Overproduction and characterization of two distinct aldehyde-oxidizing enzymes from Gluconobacter oxydans 621H

The Gluconobacter oxydans 621H genome contains two genes (gox1122 and gox0499) that encode putative cytosolic NAD(P)-dependent aldehyde dehydrogenases. Each gene was expressed in Escherichia coli, and the recombinant enzymes were purified and characterized. The native protein Gox1122 exhibited an apparent molecular mass of 50.1 kDa, and the subunit mass was 50.5 kDa, indicating a monomeric structure of the native enzyme. The preferred substrates were acetaldehyde and NADP. The enzyme also oxidized other short-chained aliphatic and aromatic aldehydes at lower rates. Recombinant protein Gox0499 was composed of a single subunit and had an apparent molecular mass of 49.5 kDa. The substrate spectrum of Gox0499 was broad with a preference for long-chained aliphatic and aromatic aldehydes. Highest activities were obtained using dodecanal and NAD as substrates. RT real-time PCR showed that genes gox0499 and gox1122 were expressed at an elevated level (about 3-fold) when the cells were exposed to ethanol and dodecanal in comparison to control cells.     

Regulation of gluconate and ketogluconate production in Gluconobacter oxydans ATCC 621-H

Gluconobacter spp. possess the enzymic potential for two pathways of direct glucose oxidation. It has been proposed that the major part of glucose is oxidized to gluconate via NADP-dependent glucose dehydrogenase and that reoxidation of NADPH under these conditions proceeds via recycling of gluconate through ketogluconates. This hypothesis was tested in experiments in which Gluconobacter oxydans ATCC 621-H was grown in glucose-yeast extract medium containing [¹⁴C]2-ketogluconate. As expected, glucose was almost quantitatively oxidized to gluconate, without further accumulation of 2- and 5-ketogluconate. Interestingly, the total amount of neither [¹⁴C]2-ketogluconate nor [¹⁴C]gluconate did change significantly during this oxidation phase, indicating that recycling of gluconate through ketogluconates did not occur. An analysis of enzyme activities in cell-free extracts of glucose-grown cells of G. oxydans ATCC 621-H showed that the membrane-bound glucose dehydrogenase was far more active than the NADP-linked glucose dehydrogenase. The activity of the latter enzyme constituted only 10–15% of that of quinoprotein glucose dehydrogenase and was far too low to match the in vivo rates of gluconate production in batch cultures of G. oxydans. It is concluded that under these conditions glucose is mainly oxidized to gluconate via the membrane-bound glucose dehydrogenase. Implications of these results for the regulation of ketogluconate formation are discussed.Abbreviations DCPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulphate - PQQ pyrrolo-quinoline quinone     

The first step in polyethylene glycol degradation by sphingomonads proceeds via a flavoprotein alcohol dehydrogenase containing flavin adenine dinucleotide.

Several Sphingomonas spp. utilize polyethylene glycols (PEGs) as a sole carbon and energy source, oxidative PEG degradation being initiated by a dye-linked dehydrogenase (PEG-DH) that oxidizes the terminal alcohol groups of the polymer chain. Purification and characterization of PEG-DH from Sphingomonas terrae revealed that the enzyme is membrane bound. The gene encoding this enzyme (pegA) was cloned, sequenced, and expressed in Escherichia coli. The purified recombinant enzyme was vulnerable to aggregation and inactivation, but this could be prevented by addition of detergent. It is as a homodimeric protein with a subunit molecular mass of 58.8 kDa, each subunit containing 1 noncovalently bound flavin adenine dinucleotide but not Fe or Zn. PEG-DH recognizes a broad variety of primary aliphatic and aromatic alcohols as substrates. Comparison with known sequences revealed that PEG-DH belongs to the group of glucose-methanol-choline (GMC) flavoprotein oxidoreductases and that it is a novel type of flavoprotein alcohol dehydrogenase related (percent identical amino acids) to other, so far uncharacterized bacterial, membrane-bound, dye-linked dehydrogenases: alcohol dehydrogenase from Pseudomonas oleovorans (46%); choline dehydrogenase from E. coli (40%); L-sorbose dehydrogenase from Gluconobacter oxydans (38%); and 4-nitrobenzyl alcohol dehydrogenase from a Pseudomonas species (35%).     

