Carbon Monoxide Dehydrogenases.

Carbon monoxide dehydrogenases (CODHs) catalyze the reversible oxidation of CO with water to CO2, two electrons, and two protons. Two classes of CODHs exist, having evolved from different scaffolds featuring active sites built from different transition metals. The basic properties of both classes are described in this overview chapter.

Structural, Biochemical, and Evolutionary Characterizations of Glyoxylate/Hydroxypyruvate Reductases Show Their Division into Two Distinct Subfamilies.

The d-2-hydroxyacid dehydrogenase (2HADH) family illustrates a complex evolutionary history with multiple lateral gene transfers and gene duplications and losses. As a result, the exact functional annotation of individual members can be extrapolated to a very limited extent. Here, we revise the previous simplified view on the classification of the 2HADH family; specifically, we show that the previously delineated glyoxylate/hydroxypyruvate reductase (GHPR) subfamily consists of two evolutionary separated GHRA and GHRB subfamilies. We compare two representatives of these subfamilies from Sinorhizobium meliloti (SmGhrA and SmGhrB), employing a combination of biochemical, structural, and bioinformatics approaches. Our kinetic results show that both enzymes reduce several 2-ketocarboxylic acids with overlapping, but not equivalent, substrate preferences. SmGhrA and SmGhrB show highest activity with glyoxylate and hydroxypyruvate, respectively; in addition, only SmGhrB reduces 2-keto-d-gluconate, and only SmGhrA reduces pyruvate (with low efficiency). We present nine crystal structures of both enzymes in apo forms and in complexes with cofactors and substrates/substrate analogues. In particular, we determined a crystal structure of SmGhrB with 2-keto-d-gluconate, which is the biggest substrate cocrystallized with a 2HADH member. The structures reveal significant differences between SmGhrA and SmGhrB, both in the overall structure and within the substrate-binding pocket, offering insight into the molecular basis for the observed substrate preferences and subfamily differences. In addition, we provide an overview of all GHRA and GHRB structures complexed with a ligand in the active site.

Four distinct types of E.C. 1.2.1.30 enzymes can catalyze the reduction of carboxylic acids to aldehydes.

Increasing demand for chemicals from renewable resources calls for the development of new biotechnological methods for the reduction of oxidized bio-based compounds. Enzymatic carboxylate reduction is highly selective, both in terms of chemo- and product selectivity, but not many carboxylate reductase enzymes (CARs) have been identified on the sequence level to date. Thus far, their phylogeny is unexplored and very little is known about their structure-function-relationship. CARs minimally contain an adenylation domain, a phosphopantetheinylation domain and a reductase domain. We have recently identified new enzymes of fungal origin, using similarity searches against genomic sequences from organisms in which aldehydes were detected upon incubation with carboxylic acids. Analysis of sequences with known CAR functionality and CAR enzymes recently identified in our laboratory suggests that the three-domain architecture mentioned above is modular. The construction of a distance tree with a subsequent 1000-replicate bootstrap analysis showed that the CAR sequences included in our study fall into four distinct subgroups (one of bacterial origin and three of fungal origin, respectively), each with a bootstrap value of 100%. The multiple sequence alignment of all experimentally confirmed CAR protein sequences revealed fingerprint sequences of residues which are likely to be involved in substrate and co-substrate binding and one of the three catalytic substeps, respectively. The fingerprint sequences broaden our understanding of the amino acids that might be essential for the reduction of organic acids to the corresponding aldehydes in CAR proteins.

Clade classification of monolignol biosynthesis gene family members reveals target genes to decrease lignin in Lolium perenne.

In monocots, lignin content has a strong impact on the digestibility of the cell wall fraction. Engineering lignin biosynthesis requires a profound knowledge of the role of paralogues in the multigene families that constitute the monolignol biosynthesis pathway. We applied a bioinformatics approach for genome-wide identification of candidate genes in Lolium perenne that are likely to be involved in the biosynthesis of monolignols. More specifically, we performed functional subtyping of phylogenetic clades in four multigene families: 4CL, COMT, CAD and CCR. Essential residues were considered for functional clade delineation within these families. This classification was complemented with previously published experimental evidence on gene expression, gene function and enzymatic activity in closely related crops and model species. This allowed us to assign functions to novel identified L. perenne genes, and to assess functional redundancy among paralogues. We found that two 4CL paralogues, two COMT paralogues, three CCR paralogues and one CAD gene are prime targets for genetic studies to engineer developmentally regulated lignin in this species. Based on the delineation of sequence conservation between paralogues and a first analysis of allelic diversity, we discuss possibilities to further study the roles of these paralogues in lignin biosynthesis, including expression analysis, reverse genetics and forward genetics, such as association mapping. We propose criteria to prioritise paralogues within multigene families and certain SNPs within these genes for developing genotyping assays or increasing power in association mapping studies. Although L. perenne was the target of the analyses presented here, this functional subtyping of phylogenetic clades represents a valuable tool for studies investigating monolignol biosynthesis genes in other monocot species.

