A novel alpha-ketoglutaric semialdehyde dehydrogenase: evolutionary insight into an alternative pathway of bacterial L-arabinose metabolism

Azospirillum brasilense possesses an alternative pathway of l-arabinose metabolism, which is different from the known bacterial and fungal pathways. In a previous paper (Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 2612-2623), we identified and characterized l-arabinose 1-dehydrogenase, which catalyzes the first reaction step in this pathway, and we cloned the corresponding gene. Here we focused on the fifth enzyme, alpha-ketoglutaric semialdehyde (alphaKGSA) dehydrogenase, catalyzing the conversion of alphaKGSA to alpha-ketoglutarate. alphaKGSA dehydrogenase was purified tentatively as a NAD(+)-preferring aldehyde dehydrogenase (ALDH) with high activity for glutaraldehyde. The gene encoding this enzyme was cloned and shown to be located on the genome of A. brasilense separately from a gene cluster containing the l-arabinose 1-dehydrogenase gene, in contrast with Burkholderia thailandensis in which both genes are located in the same gene cluster. Higher catalytic efficiency of ALDH was found with alphaKGSA and succinic semialdehyde among the tested aldehyde substrates. In zymogram staining analysis with the cell-free extract, a single active band was found at the same position as the purified enzyme. Furthermore, a disruptant of the gene did not grow on l-arabinose. These results indicated that this ALDH gene was the only gene of the NAD(+)-preferring alphaKGSA dehydrogenase in A. brasilense. In the phylogenetic tree of the ALDH family, alphaKGSA dehydrogenase from A. brasilense falls into the succinic semialdehyde dehydrogenase (SSALDH) subfamily. Several putative alphaKGSA dehydrogenases from other bacteria belong to a different ALDH subfamily from SSALDH, suggesting strongly that their substrate specificities for alphaKGSA are acquired independently during the evolutionary stage. This is the first evidence of unique "convergent evolution" in the ALDH family.     

Cloning and characterization of an adenosine kinase from Physcomitrella involved in cytokinin metabolism

Adenosine kinase (adk) from the moss Physcomitrella patens (Hedw.) B.S.G. was cloned from a cDNA library by functional complementation of an Escherichia coli purine auxotrophic strain. The length of the entire cDNA clone was 1175 bp with an open reading frame coding for a protein with a predicted molecular weight of 37.3 kDa. Southern analysis indicated the presence of a single adenosine kinase gene within the Physcomitrella genome. The deduced amino acid sequence had a 52% identity with the human adenosine kinase. The transfer of phosphate from ATP to adenosine resulting in AMP, as well as the phosphorylation of the cytokinin, isopentenyladenosine, to isopentenyladenosine monophosphate, was shown by in vitro enzyme assays using crude extracts from E. coli mutants expressing the adk cDNA clone and from Physcomitrella chloronemal tissue. Results from in vivo feeding of chloronemal tissue with tritiated isopentenyladenosine suggest that adenosine kinase plays an important role in the conversion of cytokinins towards their nucleotides in Physcomitrella.     

Eukaryotic and bacterial gene clusters related to an alternative pathway of nonphosphorylated L-rhamnose metabolism

The Entner-Doudoroff (ED) pathway is a classic central pathway of d-glucose metabolism in all three phylogenetic domains. On the other hand, Archaea and/or bacteria possess several modified versions of the ED pathway, in which nonphosphorylated intermediates are involved. Several fungi, including Pichia stipitis and Debaryomyces hansenii, possess an alternative pathway of L-rhamnose metabolism, which is different from the known bacterial pathway. Gene cluster related to this hypothetical pathway was identified by bioinformatic analysis using the metabolic enzymes involved in analogous sugar pathways to the ED pathway. Furthermore, the homologous gene cluster was found not only in many other fungi but also several bacteria, including Azotobacter vinelandii. Four putative metabolic genes, LRA1-4, were cloned, overexpressed in Escherichia coli, and purified. Substrate specificity and kinetic analysis revealed that nonphosphorylated intermediates related to L-rhamnose are significant active substrates for the purified LRA1-4 proteins. Furthermore, L-2-keto-3-deoxyrhamnonate was structurally identified as both reaction products of dehydration by LRA3 and aldol condensation by LRA4. These results suggested that the LRA1-4 genes encode L-rhamnose 1-dehydrogenase, L-rhamnono-gamma-lactonase, L-rhamnonate dehydratase, and L-KDR aldolase, respectively, by which L-rhamnose is converted into pyruvate and L-lactaldehyde through analogous reaction steps to the ED pathway. There was no evolutionary relationship between L-KDR aldolases from fungi and bacteria.     

