植物肉桂醇脱氢酶及其基因研究进展

肉桂醇脱氢酶(cinnamyl alcohol dehydrogenase,CAD)作为植物次生代谢特别是木质素合成的关键酶,与植物生长发育和抵御病原菌入侵关系密切,研究CAD基因表达调控及其与组织木质化的关系具有重要的植物生理学意义.该文综述了植物CAD的蛋白特征、酶学性质、基因分布和分类、基因结构和表达调控以及CAD表达与木质素合成的关系,为研究CAD在植物生长发育和抗病中的作用提供理论指导.     

有机相脂肪酶催化合成乙酸肉桂酯

对有机相中酶法催化合成乙酸肉桂酯的转酯化反应进行研究。结果发现:Candida anatarctic脂肪酶(Novozyme435)、根霉脂肪酶(Rhizopus niveus lipase)和荧光假单胞菌脂肪酶(Pseudomonas fluore lipase)均有较好的催化活性。同时考察各反应参数(温度、反应溶剂、体系水活度、酰化剂类型、肉桂醇与酰化剂摩尔比、肉桂醇浓度等)对脂肪酶Novozyme435合成乙酸肉桂酯反应的影响,确定了反应体系最优工艺条件:在10 mL甲基叔丁基醚中,肉桂醇200 mmol/L,n(肉桂醇)∶n(乙酸乙烯酯)=1∶1.5,初始水活度αw=0.84,温度35℃,酶加量0.02 g,反应3 h后肉桂醇转化率可达到99%,产物经质谱(MS)鉴定。固定化酶经过10个批次反应,反应转化率都保持在90%以上。     

Molecular characterisation and expression of a wound-inducible cDNA encoding a novel cinnamyl-alcohol dehydrogenase enzyme in lucerne (Medicago sativa L.)

A lucerne (alfalfa, Medicago sativa) stem cDNA library was screened with a cinnamyl-alcohol dehydrogenase (CAD) cDNA probe from tobacco (Nicotiana tabacum cv. Samsun). Two distinctly different cDNA clones (54% identical) were isolated and identified as putative CAD-encoding cDNAs by comparison of their nucleotide sequences with those of CAD-encoding DNA sequences from other plant species. One of the cDNAs, MsaCad2, was found to be 99.4% identical at the nucleotide level to the previously isolated lucerne cad cDNA which encodes a CAD isoform involved in lignin biosynthesis. The other cDNA, MsaCad1, has not been reported previously in lucerne, and encodes a protein related to the ELI3 class of elicitor-inducible defence-related plant proteins. The MsaCad1- and MsaCad2-encoded proteins were expressed in Escherichia coli and CAD1 was shown to be active with a range of cinnamyl, benzyl and aliphatic aldehyde substrates, while CAD2 was specific for the cinnamyl aldehydes only. Each of the respective genes is present as one or two copies. The MsaCad1 gene is expressed most actively in stem and floral tissue, whereas MsaCad2 is most actively expressed in stem, hypocotyl and root tissue. In stem tissue, expression of both genes occurs predominantly in internodes 4 and 5 (from the apex). MsaCad2, in contrast to MsaCad1, is not significantly expressed in the top three internodes of the stem. Both MsaCad1 and MsaCad2 are wound-inducible, and the wound-responsiveness of each gene is modulated by salicylic acid.     

