Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 1. Crystallography and site-directed mutagenesis of metal binding sites

Jenkins, John A.; Janin, Joël; F, Rey; Chiadmi, M.; H, van Tilbeurgh; Lasters, Ignace; M, De Maeyer; D, Van Belle; Wodak, Shoshana J.; Lauwereys, Marc

doi:10.1021/bi00139a005

Cited by 137 publications

(114 citation statements)

References 30 publications

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“…K i (PGH) for trypanosomal TIM is about 8 M, whereas K M is 250 or 1200 M, depending on the direction of reaction (55). XYL from Streptomyces olivochromogenes has a low affinity for its natural substrates: the K M equals 5 mM for D-xylose and 290 mM for D-glucose (56). For D-threonohydroxamic acid (THA), the K i Յ 100 nM (47).…”

Section: Comparison Of Pgi͞5pah To Complexes Of Tim and Xyl With Analmentioning

confidence: 99%

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

Arsenieva

Hardré

Salmon

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Phosphoglucose isomerase (EC 5.3.1.9) catalyzes the second step in glycolysis, the reversible isomerization of D-glucose 6-phosphate to D-fructose 6-phosphate. The reaction mechanism involves acid-base catalysis with proton transfer and proceeds through a cis-enediol(ate) intermediate. 5-Phospho-D-arabinonohydroxamic acid (5PAH) is a synthetic small molecule that resembles the reaction intermediate, differing only in that it has a nitrogen atom in place of C1. Hence, 5PAH is the best inhibitor of the isomerization reaction reported to date with a Ki of 2 ؋ 10 ؊7 M. Here we report the crystal structure of rabbit phosphoglucose isomerase complexed with 5PAH at 1.9 Å resolution. The interaction of 5PAH with amino acid residues in the enzyme active site supports a model of the catalytic mechanism in which Glu-357 transfers a proton between C1 and C2 and Arg-272 helps stabilize the intermediate. It also suggests a mechanism for proton transfer between O1 and O2. P hosphoglucose isomerase (PGI; EC 5.3.1.9) is a cytosolic glycolytic enzyme that catalyzes the reversible isomerization of D-glucose 6-phosphate (G6P) to D-fructose 6-phosphate (F6P). In addition to isomerase activity, PGI has been shown to display anomerase activity between pyranose anomers of G6P (1), between furanose anomers of F6P (2), and between those of D-mannose 6-phosphate (3), as well as C-2-epimerase activity on G6P (4). It also has roles in protein glycosylation, gluconeogenesis, and the pentose phosphate pathway (5). This central role in the metabolism of phosphorylated sugars explains the strong impact of PGI deficiency in humans (6, 7), as well as the interest as a therapeutic target in parasite metabolism (8). The PGI polypeptide chain, or perhaps a variation thereof, also has extracellular roles as neuroleukin, autocrine motility factor, and differentiation and maturation mediator (9-12). These proteins promote antibody secretion by mononuclear cells, tumor cell motility, and differentiation of leukemia cells and a promyelocytic cell line (HL-60 cells), respectively. More recently, PGI has also been identified as the antigen involved in rheumatoid arthritis of a mouse line (13), as a specific inhibitor toward myofibril-bound serine proteinase (14), and as the surface antigen involved in sperm agglutination (15). Because very little is known about how PGI is precisely involved in these various biological processes, this moonlighting protein (16) has been the subject of intense investigations.Biochemical characterization of PGI began over 50 years ago and includes inhibitor studies (1,(17)(18)(19)(20)(21)(22)(23)(24)(25), labeling studies (26-32), solvent exchange (33, 34), mutagenesis (35,36), and pH profile studies (20,37). A proposed multistep catalytic mechanism includes a ring-opening step followed by an isomerization step. The isomerization step proceeds via general acid-base catalysis with proton transfer. In the G6P to F6P direction, abstraction of the proton from C2 by an active site amino acid residue yields a 1,2-cis-enediol(ate) in...