Cloning,expression, and alternative splicing of neogenin1 in zebrafish

Nek6 and Nek7 are evolutionarily conserved murine kinases structurally related to the Aspergillus mitotic-regulator NIMA (Genomics 68 (2000) 187). Comparative in situ examination of their patterns of expression revealed that during early embryogenesis nek6 is highly expressed in primary giant trophoblast cells, while nek7 is expressed in the site of decidual reaction. Later in embryogenesis, both RNAs are almost exclusively restricted to the nervous system. nek6 is found in ventricular and sub-ventricular regions, while nek7 is highly expressed in the dorsal thalamus. In the adult brain, distinct nuclei express the two genes. The lineage- and tissue-specific patterns of expression suggest that the two NIMA-related kinases have (additional) functions that are not related to the mitotic functions of NIMA.     

Biochemical characterization of WbpA, a UDP-N-acetyl-D-glucosamine 6-dehydrogenase involved in O-antigen biosynthesis in Pseudomonas aeruginosa PAO1

WbpA (PA3159) is an enzyme involved in the biosynthesis of unusual di-N-acetyl-d-mannosaminuronic acid-derived sugar nucleotides found in the O antigen of Pseudomonas aeruginosa PAO1 (serotype O5). The wbpA gene that encodes this enzyme was cloned into pET-28a, overexpressed as a histidine-tagged fusion protein, and purified by nickel chelation chromatography. Capillary electrophoresis was used to examine substrate conversion by WbpA, and the data revealed that WbpA is a UDP-N-acetyl-D-glucosamine 6-dehydrogenase (EC 1.1.1.136), which uses NAD(+) as a coenzyme. The enzyme reaction product was purified by HPLC and analyzed using NMR spectroscopy. Our results showed unequivocally that the product of the WbpA reaction is UDP-N-acetyl-d-glucosaminuronic acid. WbpA requires either NH(4)(+) or K(+) for activity and the accompanying anions exert secondary effects on activity consistent with their ranking in the Hofmeister series. Kinetic analysis showed positive cooperativity with respect to UDP-N-acetyl-d-glucosamine binding with a K(0.5) of 94 microM, a k(cat) of 86 min(-1), and a Hill coefficient of 1.8. In addition, WbpA has a K(0.5) for NAD(+) of 220 microM, a k(cat) of 86 min(-1), and a Hill coefficient of 1.1. The oligomerization state of WbpA was analyzed by gel filtration, dynamic light scattering, and analytical ultracentrifugation, with all three techniques indicating that WbpA exists as a trimer in solution. However, tertiary structure predictions suggested a tetramer, which was supported by data from transmission electron microscopy. The electron micrograph of negatively stained WbpA samples revealed structures with 4-fold symmetry.     

Cloning and expression of a fungal L-arabinitol 4-dehydrogenase gene

L-Arabinitol 4-dehydrogenase (EC ) was purified from the filamentous fungus Trichoderma reesei (Hypocrea jecorina). It is an enzyme in the L-arabinose catabolic pathway of fungi catalyzing the reaction from L-arabinitol to L-xylulose. The amino acid sequence of peptide fragments was determined and used to identify the corresponding gene. We named the gene lad1. It is not constitutively expressed. In a Northern analysis we found it only after growth on L-arabinose. The gene was cloned and overexpressed in Saccharomyces cerevisiae, and the enzyme activity was confirmed in a cell extract. The enzyme consists of 377 amino acids and has a calculated molecular mass of 39,822 Da. It belongs to the family of zinc-binding dehydrogenases and has some amino acid sequence similarity to sorbitol dehydrogenases. It shows activity toward L-arabinitol, adonitol (ribitol), and xylitol with K(m) values of about 40 mM toward L-arabinitol and adonitol and about 180 mM toward xylitol. No activity was observed with D-sorbitol, D-arabinitol, and D-mannitol. NAD is the required cofactor with a K(m) of 180 microM. No activity was observed with NADP.     

