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
Laboratory of Biochemistry, Wageningen University and ResearchWageningen, Netherlands.
Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of GroningenGroningen, Netherlands.
Front Microbiol. 2017 Jun 16;8:1110. doi: 10.3389/fmicb.2017.01110. eCollection 2017.
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 RHA1 in the host contains a mixture of phosphatidylglycerol and phosphatidylethanolamine, which are the major constituents of cytoplasmic membrane. Here, we purified 3HB6H (HB6H) produced in the host RHA#2 by employing a newly developed actinomycete expression system. Biochemical and biophysical analysis revealed that 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 NCIMB 9867 (3HB6H) produced in supports the conclusion that 3HB6H enzymes have an intrinsic ability to bind phospholipids with different specificity, reflecting the membrane composition of their bacterial host.
3-羟基苯甲酸6-羟化酶（3HB6H，EC 1.13.14.26）是一种依赖黄素腺嘌呤二核苷酸（FAD）的单加氧酶，参与土壤微生物中芳香族化合物的分解代谢。3HB6H在黄素蛋白羟化酶中独一无二，因为它含有一种磷脂配体。从宿主中表达来自RHA1的编码3HB6H的基因所获得的纯化蛋白包含磷脂酰甘油和磷脂酰乙醇胺的混合物，它们是细胞质膜的主要成分。在此，我们通过采用新开发的放线菌表达系统纯化了在宿主RHA#2中产生的3HB6H（HB6H）。生化和生物物理分析表明，3HB6H具有与3HB6H相似的催化和结构特征，但现在含有磷脂酰肌醇，它是放线菌膜的一种特定成分。原生质体质谱分析表明，脂质辅因子稳定单体-单体接触。对在[具体宿主]中产生的来自NCIMB 9867的3HB6H（3HB6H）的脂质分析支持了这样的结论，即3HB6H酶具有以不同特异性结合磷脂的内在能力，这反映了其细菌宿主的膜组成。
