Opossum Aldehyde Dehydrogenases: Evidence for Four ALDH1A1-like Genes on Chromosome 6 and ALDH1A2 and ALDH1A3 Genes on Chromosome 1

Evidence is presented for six opossum ALDH1A genes, including four ALDH1A1-like genes on chromosome 6 and ALDH1A2- and ALDH1A3-like genes on chromosome 1. Predicted structures for the opossum aldehyde dehydrogenase (ALDH) subunits and the intron–exon boundaries for opossum ALDH genes showed a high degree of similarity with other mammalian ALDHs. Phylogenetic analyses supported the proposed designation of these opossum class 1 ALDHs as ALDH1A-like, ALDH1A2-like, and ALDH1A3-like and are therefore likely to play important roles in retinal and peroxidic aldehyde metabolism. Alignments of predicted opossum ALDH1A amino acid sequences with sheep ALDH1A1 and rat ALDH1A2 sequences demonstrated conservation of key residues previously shown to participate in catalysis and coenzyme binding. Amino acid substitution rates observed for family 1A ALDHs during vertebrate evolution indicated that ALDH1A2-like genes are evolving slower than ALDH1A1- and ALDH1A3-like genes. It is proposed that the common ancestor for ALDH1A genes predates the appearance of birds during vertebrate evolution.     

酮古龙酸菌醇醛脱氢酶基因的克隆及表达

目的：从酮古龙酸菌SCB329株中分离山梨糖生物氧化相关酶的基因并进行表达验证。方法：根据酮古龙酸菌SCB329株基因组序列设计引物,通过PCR从SCB329株基因组中扩增醇醛脱氢酶基因aadh;构建载体pBMP3-aadh并在大肠杆菌中表达,经活性染色、体外转化反应等方法考察表达产物的活性。结果：目的产物能够催化山梨糖、葡萄糖、果糖、木糖等多种含羟基及羰基化合物脱氢,并能将L-山梨糖直接转化为2-酮基-L-古龙酸。结论：从酮古龙酸菌SCB329株中分离到一种醇醛脱氢酶基因,可为该菌株糖酸转化机制的研究提供帮助。     

Characteristics and Expression Patterns of the Aldehyde Dehydrogenase (ALDH) Gene Superfamily of Foxtail Millet (Setaria italica L.)

Recent genomic sequencing of the foxtail millet, an abiotic, stress-tolerant crop, has provided a great opportunity for novel gene discovery and functional analysis of this popularly-grown grass. However, few stress-mediated gene families have been studied. Aldehyde dehydrogenases (ALDHs) comprise a gene superfamily encoding NAD (P) ⁺-dependent enzymes that play the role of “aldehyde scavengers”, which indirectly detoxify cellular ROS and reduce the effect of lipid peroxidation meditated cellular toxicity under various environmental stresses. In the current paper, we identified a total of 20 ALDH genes in the foxtail millet genome using a homology search and a phylogenetic analysis and grouped them into ten distinct families based on their amino acid sequence identity. Furthermore, evolutionary analysis of foxtail millet reveals that both tandem and segmental duplication contributed significantly to the expansion of its ALDH genes. The exon-intron structures of members of the same family in foxtail millet or the orthologous genes in rice display highly diverse distributions of their exonic and intronic regions. Also, synteny analysis shows that the majority of foxtail millet and rice ALDH gene homologs exist in the syntenic blocks between the two, implying that these ALDH genes arose before the divergence of cereals. Semi-quantitative and real-time quantitative PCR data reveals that a few SiALDH genes are expressed in an organ-specific manner and that the expression of a number of foxtail millet ALDH genes, such as, SiALDH7B1, SiALDH12A1 and SiALDH18B2 are up-regulated by osmotic stress, cold, H₂O₂, and phytohormone abscisic acid (ABA). Furthermore, the transformation of SiALDH2B2, SiALDH10A2, SiALDH5F1, SiALDH22A1, and SiALDH3E2 into Escherichia coli (E.coli) was able to improve their salt tolerance. Taken together, our results show that genome-wide identification characteristics and expression analyses provide unique opportunities for assessing the functional roles of foxtail millet ALDH genes in stress responses.     

