Abstract:
:D-Xylose isomerase (XI) is a heat-stable homotetrameric enzyme used in industry for the production of high-fructose corn syrups by isomerization of D-glucose into D-fructose. To carry out biochemical and structural studies of this enzyme and of its engineered variants, a rapid and convenient method of purification of recombinant Actinoplanes missouriensis XI produced in Escherichia coli has been developed. The availability of surface-accessible histidine residues allows adsorption of XI to immobilized metal-affinity chromatography (IMAC) columns. Knowledge of the physicochemical properties of this enzyme is shown to further warrant rational modifications in the composition of the chromatographic solvents so as to achieve high selectivity in both its interaction with and its elution from a copper-loaded Chelating Sepharose Fast Flow column, an agarose-based matrix derivatized with iminodiacetic acid (IDA) groups. Purification of XI to homogeneity can thus be accomplished in a single chromatographic step starting from crude cell lysates. IDA-Cu(II)-IMAC proves convenient, fast, and reproducible. Moreover, this method is gentle to and hence suitable for mutant enzymes with decreased stability. Its disadvantage is that XI is purified in an inactive form due to inhibition by scavenged Cu2+. This handicap is however easily overcome by means of a polishing step by chromatography on Mono-Q in the presence of the chelator, EDTA. Site-directed mutants have been constructed to assess the role of surface amino acid residues in the IMA recognition event. Substitution of lysine for histidine 41 results in a mutant with near wild-type properties. Yet, this mutation is shown to completely abolish adsorption to IDA-Cu(II). This finding is analyzed in relation to the structural surface properties of the XI enzyme to provide direct evidence for the implication of histidine 41 as the predominant protein ligand to IDA-Cu(II) in IMAC.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Mrabet NTdoi
10.1021/bi00125a009subject
Has Abstractpub_date
1992-03-17 00:00:00pages
2690-702issue
10eissn
0006-2960issn
1520-4995journal_volume
31pub_type
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