AUTHOR=Montersino Stefania , te Poele Evelien , Orru Roberto , Westphal Adrie H. , Barendregt Arjan , Heck Albert J. R. , van der Geize Robert , Dijkhuizen Lubbert , Mattevi Andrea , van Berkel Willem J. H. 

TITLE=3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1 Contains a Phosphatidylinositol Cofactor

JOURNAL=Frontiers in Microbiology

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2017.01110

DOI=10.3389/fmicb.2017.01110

ISSN=1664-302X

ABSTRACT=<p>3-Hydroxybenzoate 6-hydroxylase (3HB6H, EC 1.13.14.26) is a FAD-dependent monooxygenase involved in the catabolism of aromatic compounds in soil microorganisms. 3HB6H is unique among flavoprotein hydroxylases in that it harbors a phospholipid ligand. The purified protein obtained from expressing the gene encoding 3HB6H from <italic>Rhodococcus jostii</italic> RHA1 in the host <italic>Escherichia coli</italic> contains a mixture of phosphatidylglycerol and phosphatidylethanolamine, which are the major constituents of <italic>E. coli’s</italic> cytoplasmic membrane. Here, we purified 3HB6H (<italic>Rj</italic>HB6H) produced in the host <italic>R. jostii</italic> RHA#2 by employing a newly developed actinomycete expression system. Biochemical and biophysical analysis revealed that <italic>Rj</italic>3HB6H possesses similar catalytic and structural features as 3HB6H, but now contains phosphatidylinositol, which is a specific constituent of actinomycete membranes. Native mass spectrometry suggests that the lipid cofactor stabilizes monomer-monomer contact. Lipid analysis of 3HB6H from <italic>Pseudomonas alcaligenes</italic> NCIMB 9867 (<italic>Pa</italic>3HB6H) produced in <italic>E. coli</italic> supports the conclusion that 3HB6H enzymes have an intrinsic ability to bind phospholipids with different specificity, reflecting the membrane composition of their bacterial host.</p>