3个植物源苯甲醇酰基转移酶合成乙酸肉桂酯的研究

[背景]乙酸肉桂酯是一种重要的香料化合物,在化妆品和食品工业上具有广泛的应用,传统的生产方法主要依靠植物提取和化学合成。[目的]通过筛选不同植物源的酰基转移酶,利用大肠杆菌从头合成乙酸肉桂酯。[方法]首先,通过在苯丙氨酸高产菌BPHE中表达异源基因苯丙氨酸解氨酶(Phenylalanine Ammonia-Lyase from Arabidopsis thaliana,AtPAL)、对羟基肉桂酰辅酶A连接酶(Hydroxycinnamate:CoA Ligase from Petroselinum crispum,Pc4CL)和肉桂酰辅酶 A 还原酶(Cinnamyl-CoA Reductase from Arabidopsis thaliana,AtCCR),并结合大肠杆菌自身的内源性醇脱氢酶(Alcohol Dehydrogenases,ADHs)或醛酮还原酶(Aldo-Keto Reductases,AKRs)的催化作用构建了从苯丙氨酸到肉桂醇的生物合成途径。然后,苯甲醇苯甲酰转移酶(Benzyl Alcohol O-Benzoyltransferase from Nicotiana tabacum,ANN09798;Benzyl Alcohol O-Benzoyltransferase from Clarkia breweri,ANN09796)或苯甲醇乙酰转移酶(Benzyl Alcohol Acetyltransferase from Clarkia breweri,BEAT)被引入到上述重组大肠杆菌中发酵培养生产乙酸肉桂酯。最后,在大肠杆菌中过表达乙酰辅酶A合成酶(Acetyl Coenzyme A Synthetase,ACS)来提高底物乙酰辅酶A的量。[结果]探讨了 3个植物源苯甲醇酰基转移酶生物合成乙酸肉桂酯的能力,并应用于合成乙酸肉桂酯的细胞工厂,最终使乙酸肉桂酯最高产量达到166.9±6.6mg/L。[结论]植物源苯甲醇酰基转移酶具有一定的底物宽泛性,能以肉桂醇为底物催化合成乙酸肉桂酯。首次利用植物源的苯甲醇酰基转移酶合成乙酸肉桂酯,为微生物细胞工厂以葡萄糖作为碳源生产乙酸肉桂酯提供参考。     

Enhanced Biotransformation Capacity of <Emphasis Type="Italic">Rhodiola rosea</Emphasis> Callus Cultures for Glycosid Production

Rhodiola rosea is a promising medicinal plant that produces various glycosides. Recently we developed a successful method for cultivating it in liquid cultures of compact callus aggregates. In a previous study we reported the successful production of the glycosides of R. rosea by biotransformation of cinnamyl alcohol and tyrosol. In the present study we investigated the possibility of further increasing the yields of the biotransformation products by addition of glucose to the culture medium aside from sucrose, which was used earlier as carbon source. Surprisingly, glucose addition doubled the yield of cinnamyl alcohol glycosides. Rosavin was not produced at all when only sucrose was used. When glucose was added the accumulation dynamics of rosin and a recently described derivative glycoside (designed as compound 321) were similar. Both increased during the first days and then remained constant, while other glycoside compounds increased continuously throughout the cultivation. Rosavin reached its maximum concentration after nine days. In contrast to the beneficial effect on cinnamyl alcohol related glycosides the addition of glucose did not affect the accumulation of the tyrosol derivative salidroside.     

Characterization of cinnamyl alcohol dehydrogenase of Helicobacter pylori. An aldehyde dismutating enzyme

Cinnamyl alcohol dehydrogenases (CAD; 1.1.1.195) catalyse the reversible conversion of p-hydroxycinnamaldehydes to their corresponding alcohols, leading to the biosynthesis of lignin in plants. Outside of plants their role is less defined. The gene for cinnamyl alcohol dehydrogenase from Helicobacter pylori (HpCAD) was cloned in Escherichia coli and the recombinant enzyme characterized for substrate specificity. The enzyme is a monomer of 42.5 kDa found predominantly in the cytosol of the bacterium. It is specific for NADP(H) as cofactor and has a broad substrate specificity for alcohol and aldehyde substrates. Its substrate specificity is similar to the well-characterized plant enzymes. High substrate inhibition was observed and a mechanism of competitive inhibition proposed. The enzyme was found to be capable of catalysing the dismutation of benzaldehyde to benzyl alcohol and benzoic acid. This dismutation reaction has not been shown previously for this class of alcohol dehydrogenase and provides the bacterium with a means of reducing aldehyde concentration within the cell.     

Stereospecificity of cinnamyl alcohol dehydrogenase and synthesis of stereospecifically labelled coniferyl alcohol

Using horse liver alcohol dehydrogenase, stereospecifically tritiated (R)- and (S)-(γ-³H)-coniferyl alcohol was synthesized. Using both of these substrates it was demonstrated that cinnamyl alcohol dehydrogenase from lignifying Forsythia tissue specifically removes the pro-R-hydrogen atom of coniferyl alcohol in the oxidation to the aldehyde. This also means that in the reverse reaction the A-hydrogen of NADPH is transferred to the Re-site of coniferyl aldehyde.     

