Direct evidence for Sphingomonas sp. A1 periplasmic proteins as macromolecule-binding proteins associated with the ABC transporter: molecular insights into alginate transport in the periplasm.
A Gram-negative bacterium, Sphingomonas sp. A1, has a macromolecule (alginate) import system consisting of a pit on the cell surface and an alginate-specific ATP-binding cassette importer in the inner membrane. Transport of alginate from the pit to the ABC importer is probably mediated by two periplasmic binding protein homologues (AlgQ1 and AlgQ2). Here we describe characteristics of binding of AlgQ1 and AlgQ2 to alginate and its oligosaccharides through surface plasmon resonance biosensor analysis, UV absorption difference spectroscopy, and X-ray crystallography. Both AlgQ1 and AlgQ2 were inducibly expressed in the periplasm of alginate-grown cells of strain A1. Biosensor analysis indicated that both proteins specifically bind alginate with a high degree of polymerization (>100) and that dissociation constants for alginate with an average molecular mass of 26 kDa are 2.3 x 10(-)(7) M for AlgQ1 and 1.5 x 10(-)(7) M for AlgQ2. An in vitro ATPase assay using the membrane complex, including the alginate ABC importer, suggested that both alginate-bound forms of AlgQ1 and AlgQ2 are closely associated with the importer. X-ray crystallography showed that AlgQ1 consisted of two domains separated by a deep cleft that binds alginate oligosaccharides through a conformational change in the two domains. These results directly show that alginate-binding proteins play an important role in the efficient transport of alginate macromolecules with different degrees of polymerization in the periplasm.
Momma
Keiko
K
Laboratory of Basic and Applied Molecular Biotechnology, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Mishima
Yumiko
Y
Hashimoto
Wataru
W
Mikami
Bunzo
B
Murata
Kousaku
K
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000464:Alginates / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001693:Biological Transport, Active; D018360:Crystallography, X-Ray; D015870:Gene Expression; D005798:Genes, Bacterial; D020723:Glucuronic Acid / Q000737:chemistry / Q000378:metabolism*; D006603:Hexuronic Acids / Q000737:chemistry / Q000378:metabolism*; D006860:Hydrogen Bonding; D046911:Macromolecular Substances; D008958:Models, Molecular; D019897:Periplasm / Q000378:metabolism; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020578:Sphingomonas / Q000235:genetics / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D000464:Alginates; D001426:Bacterial Proteins; D006603:Hexuronic Acids; D046911:Macromolecular Substances; D033902:Periplasmic Binding Proteins; D011994:Recombinant Proteins; D020723:Glucuronic Acid