Identification and characterization of the flavin:NADH reductase (PrnF) involved in a novel two-component arylamine oxygenase

Two-component oxygenases catalyze a wide variety of important oxidation reactions. Recently we characterized a novel arylamine N-oxygenase (PrnD), a new member of the two-component oxygenase family (J. Lee et al., J. Biol. Chem. 280:36719-36728, 2005). Although arylamine N-oxygenases are widespread in nature, aminopyrrolnitrin N-oxygenase (PrnD) represents the only biochemically and mechanistically characterized arylamine N-oxygenase to date. Here we report the use of bioinformatic and biochemical tools to identify and characterize the reductase component (PrnF) involved in the PrnD-catalyzed unusual arylamine oxidation. The prnF gene was identified via sequence analysis of the whole genome of Pseudomonas fluorescens Pf-5 and subsequently cloned and overexpressed in Escherichia coli. The purified PrnF protein catalyzes reduction of flavin adenine dinucleotide (FAD) by NADH with a k_cat of 65 s⁻¹ (K_m = 3.2 μM for FAD and 43.1 μM for NADH) and supplies reduced FAD to the PrnD oxygenase component. Unlike other known reductases in two-component oxygenase systems, PrnF strictly requires NADH as an electron donor to reduce FAD and requires unusual protein-protein interaction with the PrnD component for the efficient transfer of reduced FAD. This PrnF enzyme represents the first cloned and characterized flavin reductase component in a novel two-component arylamine oxygenase system.     

Characterization of a novel airway epithelial cell-specific short chain alcohol dehydrogenase/reductase gene whose expression is up-regulated by retinoids and is involved in the metabolism of retinol

Multiple retinoic acid responsive cDNAs were isolated from a high density cDNA microarray membrane, which was developed from a cDNA library of human tracheobronchial epithelial cells. Five selected cDNA clones encoded the sequence of the same novel gene. The predicted open reading frame of the novel gene encoded a protein of 319 amino acids. The deduced amino acid sequence contains four motifs that are conserved in the short-chain alcohol dehydrogenase/reductase (SDR) family of proteins. The novel gene shows the greatest homology to a group of dehydrogenases that can oxidize retinol (retinol dehydrogenases). The mRNA of the novel gene was found in trachea, colon, tongue, and esophagus. In situ hybridization of airway tissue sections demonstrated epithelial cell-specific gene expression, especially in the ciliated cell type. Both all-trans-retinoic acid and 9-cis-retinoic acid were able to elevate the expression of the novel gene in primary human tracheobronchial epithelial cells in vitro. This elevation coincided with an enhanced retinol metabolism in these cultures. COS cells transfected with an expression construct of the novel gene were also elevated in the metabolism of retinol. The results suggested that the novel gene represents a new member of the SDR family that may play a critical role in retinol metabolism in airway epithelia as well as in other epithelia of colon, tongue, and esophagus.     

ATP‐dependent/‐independent enzymatic ring reductions involved in the anaerobic catabolism of naphthalene

Polycyclic aromatic hydrocarbons are among the most hazardous environmental pollutants. However, in contrast to aerobic degradation, the respective degradation pathways in anaerobes are greatly unknown which has so far prohibited many environmental investigations. In this work, we studied the enzymatic dearomatization reactions involved in the degradation of the PAH model compounds naphthalene and 2‐methylnaphthalene in the sulfate‐reducing enrichment culture N47. Cell extracts of N47 grown on naphthalene catalysed the sodium dithionite‐dependent four‐electron reduction of the key intermediate 2‐naphthoyl‐coenzyme A (NCoA) to 5,6,7,8‐tetrahydro‐2‐naphthoyl‐CoA (THNCoA). The NCoA reductase activity was independent of ATP and was, surprisingly, not sensitive to oxygen. In cell extracts in the presence of various electron donors the product THNCoA was further reduced by a two‐electron reaction to most likely a conjugated hexahydro‐2‐naphthoyl‐CoA isomer (HHNCoA). The reaction assigned to THNCoA reductase strictly depended on ATP and was oxygen‐sensitive with a half‐life time between 30 s and 1 min when exposed to air. The rate was highest with NADH as electron donor. The results indicate that two novel and completely different dearomatizing ring reductases are involved in anaerobic naphthalene degradation. While the THNCoA reducing activity shows some properties of ATP‐dependent class I benzoyl‐CoA reductases, NCoA reduction appears to be catalysed by a previously unknown class of dearomatizing aryl‐carboxyl‐CoA reductases.     