Investigation of genome structure of a cinnamyl alcohol dehydrogenase locus in a basal angiosperm hardwood species, Liriodendron tulipifera L., reveals low synteny

Basal angiosperms contain a wide diversity of floral and growth forms and gave rise to the largest recent angiosperm lineages.As none of the basal angiosperm genomes has been sequenced,examining large bacterial artificial chromosome (BAC) inserts remains the main approach to providing a first glimpse of the structure and organization of their genomes.In this study,we sequenced a 126.9-kbp BAC contig harboring a cinnamyl alcohol dehydrogenase gene (LtuCAD1) in a basal angiosperm species,Liriodendron tulipifera L.,an important timber tree species with significant ecological and economic values.A key enzyme in lignin biosynthesis,CAD catalyzes the final step in the synthesis of monolignols.We carried out phylogenetic analyses of seven full-length CAD family genes (LtuCAD1-7) obtained from a comprehensive Liriodendron expressed sequence tag dataset.The phylogenetic tree suggests that LtuCAD1 is the primary CAD gene involved in lignifications as it is the only Liriodendron CAD grouped with the bona fide CADs class.As well as the LtuCAD1,the BAC contig contained fragmented sequences for one integrase,eight hypothetical proteins,two gag-pol polyproteins,one RNase H family protein,and one chromatin binding protein.Comparative analysis with other angiosperm species suggests that the genomic segment in this BAC has undergone frequent arrangement.This study is our initial step in identifying and understanding lignin biosynthesis genes from basal angiosperm species.Such knowledge can help bridge the information gap between hardwood (angiosperm) and softwood (gymnosperm) species and benefit potential breeding and biotechnology application for enhanced production ofbiomass and digestibility in L.tulipifera.     

Down-regulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility

To improve the digestibility of the forage crop alfalfa (Medicago sativa L.), cinnamyl alcohol dehydrogenase (CAD), which catalyses the last step in the biosynthesis of the lignin monomers, was down-regulated by using an antisense approach. A subset of six transgenic lines with reduced CAD activity and control lines were analysed when grown in the greenhouse and in the field. The down-regulation of the CAD enzyme was associated with a red coloration of the stem. The lignin quantity remained unchanged, but the lignin composition, as determined by thioacidolysis, was altered. The highest reduction of CAD activity was associated with a lower syringyl/guaiacyl (S/G) ratio and a lower S+G yield, mainly because of a decreased amount of S units. An increase in in situ disappearance of dry matter and of cell wall residue was detected in one of the transgenic lines grown in the greenhouse, and for two of the lines grown in the field the rate of disappearance of dry matter slightly improved. Furthermore, these two lines had a higher solubility in alkali as shown by the lower yield of saponified residue. This study opens perspectives for improving forage crop digestibility by the modulation of enzymes involved in lignin biosynthesis.     

Apo and Holo structures of an NADPH-dependent cinnamyl alcohol dehydrogenase from Saccharomyces cerevisiae

The crystal structure of Saccharomyces cerevisiae ScAdh6p has been solved using the anomalous signal from the two zinc atoms found per subunit, and it constitutes the first structure determined from a member of the cinnamyl alcohol dehydrogenase family. ScAdh6p subunits exhibit the general fold of the medium-chain dehydrogenases/reductases (MDR) but with distinct specific characteristics. In the three crystal structures solved (two trigonal and one monoclinic), ScAdh6p molecules appear to be structural heterodimers composed of one subunit in the apo and the second subunit in the holo conformation. Between the two conformations, the relative disposition of domains remains unchanged, while two loops, Cys250-Asn260 and Ile277-Lys292, experience large movements. The apo-apo structure is disfavoured because of steric impairment involving the loop Ile277-Lys292, while in the holo-holo conformation some of the hydrogen bonds between subunits would break apart. These suggest that the first NADPH molecule would bind to the enzyme much more tightly than the second. In addition, fluorimetric analysis of NADPH binding demonstrates that only one cofactor molecule binds per dimer. Therefore, ScAdh6p appears to function according to a half-of-the-sites reactivity mechanism, resulting from a pre-existing (prior to cofactor binding) tendency for the structural asymmetry in the dimer. The specificity of ScAdh6p towards NADPH is mainly due to the tripod-like interactions of the terminal phosphate group with Ser210, Arg211 and Lys215. The size and the shape of the substrate-binding pocket correlate well with the substrate specificity of ScAdh6p towards cinnamaldehyde and other aromatic compounds. The structural relationships of ScAdh6p with other MDR structures are analysed.     

