Abstract
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 Rhodococcus jostii RHA1 in the host Escherichia coli contains a mixture of phosphatidylglycerol and phosphatidylethanolamine, which are the major constituents of E. coli’s cytoplasmic membrane. Here, we purified 3HB6H (RjHB6H) produced in the host R. jostii RHA#2 by employing a newly developed actinomycete expression system. Biochemical and biophysical analysis revealed that Rj3HB6H 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 Pseudomonas alcaligenes NCIMB 9867 (Pa3HB6H) produced in E. coli supports the conclusion that 3HB6H enzymes have an intrinsic ability to bind phospholipids with different specificity, reflecting the membrane composition of their bacterial host.
Highlights
Rhodococcus jostii RHA1 is a biotechnologically and environmentally important bacterium from the order Actinomycetales
The crystal structure showed that the inositol headgroups of the phospholipids are located at the protein surface, and that the sn-2 acyl moieties are in contact with helix 11 of the other subunit (Figure 1)
Based on MS/MS analysis, we identified the bound phospholipids as a mixture of PIs with carbon chains between 15 and 19 carbons
Summary
Rhodococcus jostii RHA1 is a biotechnologically and environmentally important bacterium from the order Actinomycetales. 3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1 cell envelope (Sutcliffe, 1998; Guerin et al, 2010; De Carvalho et al, 2014) and an impressive catabolic diversity, allowing adaptation to different carbon sources for growth (van der Geize and Dijkhuizen, 2004). We recently reported the crystal structure of R. jostii RHA1 3-hydroxybenzoate 6-hydroxylase (3HB6H), produced as a recombinant protein in Escherichia coli (Montersino et al, 2013). The crystal structure analysis revealed that 3HB6H has the conserved fold of group A flavoprotein hydroxylases (Montersino et al, 2011; Huijbers et al, 2014), but differs from the other family members in additional binding of phospholipids. The fatty acyl chains of the phospholipid ligands of 3HB6H protrude into the substrate-binding pockets, whereas the surface-exposed hydrophilic headgroups are involved in enzyme dimerization (Figure 1) (Montersino et al, 2013)
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