Modulation of ethanol stress tolerance by aldehyde dehydrogenase in the mycorrhizal fungus Tricholoma vaccinum

We report the first mycorrhizal fungal aldehyde dehydrogenase gene, ald1, which was isolated from the basidiomycete Tricholoma vaccinum. The gene, encoding a protein Ald1 of 502 amino acids, is up-regulated in ectomycorrhiza. Phylogenetic analyses using 53 specific fungal aldehyde dehydrogenases from all major phyla in the kingdom of fungi including Ald1 and two partial sequences of T. vaccinum were performed to get an insight in the evolution of the aldehyde dehydrogenase family. By using competitive and real-time RT-PCR, ald1 is up-regulated in response to alcohol and aldehyde-related stress. Furthermore, heterologous expression of ald1 in Escherichia coli and subsequent in vitro enzyme activity assay demonstrated the oxidation of propionaldehyde and butyraldehyde with different kinetics using either NAD(+) or NADP(+) as cofactors. In addition, overexpression of ald1 in T. vaccinum after Agrobacterium tumefaciens-mediated transformation increased ethanol stress tolerance. These results demonstrate the ability of Ald1 to circumvent ethanol stress, a critical function in mycorrhizal habitats.     

Identification and characterization of a Pantoea citrea gene encoding glucose dehydrogenase that is essential for causing pink disease of pineapple.

Pantoea citrea, a member of the family Enterobacteriaceae, causes pink disease of pineapple, whose symptom is characterized by the formation of pink to brown discolorations of the infected portions of the pineapple fruit cylinder upon canning. Molecular genetic approaches were applied to elucidate the mechanism responsible for this fruit discoloration. A P. citrea mutant strain, CMC6, defective in its ability to cause pink disease and fruit discoloration, was generated by nitrosoguanidine mutagenesis. A DNA fragment that restored these activities was isolated by screening a genomic cosmid library of P. citrea. A large open reading frame of 2,361 bp, identified by nucleotide sequencing of a subclone of the complementing DNA, showed high similarities to identified genes encoding glucose dehydrogenase (GDH) in Escherichia coli, Acinetobacter calcoaceticus, and Gluconobacter oxydans. The predicted amino acid sequence of GDH of P. citrea was identical to known GDHs in these bacteria by 54, 44, and 34%, respectively. GDH of P. citrea has a predicted molecular mass of 86.2 kDa, contains a conserved binding domain for the cofactor pyrroloquinoline quinone, and possesses GDH activity as demonstrated by biochemical assay. GDH is the key branch point enzyme leading to the biosynthesis of gluconate, which in turn serves as the substrate leading to the formation of 2-ketogluconate, 2,5-diketogluconate, 6-phosphogluconate, and 2-keto-6-phosphogluconate. Addition of gluconate to CMC6 restores the juice- and fruit-discoloring activity. Although the pigments formed by heating (or canning) have not been identified, it is clear that GDH is one of the enzymes required for pigment formation leading to pink disease.     

NADP+-dependent glutamate dehydrogenase in the Antarctic psychrotolerant bacterium Psychrobacter sp. TAD1. Characterization, protein and DNA sequence, and relationship to other glutamate dehydrogenases.

