TY - JOUR
T1 - A crystal structure of 2-hydroxybiphenyl 3-monooxygenase with bound substrate provides insights into the enzymatic mechanism
AU - Kanteev, Margarita
AU - Bregman-Cohen, Almog
AU - Deri, Batel
AU - Shahar, Anat
AU - Adir, Noam
AU - Fishman, Ayelet
N1 - Publisher Copyright:
© 2015 Elsevier B.V. All rights reserved.
PY - 2015/12/1
Y1 - 2015/12/1
N2 - 2-Hydroxybiphenyl 3-monooxygenase (HbpA) is an FAD dependent monooxygenase which catalyzes the ortho-hydroxylation of a broad range of 2-substituted phenols in the presence of NADH and molecular oxygen. We have determined the structure of HbpA from the soil bacterium Pseudomonas azelaica HBP1 with bound 2-hydroxybiphenyl, as well as several variants, at a resolution of 2.3-2.5 Å to investigate structure function correlations of the enzyme. An observed hydrogen bond between 2-hydroxybiphenyl and His48 in the active site confirmed the previously suggested role of this residue in substrate deprotonation. The entrance to the active site was confirmed by generating variant G255F which exhibited only 7% of the wild-type's specific activity of product formation, suggesting inhibition of substrate entrance into the active site by the large aromatic residue. Residue Arg242 is suggested to facilitate FAD movement and reduction as was previously reported in studies on the homologous protein para-hydroxybenzoate hydroxylase. In addition, it is suggested that Trp225, which is located in the active site, facilitates proper substrate entrance into the binding pocket in contrast to aklavinone-11-hydroxylase and para-hydroxybenzoate hydroxylase in which a residue at a similar position is responsible for substrate deprotonation. Structure function correlations described in this work will aid in the design of variants with improved activity and altered selectivity for potential industrial applications.
AB - 2-Hydroxybiphenyl 3-monooxygenase (HbpA) is an FAD dependent monooxygenase which catalyzes the ortho-hydroxylation of a broad range of 2-substituted phenols in the presence of NADH and molecular oxygen. We have determined the structure of HbpA from the soil bacterium Pseudomonas azelaica HBP1 with bound 2-hydroxybiphenyl, as well as several variants, at a resolution of 2.3-2.5 Å to investigate structure function correlations of the enzyme. An observed hydrogen bond between 2-hydroxybiphenyl and His48 in the active site confirmed the previously suggested role of this residue in substrate deprotonation. The entrance to the active site was confirmed by generating variant G255F which exhibited only 7% of the wild-type's specific activity of product formation, suggesting inhibition of substrate entrance into the active site by the large aromatic residue. Residue Arg242 is suggested to facilitate FAD movement and reduction as was previously reported in studies on the homologous protein para-hydroxybenzoate hydroxylase. In addition, it is suggested that Trp225, which is located in the active site, facilitates proper substrate entrance into the binding pocket in contrast to aklavinone-11-hydroxylase and para-hydroxybenzoate hydroxylase in which a residue at a similar position is responsible for substrate deprotonation. Structure function correlations described in this work will aid in the design of variants with improved activity and altered selectivity for potential industrial applications.
KW - Abbreviations HbpA 2-Hydroxybiphenyl 3-monooxygenase
UR - http://www.scopus.com/inward/record.url?scp=84944213243&partnerID=8YFLogxK
U2 - 10.1016/j.bbapap.2015.08.002
DO - 10.1016/j.bbapap.2015.08.002
M3 - Article
C2 - 26275805
AN - SCOPUS:84944213243
SN - 1570-9639
VL - 1854
SP - 1906
EP - 1913
JO - Biochimica et Biophysica Acta - Proteins and Proteomics
JF - Biochimica et Biophysica Acta - Proteins and Proteomics
IS - 12
ER -