Trimeric crystal structure of the glycoside hydrolase family 42 β-galactosidase from Thermus thermophilus A4 and the structure of its complex with galactose

Masafumi Hidaka, Shinya Fushinobu, Naomi Ohtsu, Hidemasa Motoshima, Hiroshi Matsuzawa, Hirofumi Shoun, Takayoshi Wakagi

Research output: Contribution to journalArticle

98 Citations (Scopus)

Abstract

The β-galactosidase from an extreme thermophile, Thermus thermophilus A4 (A4-β-Gal), is thermostable and belongs to the glycoside hydrolase family 42 (GH-42). As the first known structures of a GH-42 enzyme, we determined the crystal structures of free and galactose-bound A4-β-Gal at 1.6 Å and 2.2 Å resolution, respectively. A4-β-Gal forms a homotrimeric structure resembling a flowerpot. Each monomer has an active site located inside a large central tunnel. The N-terminal domain of A4-β-Gal has a TIM barrel fold, as predicted from hydrophobic cluster analysis. The putative catalytic residues of A4-β-Gal (Glu141 and Glu312) superimpose well with the catalytic residues of Escherichia coli β-galactosidase. The environment around the catalytic nucleophile (Glu312) is similar to that in the case of E. coli β-galactosidase, but the recognition mechanism for a substrate is different. Trp182 of the next subunit of the trimer constitutes a part of the active-site pocket, indicating that the trimeric structure is essential for the enzyme activity. Structural comparison with other glycoside hydrolases revealed that many features of the 4/7 superfamily are conserved in the A4-β-Gal structure. On the basis of the results of 1H NMR spectroscopy, A4-β-Gal was determined to be a "retaining" enzyme. Interestingly, the active site was similar with those of retaining enzymes, but the overall fold of the TIM barrel domain was very similar to that of an inverting enzyme, β-amylase.

Original languageEnglish
Pages (from-to)79-91
Number of pages13
JournalJournal of Molecular Biology
Volume322
Issue number1
DOIs
Publication statusPublished - 2002 Jan 1
Externally publishedYes

Keywords

  • Glycoside hydrolase family 42
  • TIM barrel
  • Trimer
  • X-ray crystallography
  • β-galactosidase

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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