The Antarctic psychrotolerant bacterium Psychrobacter sp. TAD1 contains two distinct glutamate dehydrogenases (GDH), each specific for either NADP+ or NAD+. This feature is quite unusual in bacteria, which generally have a single GDH. NADP+-dependent GDH has been purified to homogeneity and the gene encoding GDH has been cloned and expressed. The enzyme has a hexameric structure. The amino acid sequence determined by peptide and gene analyses comprises 447 residues, yielding a protein with a molecular mass of 49 285 Da. The sequence shows homology with hexameric GDHs, with identity levels of 52% and 49% with Escherichia coli and Clostridium symbiosum GDH, respectively. The coenzyme-binding fingerprint motif GXGXXG/A (common to all GDHs) has Ser at the last position in this enzyme. The overall hydrophilic character is increased and a five-residue insertion in a loop between two alpha-helices may contribute to the increase in protein flexibility. Psychrobacter sp. TAD1 GDH apparent temperature optimum is shifted towards low temperatures, whereas irreversible heat inactivation occurs at temperatures similar to those of E. coli GDH. The catalytic efficiency in the temperature range 10-30 degrees C is similar or lower than that of E. coli GDH. Unlike E. coli GDH the enzyme exhibits marked positive cooperativity towards 2-oxoglutarate and NADPH. This feature is generally absent in prokaryotic GDHs. These observations suggest a regulatory role for this GDH, the most crucial feature being the structural/functional properties required for fine regulation of activity, rather than the high catalytic efficiency and thermolability encountered in several cold-active enzymes.     

NADP-dependent isocitrate dehydrogenase from the halophilic archaeon Haloferax volcanii: cloning,sequence determination and overexpression in Escherichia coli

A gene encoding NADP-dependent Ds-threo-isocitrate dehydrogenase was isolated from Haloferax volcanii genomic DNA by using a combination of polymerase chain reaction and screening of a lambda EMBL3 library. Analysis of the nucleotide sequence revealed an open reading frame of 1260 bp encoding a protein of 419 amino acids with 45837 Da molecular mass. This sequence is highly similar to previously sequenced isocitrate dehydrogenases. In the alignment of the amino acid sequences with those from several archaeal and mesophilic NADP-dependent isocitrate dehydrogenases, the residues involved in dinucleotide binding and isocitrate binding are well conserved. We have developed methods for the expression in Escherichia coli and purification of the enzyme from H. volcanii. This expression was carried out in E. coli as inclusion bodies using the cytoplasmic expression vector pET3a. The enzyme was refolded by solubilisation in 8 M urea followed by dilution into a buffer containing EDTA, MgCl(2) and 3 M NaCl. Maximal activity was obtained after several hours incubation at room temperature.     

Cloning of a gluconate/polyol dehydrogenase gene from <Emphasis Type="Italic">Gluconobacter suboxydans</Emphasis> IFO 12528, characterisation of the enzyme and its use for the production of 5-ketogluconate in a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> strain

A 5-ketogluconate (5-KGA)-forming membrane quinoprotein, gluconate dehydrogenase, was isolated from Gluconobacter suboxydans strain IFO 12528 and partially sequenced. Partial sequences of five internal tryptic peptides were elucidated by mass spectrometry and used to isolate the two adjacent genes encoding the enzyme (EBI accession no. AJ577472). These genes share close homology with sorbitol dehydrogenase from another strain of G. suboxydans (IFO 3255). Substrate specificity of gluconate 5-dehydrogenase (GA 5-DH) turned out to be quite broad, covering many polyols, amino derivatives of carbohydrates, and simple secondary alcohols. There is a broad correlation between the substrate specificity of GA 5-DH and the empirical Bertrand-Hudson rule that predicts the specificity of oxidation of polyols by acetic acid bacteria. Escherichia coli transformed with the genes encoding gluconate dehydrogenase were able to convert gluconic acid into 5-KGA at 75% yield. Furthermore, it was found that 5-KGA can be converted into tartaric acid semialdehyde by a transketolase. These results provide a basis for designing a direct fermentation-based process for conversion of glucose into tartaric acid.     

Effect of temperature on the activity and synthesis of glucose-catabolizing enzymes in Pseudomonas fluorescens.