原核表达的人乙醛脱氢酶2(ALDH2)的活性及稳定性研究

对由原核载体表达的人乙醛脱氢酶2(Aldehyde dehydrogenase 2,简称ALDH2)纯化工艺、酶活性改善、稳定性以及保存条件分别进行了参数的优化,以期为ALDH2商品化剂型开发提供理论依据。通过NAD(P)+酶活测定法,检测不同纯化工艺、金属离子以及不同保存条件对ALDH2的酶活性的影响。通过SDS-PAGE检测ALDH2在模拟胃液和胰液中的稳定性。结果显示,低离子磷酸盐缓冲液透析有利于ALDH2酶活性的恢复,且真空冷冻干燥处理可导致ALDH2酶活性下降。K+、Zn2+、Mg2+、Mn2+、Ca2+均能提高ALDH2酶活性。ALDH2在模拟胃液中稳定性良好,但在模拟胰液中迅速被降解。与此同时,ALDH2酶液在-20℃下能良好地保持其稳定性及酶活,但在4℃和30℃下保存一个月酶活急剧下降。以上结果表明,低离子磷酸盐缓冲液透析法、K+均能提高ALDH2的酶活性,同时该酶可耐受模拟胃液的降解作用,且添加山梨酸钾的ALDH2于-20℃可良好地保持其稳定性及酶活。     

Aldehyde Dehydrogenase 3B1 (ALDH3B1): Immunohistochemical Tissue Distribution and Cellular-specific Localization in Normal and Cancerous Human Tissues

Aldehyde dehydrogenase (ALDH) enzymes are critical in the detoxification of endogenous and exogenous aldehydes. Our previous findings indicate that the ALDH3B1 enzyme is expressed in several mouse tissues and is catalytically active toward aldehydes derived from lipid peroxidation, suggesting a potential role against oxidative stress. The aim of this study was to elucidate by immunohistochemistry the tissue, cellular, and subcellular distribution of ALDH3B1 in normal human tissues and in tumors of human lung, colon, breast, and ovary. Our results indicate that ALDH3B1 is expressed in a tissue-specific manner and in a limited number of cell types, including hepatocytes, proximal convoluted tubule cells, cerebellar astrocytes, bronchiole ciliated cells, testis efferent ductule ciliated cells, and histiocytes. ALDH3B1 expression was upregulated in a high percentage of human tumors (lung > breast = ovarian > colon). Increased ALDH3B1 expression in tumor cells may confer a growth advantage or be the result of an induction mechanism mediated by increased oxidative stress. Subcellular localization of ALDH3B1 was predominantly cytosolic in tissues, with the exception of normal human lung and testis, in which localization appeared membrane-bound or membrane-associated. The specificity of ALDH3B1 distribution may prove to be directly related to the functional role of this enzyme in human tissues. (J Histochem Cytochem 58:765–783, 2010)     

Aldehyde Dehydrogenase Gene Superfamily in Populus: Organization and Expression Divergence between Paralogous Gene Pairs

