Journal:JBIC:5
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| - | 2,3-HPCD catalyzes the cleavage of the C2-C3 bond of homoprotocatechuate (HPCA) with the incorporation of both oxygen atoms derived from O2 to form ring opened, 5-carboxymethyl-2-hydroxymuconic semialdehyde (5-CHMSA). Despite the significantly higher standard redox potential of Co(III/II) (1.92 V versus SHE) relative to that of the native metal Fe(II) (0.77 V), Co-HPCD was found to have more than twice the specific activity of Fe-HPCD under saturating O2 conditions, making Co-HPCD a hyper-active enzyme. The presence of <scene name='Journal:JBIC:5/Cobalt_4_sites/1'>Co in the active sites of Co-HPCD</scene> is illustrated by the 4 strong anomalous difference peaks determined from the X-ray diffraction data set collected at the cobalt K-edge (Figure 4A, 1.6050 ≈, 7.725 keV), which shows cobalt coordinated via <scene name='Journal:JBIC:5/Hpcd_active_site/2'>2-His-1-Carboxylate facial triad</scene>. The structural comparison of the high-resolution X-ray crystal structures of Fe-HPCD (1.70 ≈, 3OJT), Mn-HPCD (1.65 ≈, 3OJN), and Co-HCPD (1.72 ≈, 3OJJ) shows <scene name='Journal:JBIC:5/Overlays/ | + | 2,3-HPCD catalyzes the cleavage of the C2-C3 bond of homoprotocatechuate (HPCA) with the incorporation of both oxygen atoms derived from O2 to form ring opened, 5-carboxymethyl-2-hydroxymuconic semialdehyde (5-CHMSA). Despite the significantly higher standard redox potential of Co(III/II) (1.92 V versus SHE) relative to that of the native metal Fe(II) (0.77 V), Co-HPCD was found to have more than twice the specific activity of Fe-HPCD under saturating O2 conditions, making Co-HPCD a hyper-active enzyme. The presence of <scene name='Journal:JBIC:5/Cobalt_4_sites/1'>Co in the active sites of Co-HPCD</scene> is illustrated by the 4 strong anomalous difference peaks determined from the X-ray diffraction data set collected at the cobalt K-edge (Figure 4A, 1.6050 ≈, 7.725 keV), which shows cobalt coordinated via <scene name='Journal:JBIC:5/Hpcd_active_site/2'>2-His-1-Carboxylate facial triad</scene>. The structural comparison of the high-resolution X-ray crystal structures of Fe-HPCD (1.70 ≈, 3OJT), Mn-HPCD (1.65 ≈, 3OJN), and Co-HCPD (1.72 ≈, 3OJJ) shows <scene name='Journal:JBIC:5/Overlays/7'>no significant structural differences in the active site</scene> environment as indicated by the RMSD values of 0.10ñ0.14 ≈ for superposition of all atoms within 15 ≈ of the metal center (Figure 5A). In addition, the presence of Co in the active site of HPCD has no structural consequences on either the mode of substrate binding (Figure SI2A) or on the conformational integrity of the active site residues in the 1st or 2nd coordination sphere environment (Figure 5B). The absence of any observable structural differences upon metal substitutions suggests that differential redox tuning of the metal centers in this dioxygenase is highly unlikely. Rather, the 2,3-HPCD enzyme can carry out the O2 activation and oxidative ring-cleavage efficiently over a large range of metal redox potentials. The structural analysis supports the proposed mechanism described above in which the ability of the enzyme to activate molecular O2 does not correlate with redox potential of the metal center. However, the current results also show that the rates of individual steps in the overall catalytic cycle can be effected by the metal present in the active site of HPCD, resulting in a earlier rate-limiting step for Co-HPCD compared to Fe-HPCD and Mn-HPCD. |
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Revision as of 15:23, 23 November 2010
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A Hyperactive Cobalt-Substituted Extradiol-Cleaving Catechol Dioxygenase
Fielding
Molecular Tour
2,3-HPCD catalyzes the cleavage of the C2-C3 bond of homoprotocatechuate (HPCA) with the incorporation of both oxygen atoms derived from O2 to form ring opened, 5-carboxymethyl-2-hydroxymuconic semialdehyde (5-CHMSA). Despite the significantly higher standard redox potential of Co(III/II) (1.92 V versus SHE) relative to that of the native metal Fe(II) (0.77 V), Co-HPCD was found to have more than twice the specific activity of Fe-HPCD under saturating O2 conditions, making Co-HPCD a hyper-active enzyme. The presence of is illustrated by the 4 strong anomalous difference peaks determined from the X-ray diffraction data set collected at the cobalt K-edge (Figure 4A, 1.6050 ≈, 7.725 keV), which shows cobalt coordinated via . The structural comparison of the high-resolution X-ray crystal structures of Fe-HPCD (1.70 ≈, 3OJT), Mn-HPCD (1.65 ≈, 3OJN), and Co-HCPD (1.72 ≈, 3OJJ) shows environment as indicated by the RMSD values of 0.10ñ0.14 ≈ for superposition of all atoms within 15 ≈ of the metal center (Figure 5A). In addition, the presence of Co in the active site of HPCD has no structural consequences on either the mode of substrate binding (Figure SI2A) or on the conformational integrity of the active site residues in the 1st or 2nd coordination sphere environment (Figure 5B). The absence of any observable structural differences upon metal substitutions suggests that differential redox tuning of the metal centers in this dioxygenase is highly unlikely. Rather, the 2,3-HPCD enzyme can carry out the O2 activation and oxidative ring-cleavage efficiently over a large range of metal redox potentials. The structural analysis supports the proposed mechanism described above in which the ability of the enzyme to activate molecular O2 does not correlate with redox potential of the metal center. However, the current results also show that the rates of individual steps in the overall catalytic cycle can be effected by the metal present in the active site of HPCD, resulting in a earlier rate-limiting step for Co-HPCD compared to Fe-HPCD and Mn-HPCD.
