Formation of unnatural novel polyketides and isoprenoids from alternate and/or nonphysiological substrate by the engineered plant enzymes

通过工程植物酶从替代和/或非生理底物形成非天然的新型聚酮化合物和类异戊二烯

• 批准号: 15310153
• 负责人: NOGUCHI Hiroshi
• 金额: $ 10.18万
• 依托单位: University of Shizuoka
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 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-15310153/
• 关键词: chalcone synthase; Hopene synthase; polyketide; squalene; Rheum palmatum; aloesone; triterpene; flavonoid

(1) Recombinant chalcone synthase (CHS) and Stilbene synthase from various plants accepted CoA esters of long-chain fatty acid (CHS up to the C12 ester, while STS up to the C14 ester) as a starter substrate, and carried out sequential condensations with malonyl-CoA, leading to formation of triketide and tetraketide a-pyrones. The catalytic diversities of the enzymes provided further mechanistic insights into the type III PKS reactions, and suggested involvement of the CHS-superfamily enzymes in the biosynthesis of long-chain alkyl polyphenols such as urushiol and ginkgolic acid in plants.A cDNA encoding a novel plant type III polyketide synthase (PKS) was cloned from rhubarb (Rheum palmatum). A recombinant enzyme expressed in E. coli accepted acetyl-CoA as a starter, carried out six successive condensations with malonyl-CoA and subsequent cyclization to yield an aromatic heptaketide, aloesone. The enzyme shares 60% amino acid sequence identity with chalcone synthases (CHSs), and mainta … More ins almost identical CoA binding site and catalytic residues conserved in the CHS superfamily enzymes. Further, homology modeling predicted that the 43-kDa protein has the same overall fold as CHS. This provides new insights into the catalytic functions of type III PKSs, and suggests further involvement in the biosynthesis of plant polyketides.(2) 1-Methylidenesqualene and 25-methylidenesqualene were converted to 30-methylidenehop-22(29)-ene by squalene: hopene cyclase from Alicyclobacillus acidacaldarius. It was remarkable that both analogues generated the same product. The hopanyl intermediate cation, stabilized by the methylidene residue, enabled a rotation of the isobutenyl group at C-21 prior to the final proton elimination. In contrast, in the formation of hop-22(29)-ene, the final proton abstraction takes place regiospecifically from the Z-methyl group, which was verified by cyclization of (1,1,1,24,24,24-2H6) squalene into (23,23,23,30,30,30-2H6) hop-22(29)-ene. Squalene: hopene cyclase (SHC) from A, acidocaldarius accepted 26-methylidenesqualene and 27-methylidenesqualene as a substrate and converted to novel pentacyclic C31 polyprenoids; a dammarene derivative with a 6.6.6.5-6 ring system and 26-methylidene-hop-22(29)-ene, respectively. The broad substrate specificity of the enzyme provided important information on the structure and function of SHC. Interestingly, 27-MS was also found to be a potent inhibitor of the bacterial SHC (IC501/45 lM), while 26-MS just showed poor enzyme inhibition.(3) A cDNA encoding adenylate isopentenyltransferase (AIPT) was cloned and sequenced from cones of hop (Humulus lupulus L.) by RT-PCR using oligonucleotide primers based on the conserved sequences of Arabidopsis thaliana AIPT isozymes (AtIPT1, AtIPT3, AtIPT4, AtIPT5, AtIPT6, AtIPT7 and AtIPT8). A full-length cDNA contained a 990-bp open reading frame encoding a molecular mass of 36,603 Da protein with 329 amino acids. Further, DNA sequencing of genomic DNA revealed absence of introns in the frame. On Southern blot analysis, a single AIPT gene was detected in H. lupulus, while RT-PCR analyses demonstrated that the gene was equally expressed in almost all tissues in the plant including roots, stems, leaves and cones. The deduced amino acid sequence shares 38-51% identity to those of A. thaliana AtIPTs. A recombinant enzyme expressed in Escherichia coli catalyzed isopentenyl transfer reaction from dimethylallyldiphosphate (DMAPP) to the N6 amino group of adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP), respectively. In contrast, other nucleotides; guanosine monophosphate (GMP), inosine monophosphate (IMP), cytosine monophosphate (CMP), uridine monophosphate (UMP), were not accepted as a substrate. Interestingly, steady-state kinetic analyses revealed that the isopentenylation of ADP and ATP were more efficient than that of AMP as previously reported for A. thaliana AtIPT4. Finally, H. lupulus AIPT contains the putative ATP/GTP binding motif at the N-terminal as in the case of other known isopentenyltransferases. Site-directed mutagenesis of a conserved Asp62, located right after the ATP/GTP binding motif, with Ala resulted in complete loss of enzyme activity. Less