Aldehyde dehydrogenases (ALDHs) constitute a superfamily of NAD(P)⁺-dependent enzymes that catalyze the irreversible oxidation of a wide range of reactive aldehydes to their corresponding nontoxic carboxylic acids. ALDHs have been studied in many organisms from bacteria to mammals; however, no systematic analyses incorporating genome organization, gene structure, expression profiles, and cis-acting elements have been conducted in the model tree species Populus trichocarpa thus far. In this study, a comprehensive analysis of the Populus ALDH gene superfamily was performed. A total of 26 Populus ALDH genes were found to be distributed across 12 chromosomes. Genomic organization analysis indicated that purifying selection may have played a pivotal role in the retention and maintenance of PtALDH gene families. The exon-intron organizations of PtALDHs were highly conserved within the same family, suggesting that the members of the same family also may have conserved functionalities. Microarray data and qRT-PCR analysis indicated that most PtALDHs had distinct tissue-specific expression patterns. The specificity of cis-acting elements in the promoter regions of the PtALDHs and the divergence of expression patterns between nine paralogous PtALDH gene pairs suggested that gene duplications may have freed the duplicate genes from the functional constraints. The expression levels of some ALDHs were up- or down-regulated by various abiotic stresses, implying that the products of these genes may be involved in the adaptation of Populus to abiotic stresses. Overall, the data obtained from our investigation contribute to a better understanding of the complexity of the Populus ALDH gene superfamily and provide insights into the function and evolution of ALDH gene families in vascular plants.     

Evidence for the Heterolobosea from Phylogenetic Analysis of Genes Encoding Glyceraldehyde-3-Phosphate Dehydrogenase

ABSTRACT The phylogenetic relationships between major slime mould groups and the identification of their unicellular relatives has been a subject of controversy for many years. Traditionally, it has been assumed that two slime mould groups, the acrasids and the dictyostelids were related by virtue of their cellular slime mould habit; a view still endorsed by at least one current classification scheme, However, a decade ago, on the basis of detailed ultrastructural resemblances, it was proposed that acrasids of the family Acrasidae were not relatives of other slime moulds but instead related to a group of mostly free-living unicellular amoebae, the Schizopyrenida. The class Heterolobosea was created to contain these organisms and has since figured in many discussions of protist evolution. We sought to test the validity of Heterolobosea by characterizing homologs of the highly conserved glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from an acrasid, Acrasis rosea ; a dictyostelid, Dictyostelium discoideum ; and the schizopyrenid Naegleria andersoni. Phylogenetic analysis of these and other GAPDH sequences, using maximum parsimony, neighbour-joining distance and maximum likelihood methods strongly supports the Heterolobosea hypothesis and discredits the concept of a cellular slime mould grouping. Moreover, all of our analyses place Dictyostelium discoideum as a relatively recently originating lineage, most closely related to the Metazoa, similar to other recently published phylogenies of protein-coding genes. However, GAPDH phylogenies do not show robust branching orders for most of the relationships between major groups. We propose that several of the incongruencies observed between GAPDH and other molecular phylogenies are artifacts resulting from substitutional saturation of this enzyme.     

Gene Cloning and Biochemical Characterization of an Alcohol Dehydrogenase from Euglena gracilis1

ABSTRACT. Euglena gracilis is a freshwater free‐living organism able to grow with ethanol as carbon source; to facilitate this metabolism several alcohol dehydrogenase (ADH) activities have been detected. We report the gene cloning, over‐expression, and biochemical characterization of a medium‐chain NAD⁺‐dependent ADH from E. gracilis (EgADH). The enzyme's amino acid sequence displayed the highest percentages of similarity and identity with ADHs of bacteria and fungi. In the predicted three‐dimensional model, all the residues involved in Zn²⁺, cofactor, and substrate binding were conserved. A conventional signal peptide for import into mitochondria could not be clearly identified. The protein of 37 kDa was over‐expressed, purified to homogeneity, and kinetically characterized. The enzyme's optimal pH was 7.0 for ethanol oxidation displaying a V_m of 11.7±3.6 U/mg protein and a K_m of 3.2±0.7 mM for this substrate. Isopropanol and isopentanol were also utilized, although with less efficiency. It showed specificity for NAD⁺ with a K_m value of 0.39±0.1 mM and Mg²⁺ or Zn²⁺ were essential for activity. The recombinant EgADH reported here may help to elucidate the roles that different ADHs have on the metabolism of short‐ and long‐chain alcohols in this microorganism.     

