Chloroflexus aurantiacus acetyl-CoA carboxylase evolves fused biotin carboxylase and biotin carboxyl carrier protein to complete carboxylation activity.

Acetyl-CoA carboxylases (ACCs) convert acetyl-CoA to malonyl-CoA, a key step in fatty acid biosynthesis and autotrophic carbon fixation pathways. Three functionally distinct components, biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT), are either separated or partially fused in different combinations, forming heteromeric ACCs. However, an ACC with fused BC-BCCP and separate CT has not been identified, leaving its catalytic mechanism unclear. Here, we identify two BC isoforms (BC1 and BC2) from Chloroflexus aurantiacus, a filamentous anoxygenic phototroph that employs 3-hydroxypropionate (3-HP) bi-cycle rather than Calvin cycle for autotrophic carbon fixation. We reveal that BC1 possesses fused BC and BCCP domains, where BCCP could be biotinylated by E. coli or C. aurantiacus BirA on Lys553 residue. Crystal structures of BC1 and BC2 at 3.2 Å and 3.0 Å resolutions, respectively, further reveal a tetramer of two BC1-BC homodimers, and a BC2 homodimer, all exhibiting similar BC architectures. The two BC1-BC homodimers are connected by an eight-stranded ß-barrel of the partially resolved BCCP domain. Disruption of ß-barrel results in dissociation of the tetramer into dimers in solution and decreased biotin carboxylase activity. Biotinylation of the BCCP domain further promotes BC1 and CTß-CTα interactions to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-HP via co-expression with a recombinant malonyl-CoA reductase in E. coli cells. This study revealed a heteromeric ACC that evolves fused BC-BCCP but separate CTα and CTß to complete ACC activity.IMPORTANCEAcetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step in fatty acid biosynthesis and autotrophic carbon fixation pathways across a wide range of organisms, making them attractive targets for drug discovery against various infections and diseases. Although structural studies on homomeric ACCs, which consist of a single protein with three subunits, have revealed the "swing domain model" where the biotin carboxyl carrier protein (BCCP) domain translocates between biotin carboxylase (BC) and carboxyltransferase (CT) active sites to facilitate the reaction, our understanding of the subunit composition and catalytic mechanism in heteromeric ACCs remains limited. Here, we identify a novel ACC from an ancient anoxygenic photosynthetic bacterium Chloroflexus aurantiacus, it evolves fused BC and BCCP domain, but separate CT components to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-hydroxypropionate (3-HP) via co-expression with recombinant malonyl-CoA reductase in E. coli cells. These findings expand the diversity and molecular evolution of heteromeric ACCs and provide a structural basis for potential applications in 3-HP biosynthesis.

Cloning and characterization of α-L-rhamnosidase from Chloroflexus aurantiacus and its application in the production of isoquercitrin from rutin.

OBJECTIVE: This study was conducted to characterize recombinant α-L-rhamnosidase from Chloroflexus aurantiacus and apply the enzyme in the production of isoquercitrin from rutin. RESULTS: The α-L-rhamnosidase from C. aurantiacus was cloned and expressed in Escherichia coli BL21 and purified