The gene carD encodes the aldehyde dehydrogenase responsible for neurosporaxanthin biosynthesis in Fusarium fujikuroi

Neurosporaxanthin (β-apo-4'-carotenoic acid) biosynthesis has been studied in detail in the fungus Fusarium fujikuroi. The genes and enzymes for this biosynthetic pathway are known until the last enzymatic step, the oxidation of the aldehyde group of its precursor, β-apo-4'-carotenal. On the basis of sequence homology to Neurospora crassa YLO-1, which mediates the formation of apo-4'-lycopenoic acid from the corresponding aldehyde substrate, we cloned the carD gene of F. fujikuroi and investigated the activity of the encoded enzyme. In vitro assays performed with heterologously expressed protein showed the formation of neurosporaxanthin and other apocarotenoid acids from the corresponding apocarotenals. To confirm this function in vivo, we generated an Escherichia coli strain producing β-apo-4'-carotenal, which was converted into neurosporaxanthin upon expression of carD. Moreover, the carD function was substantiated by its targeted disruption in a F. fujikuroi carotenoid-overproducing strain, which resulted in the loss of neurosporaxanthin and the accumulation of β-apo-4'-carotenal, its derivative β-apo-4'-carotenol, and minor amounts of other carotenoids. Intermediates accumulated in the ΔcarD mutant suggest that the reactions leading to neurosporaxanthin in Neurospora and Fusarium are different in their order. In contrast to ylo-1 in N. crassa, carD mRNA content is enhanced by light, but to a lesser extent than other enzymatic genes of the F. fujikuroi carotenoid pathway. Furthermore, carD mRNA levels were higher in carotenoid-overproducing mutants, supporting a functional role for CarD in F. fujikuroi carotenogenesis. With the genetic and biochemical characterization of CarD, the whole neurosporaxanthin biosynthetic pathway of F. fujikuroi has been established.     

Identification of the gene responsible for torulene cleavage in the Neurospora carotenoid pathway

Torulene, a C₄₀ carotene, is the precursor of the end product of the Neurospora carotenoid pathway, the C₃₅ xanthophyll neurosporaxanthin. Torulene is synthesized by the enzymes AL-2 and AL-1 from the precursor geranylgeranyl diphosphate and then cleaved by an unknown enzyme into the C₃₅ apocarotenoid. In general, carotenoid cleavage reactions are catalyzed by carotenoid oxygenases. Using protein data bases, we identified two putative carotenoid oxygenases in Neurospora, named here CAO-1 and CAO-2. A search for novel mutants of the carotenoid pathway in this fungus allowed the identification of two torulene-accumulating strains, lacking neurosporaxanthin. Sequencing of the cao-2 gene in these strains revealed severe mutations, pointing to a role of CAO-2 in torulene cleavage. This was further supported by the identical phenotype found upon targeted disruption of cao-2. The biological function was confirmed by in vitro assays using the purified enzyme, which cleaved torulene to produce β-apo-4′-carotenal, the corresponding aldehyde of neurosporaxanthin. The specificity of CAO-2 was shown by the lack of γ-carotene-cleaving activity in vitro. As predicted for a structural gene of the carotenoid pathway, cao-2 mRNA was induced by light in a WC-1 and WC-2 dependent manner. Our data demonstrate that CAO-2 is the enzyme responsible for the oxidative cleavage of torulene in the neurosporaxanthin biosynthetic pathway.     

The Arabidopsis thaliana REDUCED EPIDERMAL FLUORESCENCE1 gene encodes an aldehyde dehydrogenase involved in ferulic acid and sinapic acid biosynthesis

Recent research has significantly advanced our understanding of the phenylpropanoid pathway but has left in doubt the pathway by which sinapic acid is synthesized in plants. The reduced epidermal fluorescence1 (ref1) mutant of Arabidopsis thaliana accumulates only 10 to 30% of the sinapate esters found in wild-type plants. Positional cloning of the REF1 gene revealed that it encodes an aldehyde dehydrogenase, a member of a large class of NADP(+)-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. Consistent with this finding, extracts of ref1 leaves exhibit low sinapaldehyde dehydrogenase activity. These data indicate that REF1 encodes a sinapaldehyde dehydrogenase required for sinapic acid and sinapate ester biosynthesis. When expressed in Escherichia coli, REF1 was found to exhibit both sinapaldehyde and coniferaldehyde dehydrogenase activity, and further phenotypic analysis of ref1 mutant plants showed that they contain less cell wall-esterified ferulic acid. These findings suggest that both ferulic acid and sinapic acid are derived, at least in part, through oxidation of coniferaldehyde and sinapaldehyde. This route is directly opposite to the traditional representation of phenylpropanoid metabolism in which hydroxycinnamic acids are instead precursors of their corresponding aldehydes.     