梭梭甜菜碱醛脱氢酶基因克隆及序列分析

采用RT-PCR、RACE等方法从超旱生、耐盐植物梭梭(Haloxylon ammodendron)中扩增出BADH基因的cDNA序列(命名为HaBADH),其开放阅读框为1 503 bp,推测的氨基酸序列全长为500个氨基酸残基,并含有醛脱氢酶所具有的高度保守的十肽(VTLELGGKSP)以及与酶功能有关的半胱氨酸残基(C).其核苷酸序列与藜科几种盐生植物如盐爪爪(Kalidium foliatum)、中亚滨藜(Atriplex centralasiatica)、三角叶滨藜(Atriplex triangularis)、菠菜(Spinacia oleracea)、山菠菜(Atriplex hortensis)和甜菜(Beta vulgaris)等的相似性均在85%以上,推导编码蛋白的氨基酸序列一致性均在87%以上,表明BADH基因在藜科植物中是一种比较保守的基因.研究结果为进一步从分子水平探明梭梭的抗旱、耐盐机制,挖掘并利用植物抗逆基因奠定基础.     

Modeling-Dependent Protein Characterization of the Rice Aldehyde Dehydrogenase (ALDH) Superfamily Reveals Distinct Functional and Structural Features

The completion of the rice genome sequence has made it possible to identify and characterize new genes and to perform comparative genomics studies across taxa. The aldehyde dehydrogenase (ALDH) gene superfamily encoding for NAD(P)⁺-dependent enzymes is found in all major plant and animal taxa. However, the characterization of plant ALDHs has lagged behind their animal- and prokaryotic-ALDH homologs. In plants, ALDHs are involved in abiotic stress tolerance, male sterility restoration, embryo development and seed viability and maturation. However, there is still no structural property-dependent functional characterization of ALDH protein superfamily in plants. In this paper, we identify members of the rice ALDH gene superfamily and use the evolutionary nesting events of retrotransposons and protein-modeling–based structural reconstitution to report the genetic and molecular and structural features of each member of the rice ALDH superfamily in abiotic/biotic stress responses and developmental processes. Our results indicate that rice-ALDHs are the most expanded plant ALDHs ever characterized. This work represents the first report of specific structural features mediating functionality of the whole families of ALDHs in an organism ever characterized.     

甜菜碱醛脱氢酶(BADH)基因转化小麦及其表达

Betaine aldehyde dehydrogenase (BADH) cDNA cloned from Atriplex hortensis L. in the plasmid pABH9 containing maize ubiquitin promoter and bar gene was transferred into wheat (Triticum aestivum L.) by microprojectile bombardment with 4.1% of average frequency of transformation. From 300 young embryo calli bombarded with the plasmid, 24 transgenic plants were obtained showing BADH gene integration by both PCR and Southern blotting analysis. Among the 24 transgenic plants, 13 exhibited higher BADH activity than the control. Some transgenic plants grew normally with healthy roots on the medium containing 0.7% NaCl while the control plants had very poor roots and finally died.     

甜菜碱醛脱氢酶(BADH)基因转化小麦及其表达

采用基因枪法将山菠菜甜菜碱醛脱氢酶 (BADH)基因导入小麦 (TriticumaestivumL .)品种 ,并且得以表达。该基因由玉米Ubi1启动子控制。在盐胁迫条件下 ,多数转基因植株叶片的BADH活性比受体亲本提高 1～ 3倍 ,部分植株相对电导率比亲本明显低 ,表明转基因植株的细胞膜在胁迫时有受损较轻倾向。PCR和Southern杂交分析证实外源BADH基因已插入小麦基因组 ,平均转化频率为 4.1%。     

