2ecr
From Proteopedia
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Crystal structure of the ligand-free form of the flavin reductase component (HpaC) of 4-hydroxyphenylacetate 3-monooxygenase
Overview
The two-component enzyme, 4-hydroxyphenylacetate 3-monooxygenase, catalyzes the conversion of 4-hydroxyphenylacetate to, 3,4-dihydroxyphenylacetate. In the overall reaction, the oxygenase, component (HpaB) introduces a hydroxyl group into the benzene ring of, 4-hydroxyphenylacetate using molecular oxygen and reduced flavin, while, the reductase component (HpaC) provides free reduced flavins for HpaB. The, crystal structures of HpaC from Thermus thermophilus HB8 in the, ligand-free form, the FAD-containing form, and the ternary complex with, FAD and NAD(+) were determined. In the ligand-free form, two large grooves, are present at the dimer interface, and are occupied by water molecules. A, structural analysis of HpaC containing FAD revealed that FAD has a low, occupancy, indicating that it is not tightly bound to HpaC. This was, further confirmed in flavin dissociation experiments, showing that FAD can, be released from HpaC. The structure of the ternary complex revealed that, FAD and NAD(+) are bound in the groove in the extended and folded, conformation, respectively. The nicotinamide ring of NAD(+) is sandwiched, between the adenine ring of NAD(+) and the isoalloxazine ring of FAD. The, distance between N5 of the isoalloxazine ring and C4 of the nicotinamide, ring is about 3.3 A, sufficient to permit hydride transfer. The structures, of these three states are essentially identical, however, the side chains, of several residues show small conformational changes, indicating an, induced fit upon binding of NADH. Inactivity with respect to NADPH can be, explained as instability of the binding of NADPH with the negatively, charged 2'-phosphate group buried inside the complex, as well as a, possible repulsive effect by the dipole of helix alpha1. A comparison of, the binding mode of FAD with that in PheA2 from Bacillus, thermoglucosidasius A7, which contains FAD as a prosthetic group, reveals, remarkable conformational differences in a less conserved loop region, (Gly83-Gly94) involved in the binding of the AMP moiety of FAD. These data, suggest that variations in the affinities for FAD in the reductases of the, two-component flavin-diffusible monooxygenase family may be attributed to, difference in the interaction between the AMP moiety of FAD and the less, conserved loop region which possibly shows structural divergence. Proteins, 2008. (c) 2007 Wiley-Liss, Inc.
About this Structure
2ECR is a Single protein structure of sequence from Thermus thermophilus. Full crystallographic information is available from OCA.
Reference
Crystal structure of the flavin reductase component (HpaC) of 4-hydroxyphenylacetate 3-monooxygenase from Thermus thermophilus HB8: Structural basis for the flavin affinity., Kim SH, Hisano T, Iwasaki W, Ebihara A, Miki K, Proteins. 2007 Aug 29;70(3):718-730. PMID:17729270
Page seeded by OCA on Wed Jan 23 11:05:25 2008
