Highly conserved Asp-204 and Gly-776 are important for activity of the quinoprotein glucose dehydrogenase of Escherichia coli and for mineral phosphate solubilization.
Gram-negative bacteria membrane-bound glucose dehydrogenase (m-GDH) has pyrroloquinoline quinone [PQQ (2,7,9,-tricarboxyl-1H-pyrrolo[2,3-f]quinoline-4,5-dione)] as its prosthetic group, transferring electrons to ubiquinone (UQ) in the membrane. Based on the sequence homology of the C-terminal catalytic domain (151-796 amino acid residues) we have modeled the 3D structure of Escherichia coli GDH. The geometrical parameters of the homology model structure, validated using the Ramachandran plot, revealed 95.8% of residues in the allowed regions and 2.2% of the residues in disallowed regions. From the model, we have identified five different amino acids that are specifically involved in maintaining the PQQ in the correct configuration along with a Ca(2+) ion in the active site, and two amino acids on the surface of the protein that might be involved in UQ binding or transfer of electrons to the UQ. Site-directed mutants R201A, D204A, E217L, E217A, R266Q, R266E, E591L, E591Q, E591K, L712W, L712R, G776K, G776D and G776L lost their GDH activity, while E217Q and G776A retained their function similar to that of wild-type GDH, both in terms of specific activity and mineral phosphate solubilization. Our conclusions are consistent with those previously based on model GDH produced by a different method and using a different template X-ray structure.
Sashidhar
Burla
B
Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Central University, Hyderabad, India.
Inampudi
Krishna Kishore
KK
Guruprasad
Lalitha
L
Kondreddy
Anil
A
Gopinath
Kodetham
K
Podile
Appa Rao
AR
D019943:Amino Acid Substitution / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism; D017124:Conserved Sequence; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005948:Glucose Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008903:Minerals / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014451:Ubiquinone / Q000378:metabolism
D003067:Coenzymes; D029968:Escherichia coli Proteins; D008903:Minerals; D010710:Phosphates; D014451:Ubiquinone; D005948:Glucose Dehydrogenases; C033955:glucose dehydrogenase (pyrroloquinoline-quinone); D002118:Calcium