甘菊BADH基因cDNA的克隆及在盐胁迫下的表达

利用PCR、RT-PCR和PCR-RACE技术,从菊科植物甘菊(Dendranthema lavandulifolium)中克隆到2个甜菜碱醛脱氢酶(betaine aldehyde dehydrogenase,BADH)基因的同源基因,分别命名为DlBADH1和DlBADH2,GenBank登录号分别为DQ011151和DQ011152.DlBADH1的cDNA全长1821 bp,其开放阅读框编码503个氨基酸的蛋白质;DlBADH2全长1918 bp,编码506个氨基酸的蛋白质.两个基因核苷酸序列的同源性为97%,推导的氨基酸序列的同源性为98%.与已发表的其它植物BADH基因氨基酸序列的同源性在64%以上.在推导的氨基酸序列中,均含有醛脱氢酶所具有的高度保守的十肽(VTLELGGKSP)以及与酶功能有关的半胱氨酸残基(C).在推导的氨基酸序列的系统关系中,甘菊位于其它双子叶植物和单子叶植物之间,与其植物分类的系统关系相吻合.RT-PCR-Southern半定量表达分析表明,甘菊BADH基因家族中存在表达受盐诱导的成员.     

甘菊BADH基因cDNA的克隆及在盐胁迫下的表达

利用PCR、RT—PCR和PCR—RACE技术,从菊科植物甘菊（Dendranthema lavandulifolium）中克隆到2个甜菜碱醛脱氢酶（betaine aldehyde dehydrogenase,BADH）基因的同源基因,分别命名为DlBADH1和DlBADH2,GenBank登录号分别为DQ011151和DQ011152。DlBADH1的cDNA全长1821bp,其开放阅读框编码503个氨基酸的蛋白质;DlBADH2全长1918bp,编码506个氨基酸的蛋白质。两个基因核苷酸序列的同源性为97％,推导的氨基酸序列的同源性为98％。与已发表的其它植物BADH基因氨基酸序列的同源性在64％以上。在推导的氨基酸序列中,均含有醛脱氢酶所具有的高度保守的十肽（VTLELGGKSP）以及与酶功能有关的半胱氨酸残基（C）。在推导的氨基酸序列的系统关系中,甘菊位于其它双子叶植物和单子叶植物之间,与其植物分类的系统关系相吻合。RT—PCR—Southern半定量表达分析表明,甘菊BADH基因家族中存在表达受盐诱导的成员。     

The Activity of Class I, II, III and IV of Alcohol Dehydrogenase (ADH) Isoenzymes and Aldehyde Dehydrogenase (ALDH) in Brain Cancer

The brain being highly sensitive to the action of alcohol is potentially susceptible to its carcinogenic effects. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are the main enzymes involved in ethanol metabolism, which leads to the generation of carcinogenic acetaldehyde. Human brain tissue contains various ADH isoenzymes and possess also ALDH activity. The purpose of this study was to compare the capacity for ethanol metabolism measured by ADH isoenzymes and ALDH activity in cancer tissues and healthy brain cells. The samples were taken from 62 brain cancer patients (36 glioblastoma, 26 meningioma). For the measurement of the activity of class I and II ADH isoenzymes and ALDH activity, the fluorometric methods were used. The total ADH activity and activity of class III and IV isoenzymes were measured by the photometric method. The total activity of ADH, and activity of class I ADH were significantly higher in cancer cells than in healthy tissues. The other tested classes of ADH and ALDH did not show statistically significant differences of activity in cancer and in normal cells. Analysis of the enzymes activity did not show significant differences depending on the location of the tumor. The differences in the activity of total alcohol dehydrogenase, and class I isoenzyme between cancer tissues and healthy brain cells might be a factor for metabolic changes and disturbances in low mature cancer cells and additionally might be a reason for higher level of acetaldehyde which can intensify the carcinogenesis.     

