Sph3 Is a Glycoside Hydrolase Required for the Biosynthesis of Galactosaminogalactan in Aspergillus fumigatus.
Aspergillus fumigatus is the most virulent species within the Aspergillus genus and causes invasive infections with high mortality rates. The exopolysaccharide galactosaminogalactan (GAG) contributes to the virulence of A. fumigatus. A co-regulated five-gene cluster has been identified and proposed to encode the proteins required for GAG biosynthesis. One of these genes, sph3, is predicted to encode a protein belonging to the spherulin 4 family, a protein family with no known function. Construction of an sph3-deficient mutant demonstrated that the gene is necessary for GAG production. To determine the role of Sph3 in GAG biosynthesis, we determined the structure of Aspergillus clavatus Sph3 to 1.25 Å. The structure revealed a (β/α)8 fold, with similarities to glycoside hydrolase families 18, 27, and 84. Recombinant Sph3 displayed hydrolytic activity against both purified and cell wall-associated GAG. Structural and sequence alignments identified three conserved acidic residues, Asp-166, Glu-167, and Glu-222, that are located within the putative active site groove. In vitro and in vivo mutagenesis analysis demonstrated that all three residues are important for activity. Variants of Asp-166 yielded the greatest decrease in activity suggesting a role in catalysis. This work shows that Sph3 is a glycoside hydrolase essential for GAG production and defines a new glycoside hydrolase family, GH135.
From the Program in Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada, the Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Natalie C
NC
Bamford
the Departments of Microbiology and Immunology and Medicine, McGill University, Montréal, Québec H4A 3J1, Canada, and.
Brendan D
BD
Snarr
the Departments of Microbiology and Immunology and Medicine, McGill University, Montréal, Québec H4A 3J1, Canada, and.
Fabrice N
FN
Gravelat
From the Program in Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada, the Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Dustin J
DJ
Little
the Departments of Microbiology and Immunology and Medicine, McGill University, Montréal, Québec H4A 3J1, Canada, and.
Mark J
MJ
Lee
the Departments of Microbiology and Immunology and Medicine, McGill University, Montréal, Québec H4A 3J1, Canada, and.
Caitlin A
CA
Zacharias
the Departments of Microbiology and Immunology and Medicine, McGill University, Montréal, Québec H4A 3J1, Canada, and.
Josée C
JC
Chabot
the Departments of Microbiology and Immunology and Medicine, McGill University, Montréal, Québec H4A 3J1, Canada, and.
Alexander M
AM
Geller
the Departments of Microbiology and Immunology and Medicine, McGill University, Montréal, Québec H4A 3J1, Canada, and.
Stefanie D
SD
Baptista
From the Program in Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada.
Perrin
P
Baker
the Photon Sciences Division, Brookhaven National Laboratory, Upton, New York 11973-5000.
Howard
H
Robinson
From the Program in Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada, the Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada, howell@sickkids.ca.
P Lynne
PL
Howell
the Departments of Microbiology and Immunology and Medicine, McGill University, Montréal, Québec H4A 3J1, Canada, and don.sheppard@mcgill.ca.
Donald C
DC
Sheppard
D000595:Amino Acid Sequence; D001232:Aspergillus fumigatus / Q000201:enzymology / Q000378:metabolism* / Q000472:pathogenicity; D002384:Catalysis; D020134:Catalytic Domain; D002473:Cell Wall / Q000201:enzymology; D003046:Coccidioidin / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D011134:Polysaccharides / Q000096:biosynthesis* / Q000235:genetics; D011487:Protein Conformation; D016415:Sequence Alignment
D005656:Fungal Proteins; D011134:Polysaccharides; C062157:galactosaminogalactan; C009303:spherulin; D003046:Coccidioidin; D006026:Glycoside Hydrolases
Aspergillus; Sph3; biofilm; carbohydrate biosynthesis; crystal structure; galactosaminogalactan; glycoside hydrolase; in vivo imaging; mutagenesis in vitro; polysaccharide