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Phe161 and Arg166 variants of p-hydroxybenzoate hydroxylase. Implications for NADPH recognition and structural stability.

Eppink MH, Bunthol C, Schreuder HA, van Berkel WJ
FEBS Lett 443 251-5 (1999)
Related entries: 1cc4, 1pbb, 1pbc, 1pbd, 1pbe, 1pbf, 1pdh, 1phh, 2phh

Cited: 14 times
EuropePMC logo PMID: 10025942

Abstract

Phe161 and Arg166 of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens belong to a newly discovered sequence motif in flavoprotein hydroxylases with a putative dual function in FAD and NADPH binding [1]. To study their role in more detail, Phe161 and Arg166 were selectively changed by site-directed mutagenesis. F161A and F161G are catalytically competent enzymes having a rather poor affinity for NADPH. The catalytic properties of R166K are similar to those of the native enzyme. R166S and R166E show impaired NADPH binding and R166E has lost the ability to bind FAD. The crystal structure of substrate complexed F161A at 2.2 A is indistinguishable from the native enzyme, except for small changes at the site of mutation. The crystal structure of substrate complexed R166S at 2.0 A revealed that Arg166 is important for providing an intimate contact between the FAD binding domain and a long excursion of the substrate binding domain. It is proposed that this interaction is essential for structural stability and for the recognition of the pyrophosphate moiety of NADPH.

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Articles citing this publication (12)

  1. Protein and ligand dynamics in 4-hydroxybenzoate hydroxylase. Wang J, Ortiz-Maldonado M, Entsch B, Massey V, Ballou D, Gatti DL. Proc Natl Acad Sci U S A 99 608-613 (2002)
  2. Gene cluster on pAO1 of Arthrobacter nicotinovorans involved in degradation of the plant alkaloid nicotine: cloning, purification, and characterization of 2,6-dihydroxypyridine 3-hydroxylase. Baitsch D, Sandu C, Brandsch R, Igloi GL. J Bacteriol 183 5262-5267 (2001)
  3. Switch of coenzyme specificity of p-hydroxybenzoate hydroxylase. Eppink MH, Overkamp KM, Schreuder HA, Van Berkel WJ. J Mol Biol 292 87-96 (1999)
  4. Structure and ligand binding properties of the epoxidase component of styrene monooxygenase . Ukaegbu UE, Kantz A, Beaton M, Gassner GT, Rosenzweig AC. Biochemistry 49 1678-1688 (2010)
  5. Structure of 2,6-dihydroxypyridine 3-hydroxylase from a nicotine-degrading pathway. Treiber N, Schulz GE. J Mol Biol 379 94-104 (2008)
  6. PqsL uses reduced flavin to produce 2-hydroxylaminobenzoylacetate, a preferred PqsBC substrate in alkyl quinolone biosynthesis in Pseudomonas aeruginosa. Drees SL, Ernst S, Belviso BD, Jagmann N, Hennecke U, Fetzner S. J Biol Chem 293 9345-9357 (2018)
  7. Arg169 is essential for catalytic activity of 3-hydroxybenzoate 6-hydroxylase from Klebsiella pneumoniae M5a1. Liu DQ, Liu H, Gao XL, Leak DJ, Zhou NY. Microbiol Res 160 53-59 (2005)
  8. Conserved and non-conserved residues and their role in the structure and function of p-hydroxybenzoate hydroxylase. Suemori A. Protein Eng Des Sel 26 479-488 (2013)
  9. The Affymetrix DMET Plus platform reveals unique distribution of ADME-related variants in ethnic Arabs. Wakil SM, Nguyen C, Muiya NP, Andres E, Lykowska-Tarnowska A, Baz B, Tahir AI, Meyer BF, Morahan G, Dzimiri N. Dis Markers 2015 542543 (2015)
  10. A 4-hydroxybenzoate 3-hydroxylase mutant enables 4-amino-3-hydroxybenzoic acid production from glucose in Corynebacterium glutamicum. Nonaka K, Osamura T, Takahashi F. Microb Cell Fact 22 168 (2023)
  11. Functional and structural characterization of a flavoprotein monooxygenase essential for biogenesis of tryptophylquinone cofactor. Oozeki T, Nakai T, Kozakai K, Okamoto K, Kuroda S, Kobayashi K, Tanizawa K, Okajima T. Nat Commun 12 933 (2021)
  12. Tetracycline-modifying enzyme SmTetX from Stenotrophomonas maltophilia. Malý M, Kolenko P, Stránský J, Švecová L, Dušková J, Koval' T, Skálová T, Trundová M, Adámková K, Černý J, Božíková P, Dohnálek J. Acta Crystallogr F Struct Biol Commun 79 180-192 (2023)


Related citations provided by authors (9)

  1. Crystal structure of p-hydroxybenzoate hydroxylase reconstituted with the modified FAD present in alcohol oxidase from methylotrophic yeasts: evidence for an arabinoflavin.. van Berkel WJ, Eppink MH, Schreuder HA Protein Sci 3 2245-53 (1994)
  2. Crystal structures of wild-type p-hydroxybenzoate hydroxylase complexed with 4-aminobenzoate,2,4-dihydroxybenzoate, and 2-hydroxy-4-aminobenzoate and of the Tyr222Ala mutant complexed with 2-hydroxy-4-aminobenzoate. Evidence for a proton channel and a new binding mode of the flavin ring.. Schreuder HA, Mattevi A, Obmolova G, Kalk KH, Hol WG, van der Bolt FJ, van Berkel WJ Biochemistry 33 10161-70 (1994)
  3. Crystal structure of the reduced form of p-hydroxybenzoate hydroxylase refined at 2.3 A resolution.. Schreuder HA, van der Laan JM, Swarte MB, Kalk KH, Hol WG, Drenth J Proteins 14 178-90 (1992)
  4. Engineering of microheterogeneity-resistant p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.. Eschrich K, van Berkel WJ, Westphal AH, de Kok A, Mattevi A, Obmolova G, Kalk KH, Hol WG FEBS Lett 277 197-9 (1990)
  5. Crystal structure of the p-hydroxybenzoate hydroxylase-substrate complex refined at 1.9 A resolution. Analysis of the enzyme-substrate and enzyme-product complexes.. Schreuder HA, Prick PA, Wierenga RK, Vriend G, Wilson KS, Hol WG, Drenth J J Mol Biol 208 679-96 (1989)
  6. The coenzyme analogue adenosine 5-diphosphoribose displaces FAD in the active site of p-hydroxybenzoate hydroxylase. An x-ray crystallographic investigation.. van der Laan JM, Schreuder HA, Swarte MB, Wierenga RK, Kalk KH, Hol WG, Drenth J Biochemistry 28 7199-205 (1989)
  7. Crystal structure of p-hydroxybenzoate hydroxylase complexed with its reaction product 3,4-dihydroxybenzoate.. Schreuder HA, van der Laan JM, Hol WG, Drenth J J Mol Biol 199 637-48 (1988)
  8. Crystal structure of p-hydroxybenzoate hydroxylase.. Wierenga RK, de Jong RJ, Kalk KH, Hol WG, Drenth J J Mol Biol 131 55-73 (1979)
  9. Crystallization and preliminary x-ray investigation of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.. Drenth J, Hol WG, Wierenga RK J Biol Chem 250 5268-9 (1975)

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