3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1 Contains a Phosphatidylinositol Cofactor
Stefania Montersino1, Evelien Te Poele2, Roberto Orru3
1Laboratory of Biochemistry, Wageningen University and ResearchWageningen, Netherlands.
Frontiers in Microbiology
|July 4, 2017
Summary
3-Hydroxybenzoate 6-hydroxylase (3HB6H) enzymes bind phospholipids, with specificity reflecting their host bacterium's membrane. This lipid cofactor stabilizes the enzyme's structure, impacting aromatic compound catabolism in soil microbes.
Area of Science:
- Biochemistry
- Microbiology
- Enzymology
Background:
- 3-Hydroxybenzoate 6-hydroxylase (3HB6H) is a FAD-dependent monooxygenase crucial for aromatic compound breakdown in soil microorganisms.
- Uniquely, 3HB6H binds a phospholipid ligand, influencing its function.
- Previous studies showed 3HB6H from Rhodococcus jostii RHA1, expressed in E. coli, contained E. coli membrane lipids.
Purpose of the Study:
- To investigate the lipid-binding specificity of 3HB6H produced in its native host, Rhodococcus jostii RHA#2.
- To characterize the biochemical and biophysical properties of 3HB6H from R. jostii (Rj3HB6H).
- To explore the role of the lipid cofactor in enzyme structure and function.
Main Methods:
- Expression of 3HB6H in a newly developed actinomycete expression system.
- Purification of Rj3HB6H.
- Biochemical and biophysical analyses (including native mass spectrometry).
- Lipid analysis of purified enzymes.
Main Results:
- Rj3HB6H, produced in R. jostii, contains phosphatidylinositol, a specific actinomycete membrane lipid.
- Biochemical and biophysical analyses confirmed Rj3HB6H shares catalytic and structural features with previously studied 3HB6H.
- Native mass spectrometry indicated the lipid cofactor stabilizes monomer-monomer interactions.
- Lipid analysis of Pseudomonas alcaligenes 3HB6H (Pa3HB6H) produced in E. coli supported the intrinsic ability of 3HB6H to bind phospholipids based on host membrane composition.
Conclusions:
- 3HB6H enzymes exhibit an intrinsic ability to bind phospholipids, with specificity adapting to the host bacterium's membrane composition.
- The phospholipid ligand plays a role in stabilizing the enzyme's structure.
- This adaptability is significant for the catabolism of aromatic compounds in diverse soil microbial environments.
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