Identification of enzymes involved in anaerobic benzene degradation by a strictly anaerobic iron‐reducing enrichment culture

Anaerobic benzene degradation was studied with a highly enriched iron‐reducing culture (BF) composed of mainly Peptococcaceae‐related Gram‐positive microorganisms. The proteomes of benzene‐, phenol‐ and benzoate‐grown cells of culture BF were compared by SDS‐PAGE. A specific benzene‐expressed protein band of 60 kDa, which could not be observed during growth on phenol or benzoate, was subjected to N‐terminal sequence analysis. The first 31 amino acids revealed that the protein was encoded by ORF 138 in the shotgun sequenced metagenome of culture BF. ORF 138 showed 43% sequence identity to phenylphosphate carboxylase subunit PpcA of Aromatoleum aromaticum strain EbN1. A LC/ESI‐MS/MS‐based shotgun proteomic analysis revealed other specifically benzene‐expressed proteins with encoding genes located adjacent to ORF 138 on the metagenome. The protein products of ORF 137, ORF 139 and ORF 140 showed sequence identities of 37% to phenylphosphate carboxylase PpcD of A. aromaticum strain EbN1, 56% to benzoate‐CoA ligase (BamY) of Geobacter metallireducens and 67% to 3‐octaprenyl‐4‐hydroxybenzoate carboxy‐lyase (UbiD/UbiX) of A. aromaticum strain EbN1 respectively. These genes are proposed as constituents of a putative benzene degradation gene cluster (～17 kb) composed of carboxylase‐related genes. The identified gene sequences suggest that the initial activation reaction in anaerobic benzene degradation is probably a direct carboxylation of benzene to benzoate catalysed by putative anaerobic benzene carboxylase (Abc). The putative Abc probably consists of several subunits, two of which are encoded by ORFs 137 and 138, and belongs to a family of carboxylases including phenylphosphate carboxylase (Ppc) and 3‐octaprenyl‐4‐hydroxybenzoate carboxy‐lyase (UbiD/UbiX).     

The crystal structure of 3alpha -hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni shows a novel oligomerization pattern within the short chain dehydrogenase/reductase family

The crystal structure of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni (3alpha-HSDH) as well as the structure of its binary complex with NAD(+) have been solved at 1.68-A and 1.95-A resolution, respectively. The enzyme is a member of the short chain dehydrogenase/reductase (SDR) family. Accordingly, the active center and the conformation of the bound nucleotide cofactor closely resemble those of other SDRs. The crystal structure reveals one homodimer per asymmetric unit representing the physiologically active unity. Dimerization takes place via an interface essentially built-up by helix alphaG and strand betaG of each subunit. So far this type of intermolecular contact has exclusively been observed in homotetrameric SDRs but never in the structure of a homodimeric SDR. The formation of a tetramer is blocked in 3alpha-HSDH by the presence of a predominantly alpha-helical subdomain which is missing in all other SDRs of known structure.     

Mutualistic interaction between dichloromethane‐ and chloromethane‐degrading bacteria in an anaerobic mixed culture