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Section: Comparison Of Pgi͞5pah To Complexes Of Tim and Xyl With Analmentioning

confidence: 99%

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

Arsenieva

Hardré

Salmon

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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“…ADR1 is a DNA-binding (16), gene-specific activator ofADH2 (15). It is possible that, like the SNF2 and SNF5 proteins, CCR4 functions both in aiding gene-specific activators and in overcoming repression by nucleosomes (20,22).The CCR4 protein contains several regions which may be important to its function. The N-terminal region is rich in glutamines and asparagines, which is characteristic of a number of eukaryotic proteins involved in transcription (9, 25).…”

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“…The N-terminal region is rich in glutamines and asparagines, which is characteristic of a number of eukaryotic proteins involved in transcription (9, 25). The C terminus contains a region similar to the manganese binding site of xylose isomerases (unpublished data) (10,20). The central region of the protein (residues 350 to 473) contains five tandem copies of a leucine-rich repeat.…”

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CCR4 is a glucose-regulated transcription factor whose leucine-rich repeat binds several proteins important for placing CCR4 in its proper promoter context.

et al. 1994

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The yeast CCR4 protein is required for the expression of a number of genes involved in nonfermentative growth, including glucose-repressible ADH2, and is the only known suppressor of mutations in the SPT6 and SPT10 genes, two genes which are believed to be involved in chromatin maintenance. We show here that although CCR4 did not bind DNA under the conditions tested, it was able to activate transcription when fused to a heterologous DNA-binding domain. The transcriptional activation ability of CCR4, in contrast to that of many other activators, was glucose regulated. Two activation domains one of which was glucose responsive and encompassed a glutamine-proline-rich region similar to that found in other eukaryotic transcriptional factors were identified. The two transactivation regions, when separated from the leucine-rich repeat and the C terminus of CCR4, were unable to complement a defective ccr4 allele, suggesting that the leucine-rich repeat and the C terminus make contacts that link the activation regions to the proper gene context. Native immunoprecipitation of CCR4 revealed that CCR4 was complexed with at least four other proteins. The leucine-rich repeat of CCR4 was both necessary and sufficient for interaction with at least two of these factors. We propose that the leucine-rich repeat links CCR4 through its associated factors to its promoter context at ADH2 and other loci where it is required for proper transcriptional regulation.The general transcription factor CCR4 from Saccharomyces cerevisiae is required for the transcription of a number of genes involved in nonfermentative growth, including that of the ADH2 gene (encoding the glucose-repressible alcohol dehydrogenase II) (11). Strains containing a deletion of CCR4 also fail to grow at elevated temperatures on a nonfermentative medium, consistent with the global role played by CCR4 under these growth conditions (14). Cells lacking CCR4, however, display other phenotypes, suggesting that CCR4 is involved in processes in addition to that of controlling nonfermentative growth. ccr4 mutations display a cold sensitivity phenotype under glucose growth conditions, a phenotype observed for defects in transcription initiation complex factors such as TFIIB, RPB1, SRB2, and SRB4 (1,2,29,30,33,34,37,41). In addition, CCR4 is required for the elevated expression at the ADH2 locus and for the altered transcriptional initiation at the his4-9128 locus that results from defects in the SPT6 and SPT10 genes (14). The SPT6 and SPT10 genes encode factors that are responsible for maintaining proper transcriptional control over a wide range of genes, and SPT6 has been specifically implicated in the maintenance of chromatin structure (11,26,27,39,40). spt6 mutations, moreover, suppress defects in the global transactivators SNF2 and SNF5, which are known to be involved in maintaining proper nucleosome positioning (5,6,19,28,43 known suppressor of spt6, may be functioning to counteract the repressive effects that SPT6 has on chromatin structure. CCR4 is also required for ADR...

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“…Site-directed mutagenesis has been used to probe the functions of specific active site residues in D-xylose isomerase (27)(28)(29)(30)(31)(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.…”

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

Whitaker

Cho

Cha

et al. 1995

Journal of Biological Chemistry

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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-

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Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 1. Crystallography and site-directed mutagenesis of metal binding sites

Cited by 137 publications

References 30 publications

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

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

CCR4 is a glucose-regulated transcription factor whose leucine-rich repeat binds several proteins important for placing CCR4 in its proper promoter context.

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

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