Evolutionary analysis of the functional plasticity of Staphylococcus aureus C30 carotenoid synthase.
Most natural carotenoids have 40-carbon (C40) backbones, while some bacteria produce carotenoids with C30 backbones. Carotenoid backbone synthases, the enzyme that catalyze the first committed step in carotenoid biosynthesis, are known to be highly specific. Previously, using C30 backbone synthase (diapophytoene synthase, CrtM) from Staphylococcus aureus, we reported two size-shifting mutations, F26A and W38A, which confer C40 synthase activity at the cost of the original C30 synthase activity. In this study, we performed a directed evolution of the C40-specialist variant CrtMF26A in search of mutations that restore the original C30 synthase function. Examination of the resultant mutants, together with the site-directed mutagenesis study identified three new mutations (H12A, D27A and I240F) that affect the size specificity of this enzyme. After re-defining the reading frame, we obtained CrtM variants that are highly active in C30 and C40 carotenoid synthesis.
Department of Applied Chemistry and Biotechnology, Chiba University, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan.
Maiko
M
Furubayashi
Department of Applied Chemistry and Biotechnology, Chiba University, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan.
Kyoichi
K
Saito
Department of Applied Chemistry and Biotechnology, Chiba University, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan; Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan. Electronic address: umeno@faculty.chiba-u.jp.
Daisuke
D
Umeno
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002338:Carotenoids / Q000096:biosynthesis* / Q000235:genetics; D019020:Directed Molecular Evolution; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
D001426:Bacterial Proteins; D050505:Mutant Proteins; D002338:Carotenoids
Carotene synthase; Carotenoid; Directed evolution; Screening; Size specificity