactly as predicted earlier from theoretical considerations, for the b-elimination of water from the keto-hexose species. Addresses 1 Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, 8010, Graz, Austria 2 Centre for Synthetic Biology, Department of Biotechnology, Ghent University, 9000, Ghent, Belgium 3 Austrian Centre of Industrial Biotechnology (acib), 8010, Graz, Austria Corresponding author: Nidetzky, Bernd (bernd.nidetzky@tugraz.at) w Current Opinion in Chemical Biology 2021, 61:43–52 This review comes from a themed issue on Biocatalysis and Biotransformation Edited by Bernd Nidetzky and Tom Desmet For a complete overview see the Issue and the Editorial https://doi.org/10.1016/j.cbpa.2020.09.01


Introduction
Transient oxidationereduction is a central mechanistic principle of sugar nucleotideemodifying enzymes to achieve a variety of biosynthetically important transformations [1e5]. These transformations notably involve epimerization, decarboxylation, and dehydration ( Figure 1) [3,4,6]. The enzymes catalyzing these reactions share common membership to the short-chain dehydrogenase/reductase (SDR) protein superfamily [4]. They are often referred to as 'extended SDRs' for the additional features of structure and function they involve compared with the prototypical SDR oxidoreductase [4]. They generally use a tightly bound NAD þ or NADP þ cofactor and are equipped with the basic SDR catalytic machinery (a highly conserved Asn/Ser/ Tyr/Lys tetrad of residues) to facilitate oxidatione reduction [4]. The active sites of extended SDRs are often expanded by residues fulfilling deprotonatione protonation types of catalytic function [4]. Thus, the extended SDRs integrate unique reactivities (e.g., epimerization [2,7e10,11*,12,13**], b-elimination of water [14**,15*,16e18], aldol cleavage for ring opening [19*,20**], decarboxylation [20**,21,22**]) into a cycle of hydride transfer to and from the nicotinamide cofactor. Oxidation of the sugar nucleotide serves the essential role of activating the substrate for further transformations. Except in hexose nucleotide 4,6dehydratases as discussed later, the hydride is delivered back to the same substrate carbon from which it was abstracted. The reduction is usually stereospecific, except in C2-and C4-epimerases ( Figure 1, UGAepi reaction) that require nonstereospecific reduction of the corresponding 2- Despite this, the finely tuned control of reactivity and selectivity in several of these enzymes still remains a mystery. A biologically important and mechanistically striking example is represented by uridine 5'-diphosphate-glucuronic acid (UDP-GlcA), which is the common substrate for epimerase and decarboxylase types of extended SDRs (Figure 1  Schematic representation of the reaction mechanisms of UAXS (black/purple) [20**], UXS (black/dark blue/light blue) [ 34]) convert UDP-GlcA into different UDP-pentose products ( Figure 1).
Recent studies suggest an important role for stereoelectronic control by the enzymes to steer a perfectly selective transformation of UDP-GlcA, leading to different products from the same substrate [13**,20**,22**]. The studies also indicate that stereoelectronic constraints at the active site enable the decarboxylase enzymes to achieve a unique timing of their catalytic reaction steps [20**]. The mechanistic idea of stereo-electronic control is extended to epimerases and dehydratases that deprotonate the Ca of the transient keto-hexose intermediate. In addition, the human guanosine 5'-diphosphate (GDP)-mannose 4,6dehydratase is discussed here for the recent mechanistic insight it has provided into the elimination mechanism. Based on structure snapshots from the reaction coordinate, the enzyme was shown to use a minimal catalytic machinery comprised of just two active-site groups, exactly as predicted earlier from . Interestingly, ArnA reaction with UDP-GlcA stops at the UDP-4-ketopentose, which is then used by a different enzyme (ArnB) in a transamination reaction with L-glutamic acid to form UDP-4-amino-4-deoxy-L-arabinose [35,36]. ArnA releases the NADH formed [21]. In UXS and ArnA, a conserved glutamic acid residue is involved in proton transfer to C5 during decarboxylation and/or fixing the substrate in place for the enzymatic conversion ( Figure 2a) [21,22**]. The Glu is also conserved in hexose nucleotide 4,6dehydratases, in which it plays the mechanistically comparable role of deprotonation-protonation at C5 [14**,15*, 18,37], as discussed later.