Functional characterization of a vanillin dehydrogenase in Corynebacterium glutamicum.

Vanillin dehydrogenase (VDH) is a crucial enzyme involved in the degradation of lignin-derived aromatic compounds. Herein, the VDH from Corynebacterium glutamicum was characterized. The relative molecular mass (Mr) determined by SDS-PAGE was ~51 kDa, whereas the apparent native Mr values revealed by gel filtration chromatography were 49.5, 92.3, 159.0 and 199.2 kDa, indicating the presence of dimeric, trimeric and tetrameric forms. Moreover, the enzyme showed its highest level of activity toward vanillin at pH 7.0 and 30°C, and interestingly, it could utilize NAD(+) and NADP(+) as coenzymes with similar efficiency and showed no obvious difference toward NAD(+) and NADP(+). In addition to vanillin, this enzyme exhibited catalytic activity toward a broad range of substrates, including p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, o-phthaldialdehyde, cinnamaldehyde, syringaldehyde and benzaldehyde. Conserved catalytic residues or putative cofactor interactive sites were identified based on sequence alignment and comparison with previous studies, and the function of selected residues were verified by site-directed mutagenesis analysis. Finally, the vdh deletion mutant partially lost its ability to grow on vanillin, indicating the presence of alternative VDH(s) in Corynebacterium glutamicum. Taken together, this study contributes to understanding the VDH diversity from bacteria and the aromatic metabolism pathways in C. glutamicum.

Characterization of developmental- and stress-mediated expression of cinnamoyl-CoA reductase in kenaf (Hibiscus cannabinus L.).

Cinnamoyl-CoA reductase (CCR) is an important enzyme for lignin biosynthesis as it catalyzes the first specific committed step in monolignol biosynthesis. We have cloned a full length coding sequence of CCR from kenaf (Hibiscus cannabinus L.), which contains a 1,020-bp open reading frame (ORF), encoding 339 amino acids of 37.37 kDa, with an isoelectric point (pI) of 6.27 (JX524276, HcCCR2). BLAST result found that it has high homology with other plant CCR orthologs. Multiple alignment with other plant CCR sequences showed that it contains two highly conserved motifs: NAD(P) binding domain (VTGAGGFIASWMVKLLLEKGY) at N-terminal and probable catalytic domain (NWYCYGK). According to phylogenetic analysis, it was closely related to CCR sequences of Gossypium hirsutum (ACQ59094) and Populus trichocarpa (CAC07424). HcCCR2 showed ubiquitous expression in various kenaf tissues and the highest expression was detected in mature flower. HcCCR2 was expressed differentially in response to various stresses, and the highest expression was observed by drought and NaCl treatments.

Expression analysis of cinnamoyl-CoA reductase (CCR) gene in developing seedlings of Leucaena leucocephala: a pulp yielding tree species.

Removal of lignin is a major hurdle for obtaining good quality pulp. Leucaena leucocephala (subabul) is extensively used in paper industry in India; therefore, as a first step to generate transgenic plants with low lignin content, cDNA and genomic clones of CCR gene were isolated and characterized. The cDNA encoding CCR (EC 1.2.1.44) was designated as Ll-CCR; the sequence analysis revealed an Open Reading Frame (ORF) of 1005 bp. Phylogenetic analysis showed that Ll-CCR sequence is highly homologous to CCRs from other dicot plants. The 2992 bp genomic clone of Leucaena CCR consists of 5 exons and 4 introns. The haploid genome of L. leucocephala contains two copies as revealed by DNA blot hybridization. Ll-CCR gene was over-expressed in Escherichia coli, which showed a molecular mass of approximately 38 kDa. Protein blot analysis revealed that Ll-CCR protein is expressed at higher levels in root and in stem, but undetectable in leaf tissues. Expression of CCR gene in Leucaena increased up to 15 d in case of roots and stem as revealed by QRT-PCR studies in 0-15 d old seedlings. ELISA based studies of extractable CCR protein corroborated with QRT-PCR data. CCR protein was immuno-cytolocalized around xylem tissue. Lignin estimation and expression studies of 5, 10 and 15 d old stem and root suggest that CCR expression correlates with quantity of lignin produced, which makes it a good target for antisense down regulation for producing designer species for paper industry.