Analysis of the enzymatic formation of citral in the glands of sweet basil

Basil glands of the Sweet Dani cultivar contain high levels of citral, a mixture of geranial and its cis-isomer neral, as well as low levels of geraniol and nerol. We have previously reported the identification of a cDNA from Sweet Dani that encodes an enzyme responsible for the formation of geraniol from geranyl diphosphate in the glands, and that these glands cannot synthesize nerol directly from geranyl diphosphate. Here, we report the identification of two basil cDNAs encoding NADP+-dependent dehydrogenases that can use geraniol as the substrate. One cDNA, designated CAD1, represents a gene whose expression is highly specific to gland cells of all three basil cultivars examined, regardless of their citral content, and encodes an enzyme with high sequence similarity to known cinnamyl alcohol dehydrogenases (CADs). The enzyme encoded by CAD1 reversibly oxidizes geraniol to produce geranial (which reversibly isomerizes to neral via keto-enol tautomerization) at half the efficiency compared with its activity with cinnamyl alcohol. CAD1 does not use nerol and neral as substrates. A second cDNA, designated GEDH1, encodes an enzyme with sequence similarity to CAD1 that is capable of reversibly oxidizing geraniol and nerol in equal efficiency, and prolonged incubation of geraniol with GEDH1 in vitro produces not only geranial and neral, but also nerol. GEDH1 is also active, although at a lower efficiency, with cinnamyl alcohol. However, GEDH1 is expressed at low levels in glands of all cultivars compared with its expression in leaves. These and additional data presented indicate that basil glands may contain additional dehydrogenases capable of oxidizing geraniol.     

Production and solid-phase refolding of human glucagon-like peptide-1 using recombinant Escherichia coli

A cDNA encoding cinnamyl alcohol dehydrogenase (CAD), catalyzing conversion of cinnamyl aldehydes to corresponding cinnamyl alcohols, was cloned from secondary xylem of Leucaena leucocephala. The cloned cDNA was expressed in Escherichia coli BL21 (DE3) pLysS cells. Temperature and Zn(2+) ion played crucial role in expression and activity of enzyme, such that, at 18°C and at 2 mM Zn(2+) the CAD was maximally expressed as active enzyme in soluble fraction. The expressed protein was purified 14.78-folds to homogeneity on Ni-NTA agarose column with specific activity of 346 nkat/mg protein. The purified enzyme exhibited lowest Km with cinnamyl alcohol (12.2 μM) followed by coniferyl (18.1 μM) and sinapyl alcohol (23.8 μM). Enzyme exhibited high substrate inhibition with cinnamyl (beyond 20 μM) and coniferyl (beyond 100 μM) alcohols. The in silico analysis of CAD protein exhibited four characteristic consensus sequences, GHEXXGXXXXXGXXV; C(100), C(103), C(106), C(114); GXGXXG and C(47), S(49), H(69), L(95), C(163), I(300) involved in catalytic Zn(2+) binding, structural Zn(2+) binding, NADP(+) binding and substrate binding, respectively. Tertiary structure, generated using Modeller 9v5, exhibited a trilobed structure with bulged out structural Zn(2+) binding domain. The catalytic Zn(2+) binding, substrate binding and NADP(+) binding domains formed a pocket protected by two major lobes. The enzyme catalysis, sequence homology and 3-D model, all supported that the cloned CAD belongs to alcohol dehydrogenase family of plants.     

Characterization of Two Isozymes of Coniferyl Alcohol Dehydrogenase from Streptomyces sp. NL15-2K

We purified two isozymes of coniferyl alcohol dehydrogenase (CADH I and II) to homogeneity from cell-free extracts of Streptomyces sp. NL15-2K. The apparent molecular masses of CADH I and II were determined to be 143 kDa and 151 kDa respectively by gel filtration, whereas their subunit molecular masses were determined to be 35,782.2 Da and 37,597.7 Da respectively by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Thus, it is probable that both isozymes are tetramers. The optimum pH and temperature for coniferyl alcohol dehydrogenase activity were pH 9.5 and 45 °C for CADH I and pH 8.5 and 40 °C for CADH II. CADH I oxidized various aromatic alcohols and allyl alcohol, and was most efficient on cinnamyl alcohol, whereas CADH II exhibited high substrate specificity for coniferyl alcohol, and showed no activity as to the other alcohols, except for cinnamyl alcohol and 3-(4-hydroxy-3-methoxyphenyl)-1-propanol. In the presence of NADH, CADH I and II reduced cinnamaldehyde and coniferyl aldehyde respectively to the corresponding alcohols.     

