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Removal of a methyl group causes global changes in p-hydroxybenzoate hydroxylase.
Biochemistry. 2005 Jun 07; 44(22):8047-58.B

Abstract

p-Hydroxybenzoate hydroxylase (PHBH) is a homodimeric flavoprotein monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate to form 3,4-dihydroxybenzoate. Controlled catalysis is achieved by movement of the flavin and protein between three conformations, in, out, and open [Entsch, B., et al. (2005) Arch. Biochem. Biophys. 433, 297-311]. The open conformation is important for substrate binding and product release, the in conformation for reaction with oxygen and hydroxylation, and the out conformation for the reduction of FAD by NADPH. The open conformation is similar to the structure of Arg220Gln-PHBH in which the backbone peptide loop of residues 43-46, located on the si side of the flavin, is rotated. In this paper, we examine the structure and properties of the Ala45Gly-PHBH mutant enzyme. The crystal structure of the Ala45Gly enzyme is an asymmetric dimer, with one monomer similar (but not identical) to wild-type PHBH, while the other monomer has His72 flipped into solvent and replaced with Glu73 as one of several changes in the structure. The two structures correlate with evidence from kinetic studies for two forms of Ala45Gly-PHBH. One form of the enzyme dominates turnover and hydroxylates, while the other contributes little to turnover and fails to hydroxylate. Ala45Gly-PHBH favors the in conformation over alternative conformations. The effect of this mutation on the structure and function of PHBH illustrates the importance of the si side loop in the conformational state of PHBH and, consequently, the function of the enzyme. This work demonstrates some general principles of how enzymes use conformational movements to allow both access and egress of substrates and product, while restricting access to the solvent at a critical stage in catalysis.

Authors+Show Affiliations

Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

15924424

Citation

Cole, Lindsay J., et al. "Removal of a Methyl Group Causes Global Changes in P-hydroxybenzoate Hydroxylase." Biochemistry, vol. 44, no. 22, 2005, pp. 8047-58.
Cole LJ, Gatti DL, Entsch B, et al. Removal of a methyl group causes global changes in p-hydroxybenzoate hydroxylase. Biochemistry. 2005;44(22):8047-58.
Cole, L. J., Gatti, D. L., Entsch, B., & Ballou, D. P. (2005). Removal of a methyl group causes global changes in p-hydroxybenzoate hydroxylase. Biochemistry, 44(22), 8047-58.
Cole LJ, et al. Removal of a Methyl Group Causes Global Changes in P-hydroxybenzoate Hydroxylase. Biochemistry. 2005 Jun 7;44(22):8047-58. PubMed PMID: 15924424.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Removal of a methyl group causes global changes in p-hydroxybenzoate hydroxylase. AU - Cole,Lindsay J, AU - Gatti,Domenico L, AU - Entsch,Barrie, AU - Ballou,David P, PY - 2005/6/1/pubmed PY - 2005/8/27/medline PY - 2005/6/1/entrez SP - 8047 EP - 58 JF - Biochemistry JO - Biochemistry VL - 44 IS - 22 N2 - p-Hydroxybenzoate hydroxylase (PHBH) is a homodimeric flavoprotein monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate to form 3,4-dihydroxybenzoate. Controlled catalysis is achieved by movement of the flavin and protein between three conformations, in, out, and open [Entsch, B., et al. (2005) Arch. Biochem. Biophys. 433, 297-311]. The open conformation is important for substrate binding and product release, the in conformation for reaction with oxygen and hydroxylation, and the out conformation for the reduction of FAD by NADPH. The open conformation is similar to the structure of Arg220Gln-PHBH in which the backbone peptide loop of residues 43-46, located on the si side of the flavin, is rotated. In this paper, we examine the structure and properties of the Ala45Gly-PHBH mutant enzyme. The crystal structure of the Ala45Gly enzyme is an asymmetric dimer, with one monomer similar (but not identical) to wild-type PHBH, while the other monomer has His72 flipped into solvent and replaced with Glu73 as one of several changes in the structure. The two structures correlate with evidence from kinetic studies for two forms of Ala45Gly-PHBH. One form of the enzyme dominates turnover and hydroxylates, while the other contributes little to turnover and fails to hydroxylate. Ala45Gly-PHBH favors the in conformation over alternative conformations. The effect of this mutation on the structure and function of PHBH illustrates the importance of the si side loop in the conformational state of PHBH and, consequently, the function of the enzyme. This work demonstrates some general principles of how enzymes use conformational movements to allow both access and egress of substrates and product, while restricting access to the solvent at a critical stage in catalysis. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/15924424/Removal_of_a_methyl_group_causes_global_changes_in_p_hydroxybenzoate_hydroxylase_ L2 - https://doi.org/10.1021/bi050108x DB - PRIME DP - Unbound Medicine ER -