The microbial mixed culture RM grows with dichloromethane (DCM) as the sole energy source generating acetate, methane, chloride and biomass as products. Chloromethane (CM) was not an intermediate during DCM utilization consistent with the observation that CM could not replace DCM as a growth substrate. Interestingly, cultures that received DCM and CM together degraded both compounds concomitantly. Transient hydrogen (H₂) formation reaching a maximum concentration of 205 ± 13 ppmv was observed in cultures growing with DCM, and the addition of exogenous H₂ at concentrations exceeding 3000 ppmv impeded DCM degradation. In contrast, CM degradation in culture RM had a strict requirement for H₂. Following five consecutive transfers on CM and H₂, Acetobacterium 16S rRNA gene sequences dominated the culture and the DCM‐degrader Candidatus Dichloromethanomonas elyunquensis was eliminated, consistent with the observation that the culture lost the ability to degrade DCM. These findings demonstrate that culture RM harbours different populations responsible for anaerobic DCM and CM metabolism, and further imply that the DCM and CM degradation pathways are mechanistically distinct. H₂ generated during DCM degradation is consumed by the hydrogenotrophic CM degrader, or may fuel other hydrogenotrophic processes, including organohalide respiration, methanogenesis and H₂/CO₂ reductive acetogenesis.     

The OSBP-related proteins: a novel protein family involved in vesicle transport,cellular lipid metabolism,and cell signalling

Proteins/genes showing high sequence homology to the mammalian oxysterol binding protein (OSBP) have been identified in a variety of eukaryotic organisms from yeast to man. The unifying feature of the gene products denoted as OSBP-related proteins (ORPs) is the presence of an OSBP-type ligand binding (LB) domain. The LB domains of OSBP and its closest homologue bind oxysterols, while data on certain other family members suggest interaction with phospholipids. Many ORPs also have a pleckstrin homology (PH) domain in the amino-terminal region. The PH domains of the family members studied in detail are known to interact with membrane phosphoinositides and play an important role in the intracellular targeting of the proteins. It is plausible that the ORPs constitute a regulatory apparatus that senses the status of specific lipid ligands in membranes, using the PH and/or LB domains, and mediates information to yet poorly known downstream machineries. Functional studies carried out on the ORP proteins in different organisms indicate roles of the gene family in diverse cellular processes including control of lipid metabolism, regulation of vesicle transport, and cell signalling events.     

Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis

Plants of the order Ranunculales, especially members of the species Papaver, accumulate a large variety of benzylisoquinoline alkaloids with about 2500 structures, but only the opium poppy (Papaver somniferum) and Papaver setigerum are able to produce the analgesic and narcotic morphine and the antitussive codeine. In this study, we investigated the molecular basis for this exceptional biosynthetic capability by comparison of alkaloid profiles with gene expression profiles between 16 different Papaver species. Out of 2000 expressed sequence tags obtained from P. somniferum, 69 show increased expression in morphinan alkaloid-containing species. One of these cDNAs, exhibiting an expression pattern very similar to previously isolated cDNAs coding for enzymes in benzylisoquinoline biosynthesis, showed the highest amino acid identity to reductases in menthol biosynthesis. After overexpression, the protein encoded by this cDNA reduced the keto group of salutaridine yielding salutaridinol, an intermediate in morphine biosynthesis. The stereoisomer 7-epi-salutaridinol was not formed. Based on its similarities to a previously purified protein from P. somniferum with respect to the high substrate specificity, molecular mass and kinetic data, the recombinant protein was identified as salutaridine reductase (SalR; EC 1.1.1.248). Unlike codeinone reductase, an enzyme acting later in the pathway that catalyses the reduction of a keto group and which belongs to the family of the aldo-keto reductases, the cDNA identified in this study as SalR belongs to the family of short chain dehydrogenases/reductases and is related to reductases in monoterpene metabolism.     

Biomarker evidence for widespread anaerobic methane oxidation in Mediterranean sediments by a consortium of methanogenic archaea and bacteria. The Medinaut Shipboard Scientific Party

Although abundant geochemical data indicate that anaerobic methane oxidation occurs in marine sediments, the linkage to specific microorganisms remains unclear. In order to examine processes of methane consumption and oxidation, sediment samples from mud volcanoes at two distinct sites on the Mediterranean Ridge were collected via the submersible Nautile. Geochemical data strongly indicate that methane is oxidized under anaerobic conditions, and compound-specific carbon isotope analyses indicate that this reaction is facilitated by a consortium of archaea and bacteria. Specifically, these methane-rich sediments contain high abundances of methanogen-specific biomarkers that are significantly depleted in (13)C (delta(13)C values are as low as -95 per thousand). Biomarkers inferred to derive from sulfate-reducing bacteria and other heterotrophic bacteria are similarly depleted. Consistent with previous work, such depletion can be explained by consumption of (13)C-depleted methane by methanogens operating in reverse and as part a consortium of organisms in which sulfate serves as the terminal electron acceptor. Moreover, our results indicate that this process is widespread in Mediterranean mud volcanoes and in some localized settings is the predominant microbiological process.     