(1)多种植物的重组查尔酮合成酶（CHS）和二苯乙烯合成酶以长链脂肪酸CoA酯（CHS至C12酯，STS至C14酯）为起始底物，与丙二酰辅酶a依次缩合，生成三酮类和四酮类a-吡酮类化合物。这些酶的催化多样性为III型PKS反应提供了进一步的机制见解，并表明chs超家族酶参与了植物中漆酚和银杏酸等长链烷基多酚的生物合成。从大黄（Rheum palmatum）中克隆了一种新的植物III型聚酮合成酶（PKS） cDNA。在大肠杆菌中表达的一种重组酶以乙酰辅酶A为起始物，与丙二酰辅酶A进行了6次连续缩合，随后进行了环化，得到芳香七肽。该酶与查尔酮合成酶（CHSs）具有60%的氨基酸序列同源性，并保持了CHS超家族酶中几乎相同的CoA结合位点和催化残基。此外，同源性模型预测43-kDa蛋白与CHS具有相同的总体折叠。这为III型pks的催化功能提供了新的见解，并提示其进一步参与植物多酮类化合物的生物合成。(2)酸环芽孢杆菌中角鲨烯-藿烯环化酶将1-甲基烯烯和25-甲基烯烯转化为30-甲基烯-22(29)-烯。值得注意的是，这两种类似物产生了相同的产物。hopanyl中间阳离子被甲基残基稳定，使得异丁基在C-21位点的旋转在最终质子消除之前发生。相反，在hop-22(29)-ene的形成过程中，最终的质子抽离发生在z -甲基的特定区域，通过（1,1,1,24,24,24- 2h6）角鲨烯环化成（23,23,23,30,30,30- 2h6） hop-22(29)-ene证实了这一点。角鲨烯：来自A，酸藻的藿烯环化酶（SHC）接受26-甲基角鲨烯和27-甲基角鲨烯作为底物，转化为新型的五环C31聚丙烯类化合物；具有6.6.6.5-6环体系和26-甲基-跳跃-22(29)-烯的达玛烯衍生物。该酶广泛的底物特异性为研究SHC的结构和功能提供了重要信息。有趣的是，27-MS也被发现是细菌SHC的有效抑制剂（IC501/45 lM），而26-MS仅表现出较差的酶抑制作用。(3)基于拟南芥AIPT同工酶（AtIPT1、AtIPT3、AtIPT4、AtIPT5、AtIPT6、AtIPT7和AtIPT8）保守序列，利用寡核苷酸引物，从啤酒花（Humulus lupulus L.）球果中克隆出编码腺苷酸异戊烯基转移酶（AIPT）的cDNA，并进行了RT-PCR测序。全长cDNA包含一个990 bp的开放阅读框，编码329个氨基酸，分子量为36603 Da的蛋白。此外，基因组DNA测序显示框架中没有内含子。通过Southern blot分析，在狼疮中检测到一个AIPT基因，而RT-PCR分析表明该基因在植物的根、茎、叶和球果等几乎所有组织中都有相同的表达。推导出的氨基酸序列与拟南螺旋藻AtIPTs的同源性为38-51%。一种在大肠杆菌中表达的重组酶催化了异戊烯基从二磷酸二甲基烯基（DMAPP）到单磷酸腺苷（AMP）、二磷酸腺苷（ADP）和三磷酸腺苷（ATP）的N6氨基转移反应。相反，其他核苷酸；单磷酸鸟苷（GMP）、单磷酸肌苷（IMP）、单磷酸胞嘧啶（CMP）、单磷酸尿苷（UMP）不被接受作为底物。有趣的是，稳态动力学分析表明，ADP和ATP的异戊烯化比先前报道的拟南芥AtIPT4的AMP更有效。最后，与其他已知的异戊烯基转移酶一样，H. lupus AIPT在n端含有假定的ATP/GTP结合基序。位于ATP/GTP结合基序后的保守的Asp62与Ala的定点突变导致酶活性完全丧失。少

项目成果

Characterization of Folding Pathways of the Type-1 and Type-2 Periplasmic Binding Proteins MglB and ArgT

1 型和 2 型周质结合蛋白 MglB 和 ArgT 折叠途径的表征

• 发表时间: 2003
• 期刊: J. Biochem. 133
• 作者: Kashiwagi;K.;Shiba;K.;F.-Kobayashi;K.;Noda;T.;Nishikawa;K;Noguchi;H.
• 通讯作者: H.

Molecular cloning, expression, and characterization of adenylate isopentenyltransferase from hop (Humulus lupulus L.)

• DOI: 10.1016/j.phytochem.2004.08.006
• 发表时间: 2004-09-01
• 期刊: PHYTOCHEMISTRY
• 影响因子: 3.8
• 作者: Sakano, Y;Okada, Y;Abe, I
• 通讯作者: Abe, I

A plant type III polyketide synthase that produces pentaketide chromone

• DOI: 10.1021/ja0431206
• 发表时间: 2005-02-09
• 期刊: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
• 影响因子: 15
• 作者: Abe, I;Utsumi, Y;Noguchi, H
• 通讯作者: Noguchi, H

Enzymatic Cyclization of 22,23-Dihydro-2, 3-Oxidosqualene into Euph-7-en-3-ol and Bacchar-12-en-3-ol by Recombinant β-Amyrin Synthase

重组 β-香树脂醇合酶将 22,23-二氢-2, 3-氧化角鲨烯酶促环化为 Euph-7-en-3-ol 和 Bacchar-12-en-3-ol

• 发表时间: 2004
• 期刊: J. Am. Chem. Soc. 126(11)
• 作者: Abe;I.;Sakano;Y.;Tanaka;H.;Lou;W.;Noguchi;H.;Shibuya;M.;Ebizuka;Y.
• 通讯作者: Y.

Mechanism and stereochemistry of enzymatic cyclization of 24,30-bisnor-2,3-oxidosqualene by recombinant β-amyrin synthase

• DOI: 10.1021/ja0490368
• 发表时间: 2004-06-09
• 期刊: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
• 影响因子: 15
• 作者: Abe, I;Sakano, Y;Ebizuka, Y
• 通讯作者: Ebizuka, Y