A tomato gene expressed during fruit ripening encodes an enzyme of the carotenoid biosynthesis pathway.

In the initial stages of carotenoid biosynthesis in plants the enzyme phytoene synthase converts two molecules of geranylgeranyl diphosphate into phytoene, the first carotenoid of the pathway. We show here that a tomato (Lycopersicon esculentum) cDNA for a gene (Psy1) expressed during fruit ripening directs the in vitro synthesis of a 47-kDa protein which, upon import into isolated chloroplasts, is processed to a mature 42-kDa form. The imported protein is largely associated with membranes, but it can be easily solubilized by dilution or by treatment at high pH. A plasmid construct containing prokaryotic promoter and ribosome-binding sequences fused to the Psy1 cDNA complements the carotenoidless phenotype of a Rhodobacter capsulatus crtB mutant. We conclude that Psy1 encodes phytoene synthase and that this enzyme is a peripheral plastid membrane protein.     

Reversal of part of the aldehyde dehydrogenase reaction pathway during the hydrolysis of an ester.

An aldehyde dehydrogenase from rabbit liver, a homogeneous protein on three distinct polyacrylamide-gel systems, has an associated 4-nitrophenyl esterase activity. At pH 7.0 in the presence of 80 micrometer-NADH and 800 micrometer-4-nitrophenyl acetate the enzyme produces NAD+ and a stoicheiometric amount of an aldehyde, as well as hydrolysing the ester. On this and other evidence it is proposed that ester hydrolysis occurs at the usual active site of the enzyme.     

The Pseudomonas oleovorans alkBAC operon encodes two structurally related rubredoxins and an aldehyde dehydrogenase

The Pseudomonas oleovorans alkBAC operon encodes seven proteins, of which at least three are involved in alkane hydroxylase (alkBA) and alkanol dehydrogenase (alkC) activities. We have determined the nucleotide sequence of the 2.5-kilobase pair alkA region and analyzed the role of its translation products in alkane oxidation. The alkA region contains three coding sequences, encoding two related rubredoxins (alkF and alkG) of 14- and 18-kDa molecular mass and a 52-kDa aldehyde dehydrogenase (alkH). Deletion analysis indicated that neither the 14-kDa alkF gene product (rubredoxin 1) nor the amino-terminal part of the 18-kDa alkG gene product (rubredoxin 2) is required for alkane hydroxylase activity in vivo. The product of the alkH cistron restores growth of a P. oleovorans aldehyde dehydrogenase mutant on aliphatic alcohols and aldehydes. Its amino acid sequence shows considerable homology to previously characterized aldehyde dehydrogenases from mammalian and fungal origin. The nucleotide composition of the alk genes (47% G + C) differs considerably from the G + C content of the P. oleovorans genome suggesting that the alk regulon may originate from an unrelated organism.     

The arabidopsis DELAYED DEHISCENCE1 gene encodes an enzyme in the jasmonic acid synthesis pathway

delayed dehiscence1 is an Arabidopsis T-DNA mutant in which anthers release pollen grains too late for pollination to occur. The delayed dehiscence1 defect is caused by a delay in the stomium degeneration program. The gene disrupted in delayed dehiscence1 encodes 12-oxophytodienoate reductase, an enzyme in the jasmonic acid biosynthesis pathway. We rescued the mutant phenotype by exogenous application of jasmonic acid and obtained seed set from previously male-sterile plants. In situ hybridization studies showed that during the early stages of floral development, DELAYED DEHISCENCE1 mRNA accumulated within all floral organs. Later, DELAYED DEHISCENCE1 mRNA accumulated specifically within the pistil, petals, and stamen filaments. DELAYED DEHISCENCE1 mRNA was not detected in the stomium and septum cells of the anther that are involved in pollen release. The T-DNA insertion in delayed dehiscence1 eliminated both DELAYED DEHISCENCE1 mRNA accumulation and 12-oxophytodienoate reductase activity. These experiments suggest that jasmonic acid signaling plays a role in controlling the time of anther dehiscence within the flower.     

dim-2 encodes a DNA methyltransferase responsible for all known cytosine methylation in Neurospora