The Glu is lacking in UGAepi ( Figure 2a). Structurally, UGAepi resembles the well-characterized UDP-galactose 4-epimerase in that it provides fully formed binding pockets for NAD þ and UDP [27**]. From the perspective of molecular structure, therefore, any repositioning of the two molecules in the course of the UGAepi catalytic cycle is highly unlikely. To perform a nonstereospecific reduction of the C4 carbonyl group, UGAepi must therefore enable free rotation of the 4keto-hexuronic acid moiety. An interesting mechanistic challenge for UGAepi is to combine this free rotation requirement with proper protection of the chemically labile b-keto-acid intermediate against decarboxylation The reaction of UAXS leading to UDP-apiose is unusually complex to be carried out by a single active site. The proposed mechanism involves a retro-aldol reaction for ring opening of the 4-keto-hexuronic acid intermediate [ Both enzymes also belong to the extended SDRs. The UAXS (from Arabidopsis thaliana) was shown to not catalyze ring opening on an isolated UDP-4-ketopentopyranose intermediate [20**]. Therefore, timing of the catalytic steps, such that the ring opening occurs before the decarboxylation, appears to be essential. Ring closure establishes the ring contraction in a reversible aldol reaction. Ring closure without ring contraction appears to be effectively irreversible. Reduction leads to UDP-apiose and UDP-xylose in a ratio that seems to depend on both the enzyme and the reaction conditions used [20**].
Evidence for stereo-electronic control in reactions of UXS, UGAepi, and UAXS As shown in Figure 1, the catalytic paths of UAXS, UXS, ArnA, and UGAepi diverge at the UDP-4-ketohexuronic acid intermediate. To appreciate the enzymes' individual ways of handling the intermediate (a labile b-keto acid species), it is relevant to consider chemical requirements for decarboxylation in the context of a six-membered ring system. Generally, decarboxylation is stereo-electronically favored when the dihedral angle between the C=O bond and the cleaved CeC bond is w90 (Figure 2b) [40,41]. In the UDP-4-keto-hexuronic acid intermediate, a dihedral of w90 requires the carboxylate group to be positioned axially. Chemical studies show that optimal orbital alignment for decarboxylation involves a continuous overlap, via hyperconjugation and resonance, from the breaking CeC bond and the p-orbital of the carbonyl (C4=O) group [41e43]. In the stereo-electronically optimal case for the enzymatic decarboxylation (axial carboxylate), the breaking C5eC6 bond is roughly orthogonal to the plane of the C5eC4=O system, allowing for productive sep delocalization [43] to enable good overlap of the p-orbital system of the carbonyl carbon (C4) with the developing C5 p-orbital in the incipient 4,5-enolate [41,43] (Figure 2b). To bring the carboxylate into an axial position at the enzyme's active site, a binding energyedriven distortion from the pyranose chair conformation may be necessary. The relaxed chair conformation of the 4-keto-hexuronic acid features an equatorial carboxylate with a dihedral angle of w0 . The equatorial carboxylate thus provides stereo-electronic constraints nonoptimal for decarboxylation. For an enzymatic path to exclude (UGAepi), or to delay (UAXS), the decarboxylation, therefore, a stereo-electronic rational to maintain an equatorial carboxylate is thus provided. The mechanistic idea of differential stereo-electronic control in epimerase and decarboxylase reactions of UDP-GlcA is supported by a comparison of UXS and UGAepi. Binding of UDP-GlcA by UAXS is also consistent with the overall stereoelectronic argument. The importance of proper orbital alignment for promoting or preventing decarboxylation has been recognized over decades in studies of pyridoxal 5'-phosphate (PLP)-dependent enzymes, where the enzyme applies stereo-electronic constraints to 'choose' between decarboxylation and transamination/racemization [42,44e46].  Structures of UGAepi bound with UDP-GlcA ( Figure 3a) and UDP-GalA (Figure 3b) show the pyranose ring in an undistorted 4 C 1 conformation, which places the carboxylate group equatorially [27**]. In each structure, the carboxylate is accommodated within a tight network of hydrogen bonds. The reactive 4-OH of the substrate/product is well aligned with the catalytic residues of the enzyme. Threonine from the SDR catalytic triad orients the 4-OH for proton abstraction by tyrosine. Interestingly, the threonine is also close to the substrate/product carboxylate. Besides establishing stereo-electronic conditions to disfavor decarboxylation, binding of the carboxylate group could arguably contribute to precise positioning of the substrate for catalysis. Binding constraints from the active site pose a conundrum for UGAepi given the requirement for free rotation of the 4-keto-hexuronic acid intermediate for epimerization. Computational analysis will be important to elucidate dynamic features of the catalytic reaction. The conformational rearrangements associated with the rotation have now been captured at high resolution in the crystal [27**].