Structural and Biochemical Characterization of Aldehyde Dehydrogenase 12, the Last Enzyme of Proline Catabolism in Plants

Heterokonts, Alveolata protists, green algae from Charophyta and Chlorophyta divisions, and all Embryophyta plants possess an aldehyde dehydrogenase (ALDH) gene named ALDH12. Here, we provide a biochemical characterization of two ALDH12 family members from the lower plant Physcomitrella patens and higher plant Zea mays. We show that ALDH12 encodes an NAD⁺-dependent glutamate γ-semialdehyde dehydrogenase (GSALDH), which irreversibly converts glutamate γ-semialdehyde (GSAL), a mitochondrial intermediate of the proline and arginine catabolism, to glutamate. Sedimentation equilibrium and small-angle X-ray scattering analyses reveal that in solution both plant GSALDHs exist as equilibrium between a domain-swapped dimer and the dimer-of-dimers tetramer. Plant GSALDHs share very low-sequence identity with bacterial, fungal, and animal GSALDHs (classified as ALDH4), which are the closest related ALDH superfamily members. Nevertheless, the crystal structure of ZmALDH12 at 2.2-Å resolution shows that nearly all key residues involved in the recognition of GSAL are identical to those in ALDH4, indicating a close functional relationship with ALDH4. Phylogenetic analysis suggests that the transition from ALDH4 to ALDH12 occurred during the evolution of the endosymbiotic plant ancestor, prior to the evolution of green algae and land plants. Finally, ALDH12 expression in maize and moss is downregulated in response to salt and drought stresses, possibly to maintain proline levels. Taken together, these results provide molecular insight into the biological roles of the plant ALDH12 family.     

甘菊BADH基因cDNA的克隆及在盐胁迫下的表达

利用PCR、RT-PCR和PCR-RACE技术,从菊科植物甘菊(Dendranthema lavandulifolium)中克隆到2个甜菜碱醛脱氢酶(betaine aldehyde dehydrogenase,BADH)基因的同源基因,分别命名为DlBADH1和DlBADH2,GenBank登录号分别为DQ011151和DQ011152。DlBADH1的cDNA全长1821 bp,其开放阅读框编码503个氨基酸的蛋白质;DlBADH2全长1918 bp,编码506个氨基酸的蛋白质。两个基因核苷酸序列的同源性为97%,推导的氨基酸序列的同源性为98%。与已发表的其它植物BADH基因氨基酸序列的同源性在64%以上。在推导的氨基酸序列中,均含有醛脱氢酶所具有的高度保守的十肽(VTLELGGKSP)以及与酶功能有关的半胱氨酸残基(C)。在推导的氨基酸序列的系统关系中,甘菊位于其它双子叶植物和单子叶植物之间,与其植物分类的系统关系相吻合。RT-PCR-Southern半定量表达分析表明,甘菊BADH基因家族中存在表达受盐诱导的成员。     

Distinct Expression Levels and Patterns of Stem Cell Marker,Aldehyde Dehydrogenase Isoform 1 (ALDH1), in Human Epithelial Cancers

Aldehyde dehydrogenase isoform 1 (ALDH1) has been proved useful for the identification of cancer stem cells. However, our knowledge of the expression and activity of ALDH1 in common epithelial cancers and their corresponding normal tissues is still largely absent. Therefore, we characterized ALDH1 expression in 24 types of normal tissues and a large collection of epithelial tumor specimens (six cancer types, n = 792) by immunohistochemical staining. Using the ALDEFUOR assay, ALDH1 activity was also examined in 16 primary tumor specimens and 43 established epithelial cancer cell lines. In addition, an ovarian cancer transgenic mouse model and 7 murine ovarian cancer cell lines were analyzed. We found that the expression levels and patterns of ALDH1 in epithelial cancers are remarkably distinct, and they correlate with their corresponding normal tissues. ALDH1 protein expression levels are positively correlated with ALDH1 enzymatic activity measured by ALDEFLUOR assay. Long-term in vitro culture doesn''t significantly affect ALDH1 activity in epithelial tumor cells. Consistent with research on other cancers, we found that high ALDH1 expression is significantly associated with poor clinical outcomes in serous ovarian cancer patients (n = 439, p = 0.0036). Finally, ALDH^br tumor cells exhibit cancer stem cell properties and are resistant to chemotherapy. As a novel cancer stem cell marker, ALDH1 can be used for tumors whose corresponding normal tissues express ALDH1 in relatively restricted or limited levels such as breast, lung, ovarian or colon cancer.