The Crystal Structure of Galacto-N-biose/Lacto-N-biose I Phosphorylase
A LARGE DEFORMATION OF A TIM BARREL SCAFFOLD*
- Masafumi Hidaka‡§,
- Mamoru Nishimoto§,
- Motomitsu Kitaoka§,
- Takayoshi Wakagi‡,
- Hirofumi Shoun‡ and
- Shinya Fushinobu‡1
- ‡Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 and the §National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
- 1 To whom correspondence should be addressed. Tel./Fax: 81-3-5841-5151; E-mail: asfushi{at}mail.ecc.u-tokyo.ac.jp.
Abstract
Galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) from Bifidobacterium longum, a key enzyme for intestinal growth, phosphorolyses galacto-N-biose and lacto-N-biose I with anomeric inversion. GLNBP homologues are often found in human pathogenic and commensal bacteria, and their substrate specificities potentially define the nutritional acquisition ability of these microbes in their habitat. We report the crystal structures of GLNBP in five different ligand-binding forms. This is the first three-dimensional structure of glycoside hydrolase (GH) family 112. The GlcNAc- and GalNAc-bound forms provide structural insights into distinct substrate preferences of GLNBP and its homologues from pathogens. The catalytic domain consists of a partially broken TIM barrel fold that is structurally similar to a thermophilic β-galactosidase, strongly supporting the current classification of GLNBP homologues as one of the GH families. Anion binding induces a large conformational change by rotating a half-unit of the barrel. This is an unusual example of molecular adaptation of a TIM barrel scaffold to substrates.
Footnotes
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↵2 The abbreviations used are: GNB, galacto-N-biose; LNB, lacto-N-biose I; Gal-HexNAcP, β-1,3-galactosyl-N-acetylhexosamine phosphorylase; GLNBP, galacto-N-biose/lacto-N-biose I phosphorylase; GH, glycoside hydrolase; Gal1P, α-d-galactose 1-phosphate; GNBP, galacto-N-biose phosphorylase; LNBP, lacto-N-biose I phosphorylase; GT, glycosyltransferase; ASU, asymmetric unit; EG, ethylene glycol; r.m.s., root mean square; A4-β-galactosidase, β-galactosidase from T. thermophilus A4.
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The atomic coordinates and structure factors (codes 2ZUS, 2ZUT, 2ZUU, 2ZUV, and 2ZUW) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
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↵* This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and Japan Society for the Promotion of Science for Young Scientists Research Fellowship 17-00182 (to M. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2, Figs. S1-S4, and Movie S1.
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- Received November 10, 2008.
- Revision received December 19, 2008.
- The American Society for Biochemistry and Molecular Biology, Inc.
