A metal‐mediated hydride shift mechanism for xylose isomerase based on the 1.6 Å Streptomycs rubiginosus structure with xylitol and D‐xylose

Abstract

The crystal structure of recombinant Streptomyces rubiginosus D-xylose isomerase (D-xylose keto-isomerase, EC 5.3.1.5) solved by the multiple isomorphous replacement technique has been refined to R = 0.16 at 1.64 A resolution. As observed in an earlier study at 4.0 A (Carrell et al., J. Biol. Chem. 259: 3230-3236, 1984), xylose isomerase is a tetramer composed of four identical subunits. The monomer consists of an eight-stranded parallel beta-barrel surrounded by eight helices with an extended C-terminal tail that provides extensive contacts with a neighboring monomer. The active site pocket is defined by an opening in the barrel whose entrance is lined with hydrophobic residues while the bottom of the pocket consists mainly of glutamate, aspartate, and histidine residues coordinated to two manganese ions. The structures of the enzyme in the presence of MnCl2, the inhibitor xylitol, and the substrate D-xylose in the presence and absence of MnCl2 have also been refined to R = 0.14 at 1.60 A, R = 0.15 at 1.71 A, R = 0.15 at 1.60 A, and R = 0.14 at 1.60 A, respectively. Both the ring oxygen of the cyclic alpha-D-xylose and its C1 hydroxyl are within hydrogen bonding distance of NE2 of His-54 in the structure crystallized in the presence of D-xylose. Both the inhibitor, xylitol, and the extended form of the substrate, D-xylose, bind such that the C2 and C4 OH groups interact with one of the two divalent cations found in the active site and the C1 OH with the other cation. The remainder of the OH groups hydrogen bond with neighboring amino acid side chains. A detailed mechanism for D-xylose isomerase is proposed. Upon binding of cyclic alpha-D-xylose to xylose isomerase, His-54 acts as the catalytic base in a ring opening reaction. The ring opening step is followed by binding of D-xylose, involving two divalent cations, in an extended conformation. The isomerization of D-xylose to D-xylulose involves a metal-mediated 1,2-hydride shift. The final step in the mechanism is a ring closure to produce alpha-D-xylulose. The ring closing is the reverse of the ring opening step. This mechanism accounts for the majority of xylose isomerase's biochemical properties, including (1) the lack of solvent exchange between the 2-position of D-xylose and the 1-pro-R position of D-xylulose, (2) the chemical modification of histidine and lysine, (3) the pH vs. activity profile, and (4) the requirement for two divalent cations in the mechanism.

Structural Evidence for a Hydride Transfer Mechanism of Catalysis in Phosphoglucose Isomerase from Pyrococcus furiosus

“…In xylose isomerase, such an exchange is prevented in part by a tryptophan residue (Trp-16), which forms a hydrophobic envelope around the site of hydrogen transfer (7). Similarly, in the complex of PfPGI with 6PG, the tetrahedral density at the C2 position of the inhibitor indicates that the transferred hydrogen projects in toward the active site where it is framed by a markedly hydrophobic region.…”

The crystal structure of rabbit phosphoglucose isomerase complexed with 5-phospho- d -arabinonohydroxamic acid

“…In XYL, Lys-182 makes a hydrogen bond to O1 of the ligand. Another interesting similarity between XYL and PGI is that both enzymes have been proposed to use a histidine residue to catalyze ring-opening of the bound cyclic substrates (64) and thereafter this histidine interacts with the hydroxyl on C5 of the linear substrate. However, it should be noted that later studies of XYL challenged the involvement of a histidine in the ring-opening step (65,66).…”

“…However, it should be noted that later studies of XYL challenged the involvement of a histidine in the ring-opening step (65,66). It was also reported that the XYL active site base that transfers the proton between O1 and O2 is assumed to be a hydroxide ion ligated to one of the metal ions (64,67). This situation is analogous to what we propose to occur in PGI, for which lowering of the pKa of water423 would not be achieved by a metal ion, but by the surrounding network of water molecules.…”

Structure determination of glucose isomerase from Streptomyces murinus at 2.6 Å resolution

“…A number of laboratories have studied the structure of GI isolated from different bacterial species by X-ray crystallography, with or without inhibitors or metal ions present, in order to understand and explain its mechanism of activity: XI, Actinoplanes missouriensis, 2.8 A resolution (Rey et al, 1988); XI, Streptomyces olivochromogenes, 3.0 A resolution (Farber, Glasfeld, Tiraby, Ringe & Petsko, 1989); XI, Arthrobacter, 2.5/k resolution (Henrick, Collyer & Blow, 1989); XI, Streptomyces rubiginosis, 1.9 A resolution (Carrell et al, 1989;Collyer, Henrick & Blow, 1990); XI, Streptomyces albus, 1.65 A resolu-© 1994 International Union of Crystallography Printed in Great Britain -all rights reserved tion (Dauter, Terry, Witzel & Wilson, 1990); XI, Streptomyces rubiginosus, 1.6 A resolution (Whitlow et al, 1991). The mechanism has been suggested to involve a metal-mediated 1,2 hydride shift (Collyer, Henrick & Blow, 1990, Whitlow et al, 1991.…”