The PRC-barrel: a widespread,conserved domain shared by photosynthetic reaction center subunits and proteins of RNA metabolism

Background

The H subunit of the purple bacterial photosynthetic reaction center (PRC-H) is important for the assembly of the photosynthetic reaction center and appears to regulate electron transfer during the reduction of the secondary quinone. It contains a distinct cytoplasmic β-barrel domain whose fold has no close structural relationship to any other well known β-barrel domain.

Results

We show that the PRC-H β-barrel domain is the prototype of a novel superfamily of protein domains, the PRC-barrels, approximately 80 residues long, which is widely represented in bacteria, archaea and plants. This domain is also present at the carboxyl terminus of the pan-bacterial protein RimM, which is involved in ribosomal maturation and processing of 16S rRNA. A family of small proteins conserved in all known euryarchaea are composed entirely of a single stand-alone copy of the domain. Versions of this domain from photosynthetic proteobacteria contain a conserved acidic residue that is thought to regulate the reduction of quinones in the light-induced electron-transfer reaction. Closely related forms containing this acidic residue are also found in several non-photosynthetic bacteria, as well as in cyanobacteria, which have reaction centers with a different organization. We also show that the domain contains several determinants that could mediate specific protein-protein interactions.

Conclusions

The PRC-barrel is a widespread, ancient domain that appears to have been recruited to a variety of biological systems, ranging from RNA processing to photosynthesis. Identification of this versatile domain in numerous proteins could aid investigation of unexplored aspects of their biology.

     

The PHCCEx domain of Tiam1/2 is a novel protein‐ and membrane‐binding module

Tiam1 and Tiam2 (Tiam1/2) are guanine nucleotide‐exchange factors that possess the PH–CC–Ex (pleckstrin homology, coiled coil and extra) region that mediates binding to plasma membranes and signalling proteins in the activation of Rac GTPases. Crystal structures of the PH–CC–Ex regions revealed a single globular domain, PHCCEx domain, comprising a conventional PH subdomain associated with an antiparallel coiled coil of CC subdomain and a novel three‐helical globular Ex subdomain. The PH subdomain resembles the β‐spectrin PH domain, suggesting non‐canonical phosphatidylinositol binding. Mutational and binding studies indicated that CC and Ex subdomains form a positively charged surface for protein binding. We identified two unique acidic sequence motifs in Tiam1/2‐interacting proteins for binding to PHCCEx domain, Motif‐I in CD44 and ephrinB's and the NMDA receptor, and Motif‐II in Par3 and JIP2. Our results suggest the molecular basis by which the Tiam1/2 PHCCEx domain facilitates dual binding to membranes and signalling proteins.     

The twin-arginine translocation system: a novel means of transporting folded proteins in chloroplasts and bacteria

Protein translocases have been characterised in several membrane systems and the translocation mechanisms have been shown to differ in critical respects. Nevertheless, the majority were believed to transport proteins only in a largely unfolded state, and this widespread characteristic was viewed as a likely evolutionary effort to minimise the diameter of translocation pore required. Within the last few years, however, studies on the chloroplast thylakoid membrane have revealed a novel class of protein translocase which possesses the apparently unique ability to transport fully-folded proteins across a tightly sealed energy-transducing membrane. A related system, (the twin-arginine translocation, or Tat system) has now been characterised in the Escherichia coli plasma membrane and considerations of its substrate specificity again point to its involvement in the transport of folded proteins. The emerging data suggest a critical involvement in many membranes for the biogenesis of two types of globular protein: those that are obliged to fold prior to translocation, and those that fold too tightly or rapidly for other types of protein translocase to handle.