Structure and mechanism of GDP-mannose glycosyl hydrolase, a Nudix enzyme that cleaves at carbon instead of phosphorus.
GDP-mannose glycosyl hydrolase (GDPMH) catalyzes the hydrolysis of GDP-mannose and GDP-glucose to GDP and sugar by substitution with inversion at C1 of the sugar. The enzyme has a modified Nudix motif and requires one divalent cation for activity. The 1.3 A X-ray structure of the GDPMH-Mg(2+)-GDP complex, together with kinetic, mutational, and NMR data, suggests a mechanism for the GDPMH reaction. Several residues and the divalent cation strongly promote the departure of the GDP leaving group, supporting a dissociative mechanism. Comparison of the GDPMH structure with that of a typical Nudix hydrolase suggests how sequence changes result in the switch of catalytic activity from P-O bond cleavage to C-O bond cleavage. Changes in the Nudix motif result in loss of binding of at least one Mg(2+) ion, and shortening of a loop by 6 residues shifts the catalytic base by approximately 10 A.
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
Sandra B
SB
Gabelli
Mario A
MA
Bianchet
Hugo F
HF
Azurmendi
Zuyong
Z
Xia
Vibhor
V
Sarawat
Albert S
AS
Mildvan
L Mario
LM
Amzel
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002244:Carbon / Q000737:chemistry; D002384:Catalysis; D002412:Cations; D018360:Crystallography, X-Ray; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry; D006155:Guanosine Diphosphate Mannose / Q000737:chemistry*; D006868:Hydrolysis; D007477:Ions; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008358:Mannose / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000737:chemistry*; D010758:Phosphorus / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D002412:Cations; D007477:Ions; D006153:Guanosine Diphosphate; D010758:Phosphorus; D006155:Guanosine Diphosphate Mannose; D002244:Carbon; D009699:N-Glycosyl Hydrolases; D008274:Magnesium; D008358:Mannose