The activity of the enzymes of the oxidative non-phosphorylated pathway, glucose and gluconate dehydrogenases, were not significantly affected by changes in the assay temperature. Both enzymes demonstrated only a threefold difference in activity when compared at assay temperatures of 30 degrees C and 5 degrees C. In contrast, the enzymes involved in the direct phosphorylation and catabolism of glucose or its oxidation products, gluconate and 2-ketogluconate, exhibited a more pronounced response to decreasing assay temperatures. At least one enzyme in each pathway, involved in the direct phosphorylation and catabolism of glucose or 2-ketogluconate (2KG), demonstrated an eightfold decrease in activity with a decrease in assay temperature from 30 degrees C to 5 degrees C. A similar decrease in assay temperature resulted in a fivefold decrease in activity of the enzymes involved in the direct phosphorylation and catabolism of gluconate. The observed differential effect of temperature on the activity of the enzymes of glucose catabolism and on the accumulation of direct oxidation products during growth with glucose in P. fluorescens E-20 is discussed. Growth with glucose at 5 or 20 degrees C resulted in high induced levels of all glucose-catabolizing enzymes examined when compared with the levels of these same enzymes in pyruvate-grown cells. However, only low levels of glucose dehydrogenase were detected during growth at 30 degrees C with glucose, gluconate, or 2-KG. Similarly, only low levels of gluconate dehydrogenase were detected during growth with glucose at 30 degrees C, although a weak induction was observed during growth with gluconate or 2-KG at 30 degrees C. The levels of 2-KG kinase plus KPG reductase during growth at 30 degrees C were undetectable with glucose, weakly induced with gluconate, and fully induced with 2-KG. High induced levels of glucose dehydrogenase, gluconate dehydrogenase, and 2-KG kinase plus KPG reductase were present during growth at 20 degrees C with glucose or 2-KG. The low levels of glucose and gluconate dehydrogenases present at a growth temperature of 30 degrees C was not due to heat lability of the enzymes at this temperature. The low amounts of these two enzymes during growth with glucose at 30 degrees C probably prevented sufficient inducer(s) formation from glucose to allow induction of enzymes of 2-KG catabolism. The results demonstrated that temperature may regulate the pathways of glucose dissimilation by regulating, either directly or indirectly, the activity and synthesis of the enzymes involved in these pathways.     

Molecular characterization of tobacco mitochondrial L-galactono-gamma-lactone dehydrogenase and its expression in Escherichia coli

A cDNA clone encoding L-galactono-gamma-lactone (GAL) dehydrogenase (EC 1.3.2.3) was isolated from tobacco leaves. The cDNA clone contained an open reading frame encoding the protein of 501 amino acids with a calculated molecular mass of 56,926 Da, preceded by a putative mitochondrial targeting signal consisting of 86 amino acid residues. In fact, GAL dehydrogenase was localized in the mitochondria of tobacco cells. The deduced amino acid sequence of the cDNA showed 77 and 82% homology to cauliflower and sweet potato GAL dehydrogenases, respectively. Southern blot analysis showed that tobacco contains one copy of the gene for the enzyme. Northern blot analysis showed that GAL dehydrogenase mRNA (2.0 kb) is expressed in the leaves, stems, and roots in almost equal quantities. We introduced the cDNA clone encoding tobacco GAL dehydrogenase into a pET expression vector to overexpress this protein in Escherichia coli. The partially purified recombinant enzyme was used for comparative studies on the native enzymes from tobacco and other sources; its enzymatic properties were similar to those of other GAL dehydrogenases.     

Cloning of the xylitol dehydrogenase gene from Gluconobacter oxydans and improved production of xylitol from D-arabitol

Xylitol dehydrogenase (XDH) was purified from the cytoplasmic fraction of Gluconobacter oxydans ATCC 621. The purified enzyme reduced D-xylulose to xylitol in the presence of NADH with an optimum pH of around 5.0. Based on the determined NH2-terminal amino acid sequence, the gene encoding xdh was cloned, and its identity was confirmed by expression in Escherichia coli. The xdh gene encodes a polypeptide composed of 262 amino acid residues, with an estimated molecular mass of 27.8 kDa. The deduced amino acid sequence suggested that the enzyme belongs to the short-chain dehydrogenase/reductase family. Expression plasmids for the xdh gene were constructed and used to produce recombinant strains of G. oxydans that had up to 11-fold greater XDH activity than the wild-type strain. When used in the production of xylitol from D-arabitol under controlled aeration and pH conditions, the strain harboring the xdh expression plasmids produced 57 g/l xylitol from 225 g/l D-arabitol, whereas the control strain produced 27 g/l xylitol. These results demonstrated that increasing XDH activity in G. oxydans improved xylitol productivity.     

