title: Carboxyl-terminal domain characterization of polyene-specific P450 hydroxylase in Pseudonocardia autotrophica.
abstract: A polyene compound NPP identified in Pseudonocardia autotrophica was shown to contain an aglycone identical to nystatin, but to harbor a unique disaccharide moiety that led to higher solubility and reduced hemolytic activity. Recently, it was revealed that the final step of NPP (nystatin-like polyene) biosynthesis is C10 regio-specific hydroxylation by the cytochrome P450 hydroxylase (CYP) NppL (Kim et al. [7]). Through mutation and cross-complementation, here we found that NppL preferred a polyene substrate containing a disaccharide moiety for C10 hydroxylation, while its orthologue NysL involved in nystatin biosynthesis showed no substrate preference toward mono- and disaccharide moieties, suggesting that two homologous polyene CYPs, NppL and NysL might possess a unique domain recognizing a sugar moiety. Two hybrid NppL constructs containing the C-terminal domain of NysL exhibited no substrate preference toward 10-deoxy NPP and 10-deoxy nystatin-like NysL, implying that the C-terminal domain plays a major role in differentiating the sugar moiety responsible for substrate specificity. Further C-terminal domain dissection of NppL revealed that the last fifty amino acids play a critical role in determining substrate specificity of polyene-specific hydroxylation, setting the stage for the biotechnological application of hydroxyl diversification for novel polyene biosynthesis in actinomycetes.
authors: Department of Biological Engineering, Inha University, Incheon, 402-751, Korea.
authors: Min-Kyung
authors: MK
authors: Kim
authors: Department of Biological Engineering, Inha University, Incheon, 402-751, Korea.
authors: Hyung-Jin
authors: HJ
authors: Won
authors: Department of Biological Engineering, Inha University, Incheon, 402-751, Korea.
authors: Hye-Jin
authors: HJ
authors: Kim
authors: Department of Biological Engineering, Inha University, Incheon, 402-751, Korea.
authors: Si-Sun
authors: SS
authors: Choi
authors: Department of Biotechnology and Bioinformatics, Korea University, Chungnam, 339-700, Korea.
authors: Heung-Shick
authors: HS
authors: Lee
authors: Division of Biotechnology, The Catholic University of Korea, Kyungki, 420-743, Korea.
authors: Pil
authors: P
authors: Kim
authors: Department of Biological Engineering, Inha University, Incheon, 402-751, Korea. eungsoo@inha.ac.kr.
authors: Eung-Soo
authors: ES
authors: Kim
mesh_terms: D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004187:Disaccharides / Q000737:chemistry; D006900:Hydroxylation; D009761:Nystatin / Q000096:biosynthesis; D011090:Polyenes / Q000737:chemistry / Q000378:metabolism*; D000072417:Protein Domains; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004187:Disaccharides; D011090:Polyenes; D009761:Nystatin; D003577:Cytochrome P-450 Enzyme System
keywords: P450 hydroxylase; Polyene; Pseudonocardia; Substrate specificity