Cloning, sequencing, and expression of the Pseudomonas testosteroni gene encoding 3-oxosteroid delta 1-dehydrogenase.

Pseudomonas testosteroni ATCC 17410 is able to grow on testosterone. This strain was mutagenized by Tn5, and 41 mutants defective in the utilization of testosterone were isolated. One of them, called mutant 06, expressed 3-oxosteroid delta 1- and 3-oxosteroid delta 4-5 alpha-dehydrogenases only at low levels. The DNA region around the Tn5 insertion in mutant 06 was cloned into pUC19, and the 1-kbp EcoRI-BamHI segment neighbor to the Tn5 insertion was used to probe DNA from the wild-type strain. The probe hybridized to a 7.8-kbp SalI fragment. Plasmid pTES5, which is a pUC19 derivative containing this 7.8-kbp SalI fragment, was isolated after the screening by the 1-kbp EcoRI-BamHI probe. This plasmid expressed delta 1-dehydrogenase in Escherichia coli cells. The 2.2-kbp KpnI-KpnI segment of pTES5 was subcloned into pUC18, and pTEK21 was constructed. In E. coli containing the lacIq plasmid pRG1 and pTEK21, the expression of delta 1-dehydrogenase was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The induced level was about 40 times higher than the induced level in P. testosteroni. Delta 1-Dehydrogenase synthesized in E. coli was localized in the inner membrane fraction. The minicell experiments showed that a 59-kDa polypeptide was synthesized from pTEK21, and this polypeptide was located in the inner membrane fraction. The complete nucleotide sequence of the 2.2-kbp KpnI-KpnI segment of pTEK21 was determined. An open reading frame which encodes a 62.4-kDa polypeptide and which is preceded by a Shine-Dalgarno-like sequence was identified. The first 44 amino acids of the putative product exhibited significant sequence similarity to the N-terminal sequences of lipoamide dehydrogenases.     

Cloning, heterologous expression, and characterization of a phenylalanine aminomutase involved in Taxol biosynthesis

Biosynthesis of the N-benzoyl phenylisoserinoyl side chain of the anticancer drug Taxol starts with the conversion of 2S-alpha-phenylalanine to 3R-beta-phenylalanine by phenylalanine aminomutase (PAM). A gene cloning approach was based on the assumption that PAM would resemble the well known plant enzyme phenylalanine ammonia lyase. A phenylalanine ammonia lyase-like sequence acquired from a Taxus cuspidata cDNA library was expressed functionally in Escherichia coli and confirmed as the target aminomutase that is virtually identical to the recombinant enzyme and clone from Taxus chinensis, acquired recently by a reverse genetics approach (Bristol-Myers Squibb (August 14, 2003) U. S. Patent WO 03/066871 A2). The full-length cDNA has an open reading frame of 2094 base pairs and encodes a protein of 698 residues with a calculated molecular mass of 76,530 Da. The recombinant mutase has a pH optimum of 8.5, a k(cat) value of 0.015 s(-1), and a K(m) of 45 +/- 8 microm for 2S-alpha-phenylalanine. The stereochemical mechanism of PAM involves the removal and interchange of the pro-3S hydrogen and the amino group, which rebonds at C-3 with retention of configuration. The recombinant enzyme appears to catalyze both the forward and reverse reactions with specificity for both 2S-alpha-phenylalanine and 3S- or 3R-beta-phenylalanine substrates, respectively, whereas the related phenylpropanoids 2S-aminocyclohexanepropanoic acid, 2R-alpha-phenylalanine, and 2S-alpha-tyrosine are not converted to their beta-isomers by the mutase.     

