Engineering of microheterogeneity-resistant p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.
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.
Eschrich
K
K
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
van Berkel
W J
WJ
Westphal
A H
AH
de Kok
A
A
Mattevi
A
A
Obmolova
G
G
Kalk
K H
KH
Hol
W G
WG
D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000037:antagonists & inhibitors / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D009841:Oligonucleotides; D013439:Sulfhydryl Reagents; D015109:4-Hydroxybenzoate-3-Monooxygenase