Cloning and sequencing of the serine dehydrogenase gene from Agrobacterium tumefaciens

The structural gene for NADP+-dependent serine dehydrogenase [EC 1.1.1.-] from Agrobacterium tumefaciens ICR 1600 was cloned into Escherichia coli cells and its complete DNA sequence was analyzed. The gene encodes a polypeptide containing 249 amino acid residues. The enzyme had high sequence similarity to short-chain alcohol dehydrogenases from bacteria and unknown proteins of Haemophilus influenzae, Escherichia coli, and Saccharomyces cerevisiae.     

Molecular properties of membrane-bound FAD-containing D-sorbitol dehydrogenase from thermotolerant Gluconobacter frateurii isolated from Thailand

There are two types of membrane-bound D-sorbitol dehydrogenase (SLDH) reported: PQQ-SLDH, having pyrroloquinoline quinone (PQQ), and FAD-SLDH, containing FAD and heme c as the prosthetic groups. FAD-SLDH was purified and characterized from the PQQ-SLDH mutant strain of a thermotolerant Gluconobacter frateurii, having molecular mass of 61.5 kDa, 52 kDa, and 22 kDa. The enzyme properties were quite similar to those of the enzyme from mesophilic G. oxydans IFO 3254. This enzyme was shown to be inducible by D-sorbitol, but not PQQ-SLDH. The oxidation product of FAD-SLDH from D-sorbitol was identified as L-sorbose. The cloned gene of FAD-SLDH had three open reading frames (sldSLC) corresponding to the small, the large, and cytochrome c subunits of FAD-SLDH respectively. The deduced amino acid sequences showed high identity to those from G. oxydans IFO 3254: SldL showed to other FAD-enzymes, and SldC having three heme c binding motives to cytochrome c subunits of other membrane-bound dehydrogenases.     

Characterization of ligV essential for catabolism of vanillin by Sphingomonas paucimobilis SYK-6

The vanillin dehydrogenase gene (ligV), which conferred the ability to transform vanillin into vanillate on Escherichia coli, was isolated from Sphingomonas paucimobilis SYK-6. The ligV gene consists of a 1,440-bp open reading frame encoding a polypeptide with a molecular mass of 50,301 Da. The deduced amino acid sequence of ligV showed about 50% identity with the known vanillin dehydrogenases of Pseudomonas vanillin degraders. The gene product of ligV (LigV) produced in E. coli preferred NAD+ to NADP+ and exhibited a broad substrate preference, including vanillin, benzaldehyde, protocatechualdehyde, m-anisaldehyde, and p-hydroxybenzaldehyde, but the activity toward syringaldehyde was less than 5% of that toward vanillin. Insertional inactivation of ligV in SYK-6 indicated that ligV is essential for normal growth on vanillin. On the other hand, growth on syringaldehyde was only slightly affected by ligV disruption, indicating the presence of a syringaldehyde dehydrogenase gene or genes in SYK-6.     

