Studies on hyperthermostable aldolase : characteristics, structure and application

超热稳定醛缩酶的研究：特性、结构和应用

• 批准号: 15560677
• 负责人: SAKURABA Haruhiko
• 金额: $ 2.37万
• 依托单位: The University of Tokushima
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15560677/
• 关键词: Hyperthermophile; Aeropyrum pernix; Archaea; aldolase; DERA; D-2-deoxyribose-5-phosphate; aldol reaction; X-ray crystalographic analysis

A gene encoding a D-2-deoxyribose-5-phosphate aldolase (DERA) homologue was identified in the hyperthermophilic archaeon Aeropyrum pernix. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified and characterized. The enzyme was an extremely thermostable DERA ; the activity was not lost after incubation at 100℃ for 10 min. The enzyme had a molecular mass of about 93 kDa and consisted of four subunits with an identical molecular mass of 24 kDa. This shows the first presence of the tetrameric DERA. The three-dimensional structure of the enzyme was determined by x-ray analysis. The subunit folded into an α/β barrel. The asymmetric unit consisted of two homologous subunits, and a crystallographic 2-fold axis generated the functional tetramer. Compared with the structure of the E. coli DERA, the mainchain coordinate of the monomer of A. pernix enzyme was quite similar to that of the E. coli enzyme. A large difference in hydrophobic interactions and the number of ion pairs was not observed between the monomeric structures of the two enzymes. However, a significant difference in the quaternary structure was observed. The area of the subunit-subunit interface in the dimer of the A. pernix enzyme was much larger than the corresponding one of the E. coli enzyme. In addition, the A. pernix enzyme was 10 amino acids longer than the E. coli enzyme in the N-terminal region and exhibited an additional N-terminal helix. The N-terminal helix produced a unique dimer-dimer interface. This promotes the formation of a functional tetramer of the A. pernix enzyme and strengthens the hydrophobic inter-subunit interactions. These structural features are considered to be responsible for the extremely high stability of the A. pernix enzyme. This is the first description of the structure of hyperthermophilic DERA and of aldolase from the domain of archaea.

在嗜热古细菌Aeropyrum Pernix中发现了一个编码D-2-脱氧核糖-5-磷酸缩醛酶(DERA)同源基因。在大肠杆菌中高效表达了该基因，并对表达的酶进行了纯化和鉴定。该酶具有极高的耐热性，在10 0℃孵育10min后，酶活力不会丧失。该酶的相对分子质量约为93 kDa，由四个相同的亚基组成，相对分子质量均为24 kDa。这显示了四聚体DERA的首次存在。通过X-射线分析确定了该酶的三维结构。亚基折叠成一个α/β桶。不对称单元由两个同源亚基组成，功能四聚体由一个结晶学二重轴组成。与大肠杆菌DERA的结构相比，Pernix酶单体的主链坐标与大肠杆菌酶的主链坐标非常相似。两种酶的单体结构在疏水相互作用和离子对数目上没有很大的差异。然而，在四元结构中观察到了显著的差异。Pernix酶的二聚体的亚基-亚基界面面积比相应的大肠杆菌酶的二聚体大得多。此外，Pernix酶在N-末端比大肠杆菌酶长10个氨基酸，并显示一个额外的N-末端螺旋。N-末端螺旋产生了独特的二聚体-二聚体界面。这促进了Pernix酶功能四聚体的形成，并加强了疏水性亚基间的相互作用。这些结构特征被认为是Pernix酶具有极高稳定性的原因。这是首次描述了古生菌属中的嗜热菌DERA和醛缩酶的结构。

项目成果

The First Crystal Structure of Archaeal Aldolase : Unique Tetrameric Structure of 2-De-oxy-D-ribose-5-phosphate Aldolase from Hyperthermophilic Archaea Aeropyrum pernix

古菌醛缩酶的第一个晶体结构：来自超嗜热古菌 Aeropyrum pernix 的 2-脱氧-D-核糖-5-磷酸醛缩酶的独特四聚结构

• 发表时间: 2003
• 期刊: J Biol.Chem. 278
• 作者: H.Sakuraba;H.Sakuraba;Haruhiko Sakuraba;Haruhiko Sakuraba;H.Sakuraba;H.Sakurada;H.Sakuraba;H.Sakuraba
• 通讯作者: H.Sakuraba

Novel archaeal Alanine : Glyoxylate aminotransferase from Thermococcus litralis.

新型古细菌丙氨酸：来自柠檬热球菌的乙醛酸转氨酶。

• 发表时间: 2004
• 期刊: J Bacteriol. 186
• 作者: H.Sakuraba

The First Crystal Structure of Archaeal Aldolase : Unique Tetrameric Structure of 2-Deoxy-D-ribose-5-phosphate Aldolase from Hyperthermophilic Archaea Aeropyrum pernix

古菌醛缩酶的第一个晶体结构：来自超嗜热古菌 Aeropyrum pernix 的 2-脱氧-D-核糖-5-磷酸醛缩酶的独特四聚体结构

• 发表时间: 2003
• 期刊: J.Biol.Chem. Vol.278
• 作者: H.Sakuraba;H.Sakuraba;Haruhiko Sakuraba;Haruhiko Sakuraba;H.Sakuraba;H.Sakurada;H.Sakuraba;H.Sakuraba;H.Sakuraba;T.Ohshima;Haruhiko Sakuraba;Haruhiko Sakuraba
• 通讯作者: Haruhiko Sakuraba

H.Sakuraba et al.: "The First Crystal Structure of Archaeal Aldolase : Unique Tetrameric Structure of 2-Deoxy-D-ribose-5-phosphate Aldolase from Hyperthermophilic Archaea Aeropyrum pernix"J.Biol.Chem.. 278. 10799-10806 (2003)

H.Sakuraba 等人：“古细菌醛缩酶的第一个晶体结构：来自超嗜热古细菌 aeropyrum pernix 的 2-脱氧-D-核糖-5-磷酸醛缩酶的独特四聚结构”J.Biol.Chem.. 278. 10799-10806

H.Sakuraba et al.: "A nicotinamide mononucleotide adenylyltransferase with unique adenylyl group donor specificity from a hyperthermophilic archaeon, Pyrococcus horikoshii OT3"J.Mol.Catalys.B : Enzymatic. 23. 273-279 (2003)

H.Sakuraba 等人：“一种烟酰胺单核苷酸腺苷酸转移酶，具有来自超嗜热古菌 Pyrococcus horikoshii OT3 的独特腺苷酸基供体特异性”J.Mol.Catalys.B：酶促。