Aromatic esters from Solidago decurrens

From Solidago decurrens several new benzyl benzoates and two cinnamyl angelates were variously isolated from stems, flower and root.     

Regulatory role of cinnamyl alcohol dehydrogenase in the formation of guaiacyl and syringyl lignins

The substrate specificities of cinnamyl alcohol dehydrogenase (CAD) of angiosperms and gymnosperms were examined using coniferaldehyde and sinapaldehyd     

A novel aromatic alcohol dehydrogenase in higher plants: molecular cloning and expression

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.195) catalyses the conversion of p-hydroxy-cinnamaldehydes to the corresponding alcohols and is considered a key enzyme in lignin biosynthesis. In a previous study, an atypical form of CAD (CAD 1) was identified in Eucalyptus gunnii [12]. We report here the molecular cloning and characterization of the corresponding cDNA, CAD 1-5, which encodes this novel aromatic alcohol dehydrogenase. The identity of CAD 1-5 was unambiguously confirmed by sequence comparison of the cDNA with peptide sequences derived from purified CAD 1 protein and by functional expression of CAD 1 recombinant protein in Escherichia coli. Both native and recombinant CAD 1 exhibit high affinity towards lignin precursors including 4-coumaraldehyde and coniferaldehyde, but they do not accept sinapaldehyde. Moreover, recombinant CAD 1 can also utilize a wide range of aromatic substrates including unsubstituted and substituted benzaldehydes. The open reading frame of CAD 1-5 encodes a protein with a calculated molecular mass of 35790 Da and an isoelectric point of 8.1. Although sequence comparisons with proteins in databases revealed significant similarities with dihydroflavonol-4-reductases (DFR; EC 1.1.1.219) from a wide range of plant species, the most striking similarity was found with cinnamoyl-CoA reductase (CCR; EC 1.2.1.44), the enzyme which directly precedes CAD in the lignin biosynthetic pathway. RNA blot analysis and immunolocalization experiments indicated that CAD 1 is expressed in both lignified and unlignified tissues/cells. Based on the catalytic activity of CAD 1 in vitro and its localization in planta, CAD 1 may function as an alternative enzyme in the lignin biosynthetic pathway. However, additional roles in phenolic metabolism are not excluded.     

2-Oxo-labda-8(17),13-dien-15-OL from Ophryosporus chilca

The aerial parts of Ophryosporus chilca afforded several prenylated p-hydroxyacetophenones, two labdane derivatives and a cinnamyl ester. Ophryosporus peruvianus also contains prenylated p-hydroxyacetophenones. Three compounds were isolated for the first time; their structures were established by ¹H NMR spectroscopy.     

Specific Inhibition of Lignification in Bryonia dioica: Effects on Thigmomorphogenesis

Rubbing-induced lignification in Bryonia dioica internodes is significantly impaired by N(o-hydroxyphenyl) and N(o-aminophenyl) sulfinamoyl tertiobutyl acetate, specific inhibitors of cinnamyl alcohol dehydrogenase, an enzyme which is strictly associated with lignin monomer synthesis. Along with the reduction of lignification, these inhibitors counteract the inhibition of elongation due to rubbing. These results indicate that lignification participates in the thigmomorphogenetic growth response of Bryonia dioica internodes. In a general way, the data point to the causal role of lignification in the limitation of plant growth.     

Cofactor Recycling for Selective Enzymatic Biotransformation of Cinnamaldehyde to Cinnamyl Alcohol

The enzymatic, selective hydrogenation of cinnamaldehyde to cinnamyl alcohol is reported here. Yeast alcohol dehydrogenase was used in a substrate-coupled process with cofactor recycling. Both 100% selectivity and aldehyde conversion were achieved within 3 h. The reaction took place under very mild conditions, in the absence of toxic organic solvent. The overall process proved inexpensive and deserves further optimization studies in order to evaluate industrial applications.