Characterization of a zinc-containing alcohol dehydrogenase with stereoselectivity from the hyperthermophilic archaeon Thermococcus guaymasensis

An alcohol dehydrogenase (ADH) from hyperthermophilic archaeon Thermococcus guaymasensis was purified to homogeneity and was found to be a homotetramer with a subunit size of 40 ± 1 kDa. The gene encoding the enzyme was cloned and sequenced; this gene had 1,095 bp, corresponding to 365 amino acids, and showed high sequence homology to zinc-containing ADHs and l-threonine dehydrogenases with binding motifs of catalytic zinc and NADP(+). Metal analyses revealed that this NADP(+)-dependent enzyme contained 0.9 ± 0.03 g-atoms of zinc per subunit. It was a primary-secondary ADH and exhibited a substrate preference for secondary alcohols and corresponding ketones. Particularly, the enzyme with unusual stereoselectivity catalyzed an anti-Prelog reduction of racemic (R/S)-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol. The optimal pH values for the oxidation and formation of alcohols were 10.5 and 7.5, respectively. Besides being hyperthermostable, the enzyme activity increased as the temperature was elevated up to 95°C. The enzyme was active in the presence of methanol up to 40% (vol/vol) in the assay mixture. The reduction of ketones underwent high efficiency by coupling with excess isopropanol to regenerate NADPH. The kinetic parameters of the enzyme showed that the apparent K(m) values and catalytic efficiency for NADPH were 40 times lower and 5 times higher than those for NADP(+), respectively. The physiological roles of the enzyme were proposed to be in the formation of alcohols such as ethanol or acetoin concomitant to the NADPH oxidation.     

Molecular cloning and DNA sequencing of the Escherichia coli K-12 ald gene encoding aldehyde dehydrogenase.

The gene ald, encoding aldehyde dehydrogenase, has been cloned from a genomic library of Escherichia coli K-12 constructed with plasmid pBR322 by complementing an aldehyde dehydrogenase-deficient mutant. The ald region was sequenced, and a single open reading frame of 479 codons specifying the subunit of the aldehyde dehydrogenase enzyme complex was identified. Determination of the N-terminal amino acid sequence of the enzyme protein unambiguously established the identity and the start codon of the ald gene. Analysis of the 5'- and 3'-flanking sequences indicated that the ald gene is an operon. The deduced amino acid sequence of the ald gene displayed homology with sequences of several aldehyde dehydrogenases of eukaryotic origin but not with microbial glyceraldehyde-3-phosphate dehydrogenase.     

Biochemical and molecular characterization of a succinate semialdehyde dehydrogenase involved in the catabolism of 4-hydroxybutyric acid in Ralstonia eutropha

A succinate semialdehyde dehydrogenase gene (gabD) was identified to be disrupted in a transposon-induced mutant of Ralstonia eutropha exhibiting the phenotype 4-hydroxybutyric acid-leaky. The native gabD gene was cloned by colony hybridization using a homologous gabD-specific DNA probe. DNA sequencing revealed an 1452-bp open reading frame, and the deduced amino acid sequence showed strong similarities to NADP(+)-dependent succinate semialdehyde dehydrogenases from Escherichia coli, Rhizobium sp., Homo sapiens and Rattus norvegicus. The gabD gene was heterologously expressed in a recombinant E. coli strain harboring plasmid pSK::EE6.8. Similar to the molecular organization of the gab cluster in E. coli, additional genes encoding enzymes for the degradation of gamma-aminobutyrate are closely related to gabD in R. eutropha. Enzymatic studies indicated the existence of a second NAD(+)-dependent succinate semialdehyde dehydrogenase in R. eutropha.     

Crystalline 2-Ketogluconate Reductase from Acetobacter ascendens,the Second Instance of Crystalline Enzyme in Genus Acetobacter

Crystalline 2-ketogluconate reductase in genus Acetobacter was prepared from cell free extract of Acetobacter ascendens. Crystalline enzyme was purified 13,000-fold with a yield of 15%. Affinity chromatography on blue-dextran Sepharose 4B column successfully purified the enzyme. The enzyme was composed of three identical subunits with a molecular weight of 40,000. Substrate specificity of 2-ketogluconate reductase from two genera of acetic acid bacteria was compared using highly purified enzyme preparations, and it was confirmed that gluconate oxidation activity of the enzyme was intrinsically weak or absent in genus Acetobacter and intense in Gluconobacter. This fact must be a useful criterion for classification of acetic acid bacteria.