For UXS, the conformation of the Michaelis complex was derived from a high-resolution crystal structure of the human enzyme bound with NAD þ and UDP. The UDP-GlcA was modeled into the active site [22**]. Molecular dynamics computational studies revealed that to achieve a plausible positioning of GlcA for catalysis the pyranose 4 C 1 chair had to be distorted. A B O,3 boat conformation, placing the carboxylate in an almost perfect axial position 'ready for decarboxylation', was found to yield a seemingly proper alignment between the reactive groups on the substrate (UDP-GlcA), cofactor (NAD þ ), and the catalytic groups on the enzyme (Figure 3c) [22**]. UXS provides fewer interactions with the substrate carboxylate than UGAepi, and binding of the carboxylate lacks the direct connection to the immediate catalytic machinery. The crystal structure of ArnA bound with UDP-GlcA shows an undistorted chair conformation for the pyranose ring with the carboxylate in the equatorial position. However, the substrate is not positioned for catalytic oxidation at C4 to occur via the canonical SDR mechanism, and, therefore, a nonproductive enzymeesubstrate complex appears to have been captured in the ArnA crystal [21].
The special case of UAXS: precise timing of decarboxylation in multistep SDR catalysis Similar as in UXS, molecular dynamics simulations had to be done on the experimental enzyme crystal structure (the C100A variant of A. thaliana UAXS bound with NADH and UDP-GlcA) to identify the plausible conformation of the Michaelis complex [20**]. The substrate-bound conformation of the enzyme's active site is unusually flexible, with the pyranose ring pucker changing substantially along simulated trajectories. The enzyme complex conformations featuring a plausible positioning of UDP-GlcA for the initial catalytic step (oxidation at C4) all have the carboxylate group in a closely equatorial position, as shown in Figure 3d. This supports the particular timing of UAXS catalytic steps that delays the decarboxylation of the b-keto-acid until after the ring opening between C2 and C3 has happened. The ring-opened intermediate can then position the carboxylate moiety for suitable orbital alignment to meet the stereo-electronic requirements for efficient decarboxylation [20**].

Stereo-electronic considerations extended to other epimerases and dehydratases
In pyranose ring systems, the a-proton of a ketone is more easily abstracted (i.e., becomes more acidic) when it is positioned axially [38*,47]. Concerning the orbital alignment optimal for deprotonation, effectively the same considerations (sep delocalization) apply as for decarboxylation [41,43,47]. Structural and mechanistic studies of hexose nucleotide 4,6-dehydratases (e.g., and SQDs, respectively [15*e18]. The regioselectivity of the reduction is controlled by a fine-tuned alignment of nicotinamide ring of the NAD þ cofactor relative to the sugar ring plane. In 4,6-dehydratases a parallel alignment is observed, allowing hydride abstraction from C4 and re-donation to C6. In contrast, a nonparallel alignment is observed in TunA and SQD1 with an angle of around 15e30 to each other, favoring redonation of the hydride to C4 (Figure 4c) [14**,15*].

Conclusion
Constraining substrates into optimal conformations is an essential aspect of enzymatic catalysis [57]. Enzymes promote proper orientation of the orbitals to facilitate the desired biochemical transformation [42,57e59]. The proposed catalytic mechanism of dehydration by hGMD (a, b) and active site close ups of hGMD, TunA and SQD1 (c). The contribution of stereo-electronic effects into enzyme-catalyzed reactions has been appreciated for decades, especially in context where enzymes apply stereo-electronic control to either allow or exclude certain reaction pathways [42,57,58,60,61]. Recent studies on SDR decarboxylases (UAXS, UXS, ArnA) [19*,20**,21,22**] and epimerases (UGAepi) [13**,27**] demonstrate how structurally closely related enzymes can utilize effectively the same substrate (UDP-GlcA) to catalyze different reaction pathways (decarboxylation, epimerization, aldol cleavage for ring opening) assisted by stereo-electronic control. This fascinating concept can be expanded to NDP-sugar dehydratases (hGMD, TunA, SQD) [14**,15*,18] from the SDR superfamily, where stereo-electronic constraints contribute to the regioselectivity of the reaction. In addition, the studies from the last few years highlight how essentially the same active site of the SDR epimerases, decarboxylases, and dehydratases is fine-tuned to perform different catalytic pathways. Realizing the importance of proper orbital alignment in (bio)chemical reactions is crucial for mechanistic enzymology and will be helpful in understanding the enzymatic mechanisms in future.

Declaration of competing interest
Nothing declared.