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P-HYDROXYBENZOATE HYDROXYLASE (PHBH) MUTANT WITH CYS 116 REPLACED BY SER (C116S) AND ARG 42 REPLACED BY LYS (R42K), IN COMPLEX WITH FAD AND 4-HYDROXYBENZOIC ACIDP-HYDROXYBENZOATE HYDROXYLASE (PHBH) MUTANT WITH CYS 116 REPLACED BY SER (C116S) AND ARG 42 REPLACED BY LYS (R42K), IN COMPLEX WITH FAD AND 4-HYDROXYBENZOIC ACID
Structural highlights
Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedThe conserved Arg42 of the flavoprotein p-hydroxybenzoate hydroxylase is located at the entrance of the active site in a loop between helix H2 and sheet E1 of the FAD-binding domain. Replacement of Arg42 by Lys or Ser decreases the turnover rate of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens by more than two orders of magnitude. Rapid reaction kinetics show that the low activity of the Arg42 variants results from impaired binding of NADPH. In contrast to an earlier conclusion drawn for p-hydroxybenzoate hydroxylase from Acinetobacter calcoaceticus, substitution of Arg42 with Ser42 in the enzyme from P. fluorescens hardly disturbs the binding of FAD. Crystals of [Lys42]p-hydroxybenzoate hydroxylase complexed with 4-hydroxybenzoate diffract to 0.22-nm resolution. The structure of the Lys42 variant is virtually indistinguishable from the native enzyme with the flavin ring occupying the interior position within the active site. Lys42 in the mutant structure interacts indirectly via a solvent molecule with the 3-OH of the adenosine ribose moiety of FAD. Substrate perturbation difference spectra suggest that the Arg42 replacements influence the solvent accessibility of the flavin ring in the oxidized enzyme. In spite of this, the Arg42 variants fully couple enzyme reduction to substrate hydroxylation. Sequence-comparison studies suggest that Arg42 is involved in binding of the 2'-phosphoadenosine moiety of NADPH. Lys42 and Ser42 variants of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens reveal that Arg42 is essential for NADPH binding.,Eppink MH, Schreuder HA, van Berkel WJ Eur J Biochem. 1998 Apr 1;253(1):194-201. PMID:9578477[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences |
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