Cloning, bacterial expression, purification and structural characterization of N-terminal-repetitive domain of gamma-Gliadin

The gene encoding the repetitive domain located in the N-terminal half of gamma-Gliadin from wheat endosperm has been subcloned into a thioredoxin expression system (pET102/D-Topo). It was over-expressed as fusion protein with thioredoxin in Escherichia coli. Thioredoxin was removed by enterokinase cleavage or by acid cleavage at the respective engineered recognition sites. The soluble N-terminal half of gamma-Gliadin was purified by affinity and reverse-phase chromatography. While, the enterokinase cleavage leaded to only one species detectable by mass spectroscopy, the acid cleavage resulted in a three different length polypeptides, due to the presence of the same number of acid cleavage sites. The secondary structure of the purified protein domain was analysed by circular dichroism, showing an spectral shape common to a Poly(Pro) II conformation. The spectrum is dominated by a large negative peak centred around 201 nm and a broad shoulder centred around 225 nm. Also, the temperature denaturation process was studied. The differences observed in the spectra show two main tendencies, the increment of the shoulder intensity, and the drop of the intensity of the peak around 201. When the sample was cooled down, the change on intensity of the shoulder around 225 was completely reversible and that around the 201 nm peak reached a reversibility of 90%. Such structure and thermal behaviour are characteristic of the repetitive domains of the wheat prolamins.     

芽孢杆菌N1碱性蛋白酶的克隆表达及酶学性质

【背景】蛋白酶广泛应用于制革行业中,酶法脱毛对环境污染较小,但蛋白酶对化学试剂的不稳定性及胶原降解活性限制了其工业应用。【目的】克隆芽孢杆菌(Bacillussp.)N1基因组的碱性蛋白酶基因,实现其在大肠杆菌中的异源表达,并对重组酶酶学性质及脱毛作用进行研究。【方法】利用基因组文库法克隆获得蛋白酶基因aprG,构建重组大肠杆菌(Escherichiacoli)BL21(DE3)pLysS/pET-28a-aprG。异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达该重组酶,以福林酚显色法对其酶学性质进行研究,并将AprG作用于羊皮、兔皮和羽毛。【结果】克隆得到蛋白酶基因aprG,并实现其在大肠杆菌中的表达。重组酶AprG最适反应温度为50°C,最适反应pH为10.0。各种金属离子对AprG活性影响较小,且AprG对表面活性剂和氧化剂、还原剂的耐受性较强。底物特异性分析表明,该酶胶原活性较低。AprG对羊皮和兔皮作用显著,且降解羽毛效果明显。【结论】蛋白酶AprG在制革行业中具有良好的应用前景。     

Cloning, expression and characterization of gene sgcD involved in the biosynthesis of novel antitumor lidamycin

Lidamycin, an antitumor antibiotic composed of a macro-molecule peptide and enediyne chromophore[1] and originally named C1027, is produced by Streptomyces globisporus C1027 isolated from the soil in Qianjiang County, Hubei Province, China. It has extremely high antitu- mor activity, which has been proved to be the highest among antitumor compounds[2], being 1000- fold higher than that of adriamycin commonly used in clinic. The structure of lidamycin consists of an acid apoprotein and a chr…     

Cloning, expression, and preliminary structural characterization of RTN-1C

Reticulons (RTNs) are endoplasmic reticulum-associated proteins widely distributed in plants, yeast, and animals. They are characterized by unique N-terminal parts and a common 200 amino acid C-terminal domain containing two long hydrophobic sequences. Despite their implication in many cellular processes, their molecular structure and function are still largely unknown. In this study, the reticulon family member RTN-1C has been expressed and purified in Escherichia coli and its molecular structure has been analysed by fluorescence and CD spectroscopy in different detergents in order to obtain a good solubility and a relative stability. The isotopically enriched protein has been also produced to perform structural studies by NMR spectroscopy. The preliminary results obtained showed that RTN-1C protein possesses helical transmembrane segments when a membrane-like environment is produced by detergents. Moreover, fluorescence experiments indicated the exposure of tryptophan side chains as predicted by structure prediction programs. We also produced the isotopically labelled protein and the procedure adopted allowed us to plan future NMR studies to investigate the biochemical behaviour of reticulon-1C and of its peptides spanning out from the membrane.