Engineering of microheterogeneity-resistant p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens

K Eschrich; W J van Berkel; A H Westphal; A de Kok; A Mattevi; G Obmolova; K H Kalk; W G Hol

doi:10.1016/0014-5793(90)80843-8

Engineering of microheterogeneity-resistant p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens

FEBS Lett. 1990 Dec 17;277(1-2):197-9. doi: 10.1016/0014-5793(90)80843-8.

Authors

K Eschrich¹, W J van Berkel, A H Westphal, A de Kok, A Mattevi, G Obmolova, K H Kalk, W G Hol

Affiliation

¹ Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.

PMID: 2269354
DOI: 10.1016/0014-5793(90)80843-8

Abstract

By site-directed mutagenesis, Cys-116 was converted to Ser-116 in p-hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens. In contrast to wild-type enzyme, the C116S mutant is no longer susceptible to oxidation by hydrogen peroxide and shows no reactivity towards 5,5'-dithiobis(2-nitrobenzoate). Crystals of the C116S mutant are isomorphous with the crystal form of wild-type enzyme. A difference electron density confirms the mutation made.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

4-Hydroxybenzoate-3-Monooxygenase / antagonists & inhibitors
4-Hydroxybenzoate-3-Monooxygenase / genetics*
Base Sequence
Cloning, Molecular
DNA Mutational Analysis
Models, Molecular
Molecular Sequence Data
Oligonucleotides / chemistry
Protein Conformation
Pseudomonas / enzymology*
Sulfhydryl Reagents / pharmacology

Substances

Oligonucleotides
Sulfhydryl Reagents
4-Hydroxybenzoate-3-Monooxygenase