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)来自各种植物的重组查耳酮合酶（CHS）和芪合酶接受长链脂肪酸的CoA酯（CHS至C12酯，而STS至C14酯）作为起始底物，并与丙二酰CoA进行连续缩合，导致形成三酮和四酮α-吡喃酮。从大黄（Rheum palmatum）中克隆了一个新的植物III型聚酮合成酶（PKS）的cDNA序列，该序列的构建为进一步研究III型PKS反应提供了理论依据。在E.大肠杆菌接受乙酰辅酶A作为起始剂，与丙二酰辅酶A进行六次连续缩合，随后环化，得到芳香族七肽芦荟苦素。该酶与查耳酮脱氢酶（CHS）有60%的氨基酸序列相同性， ...更多信息 在CHS超家族酶中保守的几乎相同的CoA结合位点和催化残基。此外，同源性建模预测，43-kDa的蛋白质具有相同的整体折叠CHS。这为III型PKS的催化功能提供了新的见解，并建议进一步参与植物聚酮化合物的生物合成。(2)1-亚甲基角鲨烯和25-亚甲基角鲨烯被产自酸热脂环芽孢杆菌的角鲨烯：霍烯环化酶转化为30-亚甲基霍-22（29）-烯。值得注意的是，两种类似物产生相同的产物。由亚甲基残基稳定的庚烷基中间体阳离子，使异丁烯基在C-21处的旋转在最终质子消除之前成为可能。相比之下，在形成啤酒花-22（29）-烯时，最终的质子提取发生区域特异性地从Z-甲基基团，这通过将（1，1，1，24，24，24 - 2 H6）角鲨烯环化成（23，23，23，30，30，30 - 2 H6）啤酒花-22（29）-烯来验证。角鲨烯：热酸放线菌（A. acidocaldarius）的萜烯环化酶（SHC）以26-亚甲基角鲨烯和27-亚甲基角鲨烯为底物，分别转化为新的五环C31多萜类化合物：6.6.6.5-6环的达玛烯衍生物和26-亚甲基-蛇麻烯-22（29）-烯。该酶广泛的底物特异性为SHC的结构和功能提供了重要信息。有趣的是，还发现27-MS是细菌SHC的有效抑制剂（IC 501/45 1 M），而26-MS仅显示差的酶抑制。(3)从啤酒花（HumuluslupulusL.）利用基于拟南芥AIPT同工酶（AtIPT 1、AtIPT 3、AtIPT 4、AtIPT 5、AtIPT 6、AtIPT 7和AtIPT 8）的保守序列的寡核苷酸引物通过RT-PCR。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36，603 Da。此外，基因组DNA的DNA测序显示框架中不存在内含子。Southern杂交分析表明，在H. RT-PCR分析表明，该基因在植物的根、茎、叶和球花中均有表达。推导的氨基酸序列与A.拟南芥AtIPT。大肠杆菌中表达的重组酶催化异戊烯基转移反应，从二甲基烯丙基二磷酸（DMAPP）分别转移到一磷酸腺苷（AMP）、二磷酸腺苷（ADP）和三磷酸腺苷（ATP）的N6氨基上。相反，其他核苷酸;鸟苷一磷酸（GMP），肌苷一磷酸（IMP），胞嘧啶一磷酸（CMP），尿苷一磷酸（UMP），不被接受为底物。有趣的是，稳态动力学分析表明，ADP和ATP的异戊烯化比AMP更有效，如以前报道的A。拟南芥AtIPT 4.最后，对H.与其它已知的异戊烯基转移酶一样，啤酒花AIPT在N-末端含有推定的ATP/GTP结合基序。一个保守的Asp 62，位于后的ATP/GTP结合基序，与Ala的定点突变导致酶活性的完全丧失。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