To understand better the control of DNA methylation, we cloned and characterized the dim-2 gene of Neurospora crassa, the only eukaryotic gene currently known in which mutations appear to eliminate DNA methylation. The dim-2 gene is responsible for methylation in both symmetrical and asymmetrical sites. We mapped dim-2 between wc-1 and un-10 on linkage group (LG) VIIR and identified the gene by RFLP mapping and genetic complementation. Dim-2 encodes a 1454 amino acid protein including a C-terminal domain homologous to known DNA methyltransferases (MTases) and a novel N-terminal domain. Neither a deletion that removed the first 186 amino acids of the protein nor a mutation in a putative nucleotide binding site abolished function, but a single amino acid substitution in the predicted catalytic site did. Tests for repeat-induced point mutation (RIP) indicated that dim-2 does not play a role in this process, i.e. duplicated sequences are mutated in dim-2 strains, as usual, but the mutated sequences are not methylated, unlike the situation in dim-2+ strains. We conclude that dim-2 encodes an MTase that is responsible for all DNA methylation in vegetative tissues of NEUROSPORA:     

The Bacillus subtilis yqjI gene encodes the NADP+-dependent 6-P-gluconate dehydrogenase in the pentose phosphate pathway

Despite the importance of the oxidative pentose phosphate (PP) pathway as a major source of reducing power and metabolic intermediates for biosynthetic processes, almost no direct genetic or biochemical evidence is available for Bacillus subtilis. Using a combination of knockout mutations in known and putative genes of the oxidative PP pathway and 13C-labeling experiments, we demonstrated that yqjI encodes the NADP+-dependent 6-P-gluconate dehydrogenase, as was hypothesized previously from sequence similarities. Moreover, YqjI was the predominant isoenzyme during glucose and gluconate catabolism, and its role in the oxidative PP pathway could not be played by either of two homologues, GntZ and YqeC. This conclusion is in contrast to the generally held view that GntZ is the relevant isoform; hence, we propose a new designation for yqjI, gndA, the monocistronic gene encoding the principal 6-P-gluconate dehydrogenase. Although we demonstrated the NAD+-dependent 6-P-gluconate dehydrogenase activity of GntZ, gntZ mutants exhibited no detectable phenotype on glucose, and GntZ did not contribute to PP pathway fluxes during growth on glucose. Since gntZ mutants grew normally on gluconate, the functional role of GntZ remains obscure, as does the role of the third homologue, YqeC. Knockout of the glucose-6-P dehydrogenase-encoding zwf gene was primarily compensated for by increased glycolytic fluxes, but about 5% of the catabolic flux was rerouted through the gluconate bypass with glucose dehydrogenase as the key enzyme.     

大豆醛脱氢酶基因GmALDH3-1的克隆及在生殖器官中的表达

通过基因芯片技术,从大豆中鉴定了一个花优势表达基因,其在大豆花中的表达量为叶片中的85倍。通过生物信息学方法,拼接了该基因的全长序列,并通过RT-PCR克隆了该基因。BLAST检索分析表明该基因编码醛脱氢酶,命名为GmALDH3-1。GmALDH3-1包含一个1485 bp的开放阅读框,编码494个氨基酸残基。GmADLH3-1与白杨的醛脱氢酶PtALDH3相似性最高（氨基酸相似率83%,一致率为68%）,而与来自于人的ALDH3B的氨基酸一致率和相似率分别为39%和59%。系统发生分析表明GmALDH3-1与其它植物ALDH3亚家族成员位于一个分支,且与白杨PtALDH3和拟南芥AtALDH3F1亲缘关系最近。采用实时定量RT-PCR检测了GmALDH3-1基因在大豆叶、根和花中的表达,结果表明GmALDH3-1基因在花中高丰度表达,在根和叶中未检测到表达。运用基因芯片信息分析了GmALDH3-1在种子发育过程中的表达情况,结果表明GmALDH3-1在种子发育过程中的外表皮、内表皮、外胚珠和种脐中表达量较高。文章讨论了GmALDH3-1基因在大豆生殖器官发育中可能发挥的作用。     

The structural gene for the mitochondrial aldehyde dehydrogenase maps to human chromosome 12

Summary A cloned 850 bp cDNA fragment corresponding to the 3-coding part of human ALDHI-mRNA was used as a probe for the chromosomal assignment of the ALDHI gene. Southern blot analysis of human-rodent somatic cell hybrids indicates that the human ALDHI gene resides on chromosome 12.Dedicated to Prof. Dr. H. Holzer on the occasion of his 65. birthday     

Elucidating the reaction mechanism of the benzoate oxidation pathway encoded aldehyde dehydrogenase from Burkholderia xenovorans LB400