Fatty acyl-CoA reductase and wax synthase from Euglena gracilis in the biosynthesis of medium-chain wax esters.

Euglena gracilis, a unicellular phytoflagellate, can accumulate a large amount of medium-chain wax esters under anaerobic growth conditions. Here we report the identification and characterization of two genes involved in the biosynthesis of wax esters in E. gracilis. The first gene encodes a fatty acyl-CoA reductase (EgFAR) involved in the conversion of fatty acyl-CoAs to fatty alcohols and the second gene codes for a wax synthase (EgWS) catalyzing esterification of fatty acyl-CoAs and fatty alcohols, yielding wax esters. When expressed in yeast (Saccharomyces cerevisiae), EgFAR converted myristic acid (14:0) and palmitic acid (16:0) to their corresponding alcohols (14:0Alc and 16:0Alc) with myristic acid as the preferred substrate. EgWS utilized a broad range of fatty acyl-CoAs and fatty alcohols as substrates with the preference towards myristic acid and palmitoleyl alcohol. The wax biosynthetic pathway was reconstituted by co-expressing EgFAR and EgWS in yeast. When myristic acid was fed to the yeast, myristyl myristate (14:0-14:0), myristyl palmitoleate (14:0-16:1), myristyl palmitate (14:0-16:0) and palmityl myristate (16:0-14:0) were produced. These results indicate EgFAR and EgWS are likely the two enzymes involved in the biosynthesis of medium-chain wax esters in E. gracilis.

Carbon-dependent control of electron transfer and central carbon pathway genes for methane biosynthesis in the Archaean, Methanosarcina acetivorans strain C2A.

BACKGROUND: The archaeon, Methanosarcina acetivorans strain C2A forms methane, a potent greenhouse gas, from a variety of one-carbon substrates and acetate. Whereas the biochemical pathways leading to methane formation are well understood, little is known about the expression of the many of the genes that encode proteins needed for carbon flow, electron transfer and/or energy conservation. Quantitative transcript analysis was performed on twenty gene clusters encompassing over one hundred genes in M. acetivorans that encode enzymes/proteins with known or potential roles in substrate conversion to methane. RESULTS: The expression of many seemingly "redundant" genes/gene clusters establish substrate dependent control of approximately seventy genes for methane production by the pathways for methanol and acetate utilization. These include genes for soluble-type and membrane-type heterodisulfide reductases (hdr), hydrogenases including genes for a vht-type F420 non-reducing hydrogenase, molybdenum-type (fmd) as well as tungsten-type (fwd) formylmethanofuran dehydrogenases, genes for rnf and mrp-type electron transfer complexes, for acetate uptake, plus multiple genes for aha- and atp-type ATP synthesis complexes. Analysis of promoters for seven gene clusters reveal UTR leaders of 51-137 nucleotides in length, raising the possibility of both transcriptional and translational levels of control. CONCLUSIONS: The above findings establish the differential and coordinated expression of two major gene families in M. acetivorans in response to carbon/energy supply. Furthermore, the quantitative mRNA measurements demonstrate the dynamic range for modulating transcript abundance. Since many of these gene clusters in M. acetivorans are also present in other Methanosarcina species including M. mazei, and in M. barkeri, these findings provide a basis for predicting related control in these environmentally significant methanogens.

A novel nitroreductase of Staphylococcus aureus with S-nitrosoglutathione reductase activity.

In this report we show that inactivation of the putative nitroreductase SA0UHSC_00833 (ntrA) increases the sensitivity of Staphylococcus aureus to S-nitrosoglutathione (GSNO) and augments its resistance to nitrofurans. S. aureus NtrA is a bifunctional enzyme that exhibits nitroreductase and GSNO reductase activity. A phylogenetic analysis suggests that NtrA is a member of a novel family of nitroreductases that seems to play a dual role in vivo, promoting nitrofuran activation and protecting the cell against transnitrosylation.

S-Nitrosogluthathione reductase activity of amphioxus ADH3: insights into the nitric oxide metabolism.

