Haloarchaeal-Type β-Ketothiolases Involved in Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthesis in Haloferax mediterranei
Haloarchaeal-Type β-Ketothiolases Involved in Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthesis in Haloferax mediterranei
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DOI：
10.1128/aem.01370-13
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发表时间：
2013-09-01
影响因子：
4.4
通讯作者：
Xiang, Hua
中科院分区：
文献类型：
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作者：
Hou, Jing;Feng, Bo;Xiang, Hua
除了β-酮硫解酶（β-ketothiolase）外，盐古菌中聚（3-羟基丁酸-co-3-羟基戊酸）（PHBV）生物合成的关键酶已被鉴定，β-酮硫解酶将两个乙酰辅酶A（acetyl-CoA）分子缩合为乙酰乙酰辅酶A，或将一个乙酰辅酶A和一个丙酰辅酶A缩合为3-酮戊酰辅酶A。全基因组分析表明，盐古菌Haloferax medicinei中存在8个潜在的β-酮硫解酶基因，其中PHBV特异性的BktB和PhaA基因通过基因敲除和互补分析得到鉴定。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（PhaA α和PhaA β）组成，并由共转录的HFX_1023（phaA α）和HFX_1022（phaA β）基因编码。类似地，参与产生乙酰乙酰辅酶A和3-酮戊酰辅酶A的BktB也由两种不同类型的亚基（BktB α和BktB β）组成，并由共转录的HFX_6004（bktB α）和HFX_6003（bktB β）编码。BktB α和PhaA α是催化亚基，分别决定BktB和PhaA的底物特异性。它们的催化三联体“Ser-His-His”不同于细菌的“Cys-HisCys”。“BktB β和PhaA β都含有寡糖结合折叠结构域，这对β-酮硫解酶活性至关重要。有趣的是，BktB β和PhaA β在功能上是可互换的，尽管PhaA β更喜欢与PhaA α一起发挥作用。此外，BktB显示出在卤古菌中用所需的3-羟基戊酸部分生产PHBV的生物技术潜力。这是第一次报告的pHBV-特异性β-酮硫解酶，这是不同于它们的细菌对应物的亚基组成和催化残基的haloarchaeal类型。
The key enzymes for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis in haloarchaea have been identified except the beta-ketothiolase(s), which condense two acetyl coenzyme A (acetyl-CoA) molecules to acetoacetyl-CoA, or one acetylCoA and one propionyl-CoA to 3-ketovaleryl-CoA. Whole-genome analysis has revealed eight potential beta-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 (PhaA alpha and PhaA beta) and encoded by the cotranscribed HFX_1023 (phaA alpha) and HFX_1022 (phaA beta) 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 (BktB alpha and BktB beta) and encoded by cotranscribed HFX_6004 (bktB alpha) and HFX_6003 (bktB beta). BktB alpha and PhaA alpha 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-HisCys." BktB beta and PhaA beta both contained an oligosaccharide-binding fold domain, which was essential for the beta-ketothiolase activity. Interestingly, BktB beta and PhaA beta were functionally interchangeable, although PhaA beta preferred functioning with PhaA alpha. 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 beta-ketothiolases, which are distinct from their bacterial counterparts in both subunit composition and catalytic residues.