Oxidation of cis-3,4-dehydroadipyl-CoA semialdehyde to cis-3,4-dehydroadipyl-CoA by the aldehyde dehydrogenase, ALDH(C) (EC.1.2.1.77), is an essential step in the metabolism of benzoate in Burkholderia xenovorans LB400. In a previous study, we established a structural blueprint for this novel group of ALDH enzymes. Here, we build significantly on this initial work and propose a detailed reaction mechanism for ALDH(C) based on comprehensive structural and functional investigations of active site residues. Kinetic analyses reveal essential roles for C296 as the nucleophile and E257 as the associated general base. Structural analyses of E257Q and C296A variants suggest a dynamic charge repulsion relationship between E257 and C296 that contributes to the inherent flexibility of E257 in the native enzyme, which is further regulated by E496 and E167. A proton relay network anchored by E496 and supported by E167 and K168 serves to reset E257 for the second catalytic step. We also propose that E167, which is unique to ALDH(C) and its homologs, serves a critical role in presenting the catalytic water to the newly reset E257 such that the enzyme can proceed with deacylation and product release. Collectively, the reaction mechanism proposed for ALDH(C) promotes a greater understanding of these novel ALDH enzymes, the ALDH super-family in general, and benzoate degradation in B. xenovorans LB400.     

Developmental regulation of the gene for formate dehydrogenase in Neurospora crassa.

We have isolated and characterized a gene, fdh, from Neurospora crassa which is developmentally regulated and which produces formate dehydrogenase activity when expressed in Escherichia coli. The gene is closely linked (less than 0.6 kb apart) to the leu-5 gene encoding mitochondrial leucyl-tRNA synthetase; the two genes are transcribed convergently from opposite strands. The expression patterns of these genes differ: fdh mRNA is found only during conidiation and early germination and is not detectable during mycelial growth, while leu-5 mRNA appears during germination and mycelial growth. The structure of the fdh gene was determined from the sequence of cDNA and genomic DNA clones and from mRNA mapping studies. The gene encodes a 375-amino-acid-long protein with sequence similarity to NAD-dependent dehydrogenases of the E. coli 3-phosphoglycerate dehydrogenase (serA gene product) subfamily. In particular, there is striking sequence similarity (52% identity) to formate dehydrogenase from Pseudomonas sp. strain 101. All of the residues thought to interact with NAD in the crystal structure of the Pseudomonas enzyme are conserved in the N. crassa enzyme. We have further shown that expression of the N. crassa gene in E. coli leads to the production of formate dehydrogenase activity, indicating that the N. crassa gene specifies a functional polypeptide.     

The Neurospora aab-1 gene encodes a CCAAT binding protein homologous to yeast HAP5.

The expression of the am (glutamate dehydrogenase) gene is dependent upon two upstream activating sequences, designated URSam(alpha) and URSam(beta). A heteromeric nuclear protein Am Alpha Binding protein (AAB) binds specifically to a CCAAT box within the URSam(alpha) element. AAB appears to be composed of three components. We used polyclonal antiserum raised against the highly purified AAB1 subunit to isolate a partial aab-1 cDNA clone, which was then used to isolate a full-length cDNA and a genomic clone. The full-length cDNA has the potential to encode a 272 amino acid protein with a calculated molecular weight of 30 kD. Amino acid sequence obtained by Edman analysis of the AAB1 protein confirmed that the aab-1 gene had been cloned. AAB-1 shows similarity to the HAP5 protein of yeast and the CBF-C protein of rat. Each of these proteins is an essential subunit of their respective heteromeric CCAAT binding proteins. The aab1 gene maps on linkage group III of Neurospora crassa near the trp-1 locus. Disruption of the aab-1 gene results in pleiotropic effects on growth and development as well as a 50% reduction in glutamate dehydrogenase levels. Transformation of the aab-1 disruption mutant strain with the cloned genomic copy of the aab-1 gene rescued all of the phenotypic alterations associated with the aab-1 mutation.     

The PSO4 gene is responsible for an error-prone recombinational DNA repair pathway in Saccharomyces cerevisiae

Summary The induction of mitotic gene conversion and crossing-over inSaccharomyces cerevisiae diploid cells homozygous for thepso4-1 mutation was examined in comparison to the corresponding wild-type strain. Thepso4-1 mutant strain was found to be completely blocked in mitotic recombination induced by photoaddition of mono- and bifunctional psoralen derivatives as well as by mono- (HN1) and bifunctional (HN2) nitrogen mustards or 254 nm UV radiation in both stationary and exponential phases of growth. Concerning the lethal effect, diploids homozygous for thepso4-1 mutation are more sensitive to all agents tested in any growth phase. However, this effect is more pronounced in the G2 phase of the cell cycle. These results imply that the ploidy effect and the resistance of budding cells are under the control of thePSO4 gene. On the other hand, thepso4-1 mutant is mutationally defective for all agents used. Therefore, thepso4-1 mutant has a generalized block in both recombination and mutation ability. This indicates that thePSO4 gene is involved in an error-prone repair pathway which relies on a recombinational mechanism, strongly suggesting an analogy between thepso4-1 mutation and theRecA orLexA mutation ofEscherichia coli.