State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Appl Environ Microbiol. 2013 Sep;79(17):5104-11. doi: 10.1128/AEM.01370-13. Epub 2013 Jun 21.
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.
除了缩合两个乙酰辅酶 A（乙酰辅酶 A）分子形成乙酰乙酰辅酶 A，或一个乙酰辅酶 A 和一个丙酰辅酶 A 形成 3-酮戊酰辅酶 A 的β-酮硫解酶外，用于聚（3-羟基丁酸酯-共-3-羟基戊酸酯）（PHBV）生物合成的关键酶在盐杆菌中已被确定。全基因组分析显示，在嗜盐古菌盐沼盐杆菌中有 8 个潜在的β-酮硫解酶基因，其中 PHBV 特异性的 BktB 和 PhaA 通过基因敲除和互补分析得以鉴定。与所有已知的由单个基因编码的细菌对应物不同，参与乙酰乙酰辅酶 A 生成的盐古菌 PhaA 由两种不同类型的亚基（PhaAα和 PhaAβ）组成，并由共转录的 HFX_1023（phaAα）和 HFX_1022（phaAβ）基因编码。同样，参与生成乙酰乙酰辅酶 A 和 3-酮戊酰辅酶 A 的 BktB 也由两种不同类型的亚基（BktBα和 BktBβ）组成，并由共转录的 HFX_6004（bktBα）和 HFX_6003（bktBβ）基因编码。BktBα和 PhaAα是催化亚基，分别决定了 BktB 和 PhaA 的底物特异性。它们的催化三联体“Ser-His-His”与细菌的“Cys-His-Cys”不同。BktBβ和 PhaAβ都含有寡糖结合折叠结构域，这对于β-酮硫解酶的活性是必需的。有趣的是，尽管 PhaAβ更喜欢与 PhaAα一起发挥作用，但 BktBβ和 PhaAβ在功能上是可互换的。此外，BktB 在生产聚（3-羟基丁酸酯-共-3-羟基戊酸酯）方面具有生物技术潜力，可以在盐杆菌中获得所需的 3-羟基戊酸酯部分。这是首次报道盐古菌型 PHBV 特异性β-酮硫解酶，其在亚基组成和催化残基方面与细菌对应物不同。