Nitric oxide (NO) is a signalling molecule involved in many physiological functions. An important via of NO action is through the S-nitrosylation of proteins, a post-translational modification that regulates the activity of enzymes, protein-protein interactions and signal transduction pathways. Alcohol dehydrogenase class III (ADH3) recognises S-nitrosoglutathione (GSNO), the main reservoir of non-protein S-nitrosothiol, and functions as an effective GSNO reductase (GSNOR) and as a safeguard against nitrosative stress. To investigate the evolutionary conservation of this metabolic role, we have produced recombinant Branchiostoma floridae ADH3. Pure preparations of ADH3 showed 2-fold higher activity as GSNOR than as formaldehyde dehydrogenase, the previously assumed main role for ADH3. To correlate ADH3 expression in the gut with areas of NO production, we analysed the tissue distribution of the nitric oxide synthase (NOS) enzyme in amphioxus larvae. Immunostaining of the NOS enzyme revealed expression in the gut and in the dorsal region of the club-shaped gland. Co-localization in the gut supports the ADH3 and NOS joint contribution to the NO/SNO homeostasis.

Localization of retinaldehyde dehydrogenases and retinoid binding proteins to sustentacular cells, glia, Bowman's gland cells, and stroma: potential sites of retinoic acid synthesis in the postnatal rat olfactory organ.

Work from our laboratory suggests that retinoic acid (RA) influences neuron development in the postnatal olfactory epithelium (OE). The studies reported here were carried out to identify and localize retinaldehyde dehydrogenase (RALDH) expression in postnatal rat OE to gain a better understanding of potential in vivo RA synthesis sites in this continuously regenerating tissue. RALDH 1, 2, and 3 mRNAs were detected in postnatal rat olfactory tissue by RT-PCR analysis, but RALDH 1 and 2 transcripts were predominant. RALDH 1 immunoreactivity was localized to sustentacular cells in the OE and to Bowman's gland cells, and GFAP(+)/p75(-) olfactory ensheathing cells (OECs) in the underlying lamina propria (LP). RALDH 2 did not colocalize with RALDH 1, but appeared to be expressed in GFAP(-)/RALDH 1(-) OECs as well as in unidentified structures in the LP. Cellular RA binding protein (CRABP II) colocalized with RALDH 1. Cellular retinol/retinaldehyde binding protein (CRBP I) was localized to RALDH 1(+) sites in the OE and LP and RALDH 2(+) sites, primarily surrounding nerve fiber bundles in the LP. Vitamin A deficiency altered RALDH 1, but not RALDH 2 protein expression. The isozymes and binding proteins exhibited random variability in levels and areas of expression both within and between animals. These findings support the hypothesis that RA is synthesized in the postnatal OE (catalyzed by RALDH 1) and underlying LP (differentially catalyzed by RALDH 1 and RALDH 2) at sites that could influence the development, maturation, targeting, and/or turnover of olfactory receptor neurons throughout the olfactory organ.

Mg-protoporphyrin IX and heme control HEMA, the gene encoding the first specific step of tetrapyrrole biosynthesis, in Chlamydomonas reinhardtii.

HEMA encodes glutamyl-tRNA reductase (GluTR), which catalyzes the first step specific for tetrapyrrole biosynthesis in plants, archaea, and most eubacteria. In higher plants, GluTR is feedback inhibited by heme and intermediates of chlorophyll biosynthesis. It plays a key role in controlling flux through the tetrapyrrole biosynthetic pathway. This enzyme, which in Chlamydomonas reinhardtii is encoded by a single gene (HEMA), exhibits homology to GluTRs of higher plants and cyanobacteria. HEMA mRNA accumulation was inducible not only by light but also by treatment of dark-adapted cells with Mg-protoporphyrin IX (MgProto) or hemin. The specificity of these tetrapyrroles as inducers was demonstrated by the absence of induction observed upon the feeding of protoporphyrin IX, the precursor of both heme and MgProto, or chlorophyllide. The HEMA mRNA accumulation following treatment of cells with light and hemin was accompanied by increased amounts of GluTR. However, the feeding of MgProto did not suggest a role for Mg-tetrapyrroles in posttranscriptional regulation. The induction by light but not that by the tetrapyrroles was prevented by inhibition of cytoplasmic protein synthesis. Since MgProto is synthesized exclusively in plastids and heme is synthesized in plastids and mitochondria, the data suggest a role of these compounds as organellar signals that control expression of the nuclear HEMA gene.

Molecular cloning and characterization of cDNAs encoding cinnamoyl CoA reductase (CCR) from barley (Hordeum vulgare) and potato (Solanum tuberosum).

