Probing the Roles of Active Site Residues in D-Xylose Isomerase

Abstract

Initially, a cis-enediol mechanism was proposed for D-xylose isomerase (14,22), similar to the mechanism of triose-phosphate isomerase. However, isotope exchange experiments (23) and crystallographic analyses (15, 16) with various substrates and inhibitors suggests that the reaction proceeds via a metalmediated hydride shift (Fig. 1a). The currently accepted pathway for the reaction involves the preferential binding of ␣-Dxylopyranose (24, 25) followed by ring opening (25), extension of the substrate, and then the hydride shift (15,16,26). Recently, Meng et al. (27) have proposed that the hydride shift occurs on the cyclic form of sugar (Fig. 1b).Site-directed mutagenesis has been used to probe the functions of specific active site residues in D-xylose isomerase (27-32), however, only a few structures of mutant enzymes have been reported (19,(33)(34)(35). Kinetic data can be misleading if the substitutions affect the properties of catalytically important residues other than those changed by mutagenesis. For this reason, we have shifted our mutagenic studies from the Escherichia coli D-xylose isomerase (28, 29), which has not been successfully crystallized, to the S. rubiginosus enzyme which readily forms crystals diffracting x-rays beyond 2.0 Å (15,34,36 MATERIALS AND METHODSBacterial Strains and Plasmids-S. rubiginosus (ATCC 21175) was obtained from the American Type Culture Collection. E. coli BL21(DE3) (F Ϫ ompT r B Ϫm B Ϫ ( cTts857 ind1 Sam7 nin5 lacUV5-T7 gene 1)) was from Novagen. E. coli TG1 (supE hsd ⌬5 thi ⌬(lac-proAB) FЈ (traD36 proAB ϩ lacI q lacZ ⌬M15)) was used to propagate plasmid and bacteriophage M13. M13 mp18 and M13 mp19 DNA were purchased from U. S. Biochemical Corp. and pET11d plasmid DNA was from Novagen. S. rubiginosus xylA cloned into pET11d is called pRDW100 (its construction is described below) and is regulated by T7 RNA polymerase and LacZ, using the strain BL21(DE3) as a host.Biochemical Reagents-All compounds were reagent grade and purchased from Sigma, except acetaldehyde and 5-thio-␣-D-glucopyranose (THG), 1 which were from Aldrich. The sugar 5-deoxy-D-xylulose was synthesized by aldol condensation of dihydroxyacetone and acetaldehyde, using rabbit muscle aldolase with the cofactor sodium arsenate as a catalyst, and was purified by ion-exchange chromatography, 2 using a scheme similar to that described by Durrwachter et al. (37) for the synthesis of 5-deoxy-D-fructose.DNA Isolation, Transformation, and Manipulations-S. rubiginosus was grown in yeast and maltose extract medium supplemented with 34% (w/v) sucrose and chromosomal DNA was isolated as Hopwood et al. (38). Both plasmid and bacteriophage DNA were isolated from cul-

Xylose Isomerase in Substrate and Inhibitor Michaelis States: Atomic Resolution Studies of a Metal-Mediated Hydride Shift,

“…However, studies of H53A, H53D, and H53N mutants show that these mutations affect activity approximately 10-fold (although isomerization is still 3-4 orders of magnitude faster than solution rates of mutarotation (37)), while H53F, H53Y, and H53R mutations eliminate all measurable activity, suggesting a dependence on size of the introduced mutation (4, 10, 23,38,39). We propose that this phenomenon arises from an activated water species taking on the role of His53 if a large pocket is introduced by the mutation (e.g., H53A and H53D), whereas bulky side chains block this possibility and thus the ability of the enzyme to open pyranose sugars.…”

Dissolution of Xylose Metabolism in Lactococcus lactis

“…All of the group II XylAs conserved 17 active-site residues (Fig. 2) determined for the group I Streptomyces rubiginosus XylA (50)(51)(52). Although there are conserved residues throughout the group II protein, the most highly conserved region, aa 179 to 345, is in the middle of the protein.…”

Role of electrostatics at the catalytic metal binding site in xylose isomerase action: Ca2+-inhibition and metal competence in the double mutant D254E/D256E

“…Moreover, this position corresponds to Site 2 2 in the wild-type XI, suggested to be catalytically relevant in the course of the enzyme action. 10,41 Despite the similar stereochemistry, Mg 21 and Mn 21 are active with the mutant enzyme but Ca 21 is not, indicating that it is not steric factors that determine catalytic competence. This is further supported by results with Be 21 .…”