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, 96 (21), 11787-91

Active-site Structure of the Soluble Quinoprotein Glucose Dehydrogenase Complexed With Methylhydrazine: A Covalent Cofactor-Inhibitor Complex

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Active-site Structure of the Soluble Quinoprotein Glucose Dehydrogenase Complexed With Methylhydrazine: A Covalent Cofactor-Inhibitor Complex

A Oubrie et al. Proc Natl Acad Sci U S A.

Abstract

Soluble glucose dehydrogenase (s-GDH) from the bacterium Acinetobacter calcoaceticus is a classical quinoprotein. It requires the cofactor pyrroloquinoline quinone (PQQ) to catalyze the oxidation of glucose to gluconolactone. The precise catalytic role of PQQ in s-GDH and several other PQQ-dependent enzymes has remained controversial because of the absence of comprehensive structural data. We have determined the crystal structure of a ternary complex of s-GDH with PQQ and methylhydrazine, a competitive inhibitor of the enzyme. This complex, refined at 1.5-A resolution to an R factor of 16.7%, affords a detailed view of a cofactor-binding site of s-GDH. Moreover, it presents the first direct observation of covalent PQQ adduct in the active-site of a PQQ-dependent enzyme, thereby confirming previous evidence that the C5 carbonyl group of the cofactor is the most reactive moiety of PQQ.

Figures

Figure 1
Figure 1
Structure of PQQ. Atom nomenclature is indicated.
Figure 2
Figure 2
Possible reaction mechanisms for s-GDH. (A) The addition-elimination mechanism. (B) Mechanism comprising direct hydride transfer and tautomerization to PQQH2 (upper right structure).
Figure 3
Figure 3
(A) 2Fo-Fc electron density, contoured at 1.5 σ, for PQQ, refined as a completely planar molecule. (B) Density (3 σ Fo-Fc) (light grey) and −3 σ Fo-Fc density (dark grey) for planar PQQ. (C) Electron density (2Fo-Fc), contoured at 1 σ, for the final model of the carbinolamine type complex in monomer B. (D) Electron density (2Fo-Fc), contoured at 1 σ, for the final model of PQQ and noncovalently bound MH in monomer A. This picture was created with bobscript (30).
Figure 4
Figure 4
Ribbon diagram of one s-GDH monomer. PQQ and MH are shown in ball-and-stick representation. The calcium ion is shown as a large grey sphere. Oxygen, nitrogen, and carbon atoms are shown in white, grey, and black, respectively. This picture was produced with molscript (31).
Figure 5
Figure 5
Hydrogen bonding interactions in the active site of s-GDH. Distances are given in Ångstroms. Oxygen, nitrogen and carbon atoms are shown in white, grey and black, respectively. This picture was created with molscript (31).
Figure 6
Figure 6
(A) Conformation of unligated PQQ in monomer A. (B) Conformation of the C5 atom of the carbinolamine type complex in monomer B. Angles are given in degrees. Oxygen, nitrogen, and carbon atoms are shown in white, grey, and black, respectively. This picture was created with molscript (31).
Figure 7
Figure 7
Structure of the adduct of PQQ and MH. Atom nomenclature is indicated for the MH atoms and for the carbon atoms of the ortho-quinone group of PQQ.

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