title: Haloarchaeal-type β-ketothiolases involved in Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in Haloferax mediterranei.
abstract: The key enzymes for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis in haloarchaea have been identified except the β-ketothiolase(s), which condense two acetyl coenzyme A (acetyl-CoA) molecules to acetoacetyl-CoA, or one acetyl-CoA and one propionyl-CoA to 3-ketovaleryl-CoA. Whole-genome analysis has revealed eight potential β-ketothiolase genes in the haloarchaeon Haloferax mediterranei, among which the PHBV-specific BktB and PhaA were identified by gene knockout and complementation analysis. Unlike all known bacterial counterparts encoded by a single gene, the haloarchaeal PhaA that was involved in acetoacetyl-CoA generation, was composed of two different types of subunits (PhaAα and PhaAβ) and encoded by the cotranscribed HFX_1023 (phaAα) and HFX_1022 (phaAβ) genes. Similarly, the BktB that was involved in generation of acetoacetyl-CoA and 3-ketovaleryl-CoA, was also composed of two different types of subunits (BktBα and BktBβ) and encoded by cotranscribed HFX_6004 (bktBα) and HFX_6003 (bktBβ). BktBα and PhaAα were the catalytic subunits and determined substrate specificities of BktB and PhaA, respectively. Their catalytic triad "Ser-His-His" was distinct from the bacterial "Cys-His-Cys." BktBβ and PhaAβ both contained an oligosaccharide-binding fold domain, which was essential for the β-ketothiolase activity. Interestingly, BktBβ and PhaAβ were functionally interchangeable, although PhaAβ preferred functioning with PhaAα. In addition, BktB showed biotechnological potential for the production of PHBV with the desired 3-hydroxyvalerate fraction in haloarchaea. This is the first report of the haloarchaeal type of PHBV-specific β-ketothiolases, which are distinct from their bacterial counterparts in both subunit composition and catalytic residues.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Hou
authors.forename: Bo
authors.initials: B
authors.lastname: Feng
authors.forename: Jing
authors.initials: J
authors.lastname: Han
authors.forename: Hailong
authors.initials: H
authors.lastname: Liu
authors.forename: Dahe
authors.initials: D
authors.lastname: Zhao
authors.forename: Jian
authors.initials: J
authors.lastname: Zhou
authors.forename: Hua
authors.initials: H
authors.lastname: Xiang
mesh_terms: D000102:Acetyl-CoA C-Acyltransferase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000235:genetics; D019295:Computational Biology; D015870:Gene Expression; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D020745:Genome, Archaeal; D019623:Haloferax mediterranei / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009876:Operon; D010802:Phylogeny; D011091:Polyesters / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity; D014158:Transcription, Genetic; D000094122:Polyhydroxybutyrates
chemical_list: D000102:Acetyl-CoA C-Acyltransferase; D011091:Polyesters; D021122:Protein Subunits; C052620:poly(3-hydroxybutyrate)-co-(3-hydroxyvalerate); D000094122:Polyhydroxybutyrates