Cinnamoyl CoA reductase catalyzes the reduction of cinnamic acid CoA esters into their corresponding aldehydes, the first step of the phenylpropanoid pathway specifically dedicated to monolignol biosynthesis. Two cDNA clones encoding cinnamoyl CoA reductase (CCR) have been isolated from Hordeum vulgare (HvCCR) and Solanum tuberosum (StCCR1). Amino acid sequence alignment revealed the motif, NWYCY, representing the active site of CCR, conserved in both the encoded HvCCR and StCCR1 amino acid sequences. The HvCCR encoded protein possesses substantial homology with CCRs isolated and cloned form other sources; the highest identity (89%) was observed with CCR from fescue (Festuca arundicinea). Expression analysis by RT-PCR demonstrated that the HvCCR gene is expressed in barley stem and root tissue while no expression is detected in leaves and flowers. This expression pattern suggests that HvCCR is involved in constitutive lignification. Transcripts of StCCR1 were detected in potato root, leaf and also in stem although at a very low level.

Distinct but parallel evolutionary patterns between alcohol and aldehyde dehydrogenases: addition of fish/human betaine aldehyde dehydrogenase divergence.

Alcohol dehydrogenases (ADHs) of the MDR type (medium-chain dehydrogenases/reductases) have diverged into two evolutionary groups in eukaryotes: a set of 'constant' enzymes (class III) typical of basal enzymes, and a set of 'variable' enzymes (remaining classes) suggesting 'evolving' forms. The variable set has larger overall variability, different segment variability, and variability also in functional segments. Using a major aldehyde dehydrogenase (ALDH) from cod liver and fish ALDHs deduced from the draft genome sequence of Fugu rubripes (Japanese puffer fish), we found that ALDHs form more complex patterns than the ADHs. Nevertheless, ALDHs also group into 'constant' and 'variable' sets, have separate segment variabilities, and distinct functions. Betaine ALDH (class 9 ALDH) is 'constant,' has three segments of variability, all non-functional, and a limited fish/human divergence, reminiscent of the ADH class III pattern. Enzymatic properties of fish betaine ALDH were also determined. Although all ALDH patterns are still not known, overall patterns are related to those of ADH, and group separations may be distinguished. The results can be interpreted functionally, support ALDH isozyme distinctions, and assign properties to the multiplicities of the ADH and ALDH enzymes.

Molecular evolution of adenylating domain of aminoadipate reductase.

BACKGROUND: Aminoadipate reductase (Lys2) is a fungal-specific protein. This enzyme contains an adenylating domain. A similar primary structure can be found in some bacterial antibiotic/peptide synthetases. In this study, we aimed to determine which bacterial adenylating domain is most closely related to Lys2. In addition, we analyzed the substitution rate of the adenylating domain-encoding region. RESULTS: Some bacterial proteins contain more than two similar sequences to that of the adenylating domain of Lys2. We compared 67 amino acid sequences from 37 bacterial and 10 fungal proteins. Phylogenetic trees revealed that the lys2 genes are monophyletic; on the other hand, bacterial antibiotic/peptide synthase genes were not found to be monophyletic. Comparative phylogenetic studies among closely related fungal lys2 genes showed that the rate of insertion/deletion in these genes was lower and the nucleotide substitution rate was higher than that in the internal transcribed spacer (ITS) regions. CONCLUSIONS: The lys2 gene is one of the most useful tools for revealing the phylogenetic relationships among fungi, due to its low insertion/deletion rate and its high substitution rate. Lys2 is most closely related to certain bacterial antibiotic/peptide synthetases, but a common ancestor of Lys2 and these synthetases evolutionarily branched off in the distant past.

Mouse type-2 retinaldehyde dehydrogenase (RALDH2): genomic organization, tissue-dependent expression, chromosome assignment and comparison to other types.

Retinaldehyde dehydrogenase (RALDH) isozymes catalyze the formation of an essential developmental modulator, retinoic acid. We determined the structural organization of mouse type-2 Raldh2 by isolation of overlapping genomic DNA clones from a phage library. The gene consists of 14 exons spanning more than 70 kb of genomic DNA. It was localized to mouse chromosome 6. Northern blot analysis revealed testis-specific expression. The RALDH genes belong to the aldehyde dehydrogenase (ALDH) multi-gene family. Three types of RALDH genes (e.g. human ALDH1/mouse Ahd2/rat RalDH(I), human ALDH11/mouse Raldh2/rat RalDH(II) and human ALDH6) are highly conserved during evolution, sharing about 70% identity at the amino acid level between any two gene types and 90% identity between any two mammalian genes of the same type. Different RALDH types show specific tissue and developmental expression patterns, suggesting (i) a regulatory mechanism of retinoic acid synthesis via different promoters of RALDH genes, and (ii) distinctive biological roles of different isozymes in embryogenesis and organogenesis.