title: Deciphering the Biosynthetic Origin of L-allo-Isoleucine.
abstract: The nonproteinogenic amino acid L-allo-isoleucine (L-allo-Ile) is featured in an assortment of life forms comprised of, but not limited to, bacteria, fungi, plants and mammalian systems including Homo sapiens. Despite its ubiquity and functional importance, the specific origins of this unique amino acid have eluded characterization. In this study, we describe the discovery and characterization of two enzyme pairs consisting of a pyridoxal 5'-phosphate (PLP)-linked aminotransferase and an unprecedented isomerase synergistically responsible for the biosynthesis of L-allo-Ile from L-isoleucine (L-Ile) in natural products. DsaD/DsaE from the desotamide biosynthetic pathway in Streptomyces scopuliridis SCSIO ZJ46, and MfnO/MfnH from the marformycin biosynthetic pathway in Streptomyces drozdowiczii SCSIO 10141 drive L-allo-Ile generation in each respective system. In vivo gene inactivations validated the importance of the DsaD/DsaE pair and MfnO/MfnH pair in L-allo-Ile unit biosynthesis. Inactivation of PLP-linked aminotransferases DsaD and MfnO led to significantly diminished desotamide and marformycin titers, respectively. Additionally, inactivation of the isomerase genes dsaE and mfnH completely abolished production of all L-allo-Ile-containing metabolites in both biosynthetic pathways. Notably, in vitro biochemical assays revealed that DsaD/DsaE and MfnO/MfnH each catalyze a bidirectional reaction between L-allo-Ile and L-Ile. Site-directed mutagenesis experiments revealed that the enzymatic reaction involves a PLP-linked ketimine intermediate and uses an arginine residue from the C-terminus of each isomerase to epimerize the amino acid β-position. Consequently, these data provide important new insight into the origins of L-allo-Ile in natural products with medicinal potential and illuminate new possibilities for biotool development.
authors.affiliation: CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences , Guangzhou 510301, China.
authors.forename: Qinglian
authors.initials: Q
authors.lastname: Li
authors.affiliation: CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences , Guangzhou 510301, China.
authors.forename: Xiangjing
authors.initials: X
authors.lastname: Qin
authors.affiliation: CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences , Guangzhou 510301, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.affiliation: CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences , Guangzhou 510301, China.
authors.forename: Chun
authors.initials: C
authors.lastname: Gui
authors.affiliation: CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences , Guangzhou 510301, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Wang
authors.affiliation: CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences , Guangzhou 510301, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Li
authors.affiliation: CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences , Guangzhou 510301, China.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Ju
mesh_terms: D002384:Catalysis; D019295:Computational Biology; D006801:Humans; D007532:Isoleucine / Q000096:biosynthesis*; D016297:Mutagenesis, Site-Directed
chemical_list: D007532:Isoleucine