Studies on the structure-function relationship of the HNK-1 associated glucuronyltransferase, GlcAT-P, by computer modeling and site-directed mutagenesis.
All members of a glucuronyltransferase (GlcAT) gene family cloned to date contain four conserved regions (modules I-IV), which are widely located in the catalytic domain. In order to understand the biological significance of these modules, we investigated the structure-function relationship of GlcAT-P by means of the combination of site-directed mutagenesis and computer aided three-dimensional modeling. The wild-type and mutant GlcAT-Ps were expressed in Escherichia coli as glutathione-S-transferase (GST)-fused soluble proteins. Most of the mutants in which a polar amino acid within the modules was replaced with alanine lost their transferase activity almost completely, while all of the mutants in which the replacement was outside these modules retained the original catalytic activity. A three-dimensional (3-D) model of GlcAT-P was constructed by computer simulation with the three-dimensional structure of adenylate kinase (1AKE) as a template. This model predicted that the large catalytic domain of GlcAT-P forms a globular shape with a Rossmann-fold motif consisting of five alpha-helix and beta-sheet repeats. The putative catalytic pocket consisting mainly of modules I-III is surrounded by a cluster of polar amino acids, which are essential for the transferase activity and also for the binding to the acceptor substrate (essential amino acids), asialo-orosomucoid. There is the second cluster of essential amino acids almost on the opposite surface of the molecule, in which an aspartic acid repeat (DDD) is located. The biological significance of the second cluster is currently not clear but it may be associated with the interaction of the enzyme with modulation molecules, manganese and membrane phospholipids.
Ohtsubo
K
K
Department of Biological Chemistry and CREST (Core Research for Educational Science and Technology) Project, Japan Science and Technology Corporation, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
Imajo
S
S
Ishiguro
M
M
Nakatani
T
T
Oka
S
S
Kawasaki
T
T
D000595:Amino Acid Sequence; D001212:Asialoglycoproteins / Q000737:chemistry; D018959:CD57 Antigens / Q000737:chemistry*; D020134:Catalytic Domain; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009961:Orosomucoid / Q000031:analogs & derivatives* / Q000737:chemistry; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship
D001212:Asialoglycoproteins; D018959:CD57 Antigens; D009961:Orosomucoid; D011993:Recombinant Fusion Proteins; C017030:asialoorosomucoid; C111364:galactosylgalactoylxylosylprotein 3-beta-glucuronosyltransferase; D014453:Glucuronosyltransferase; D005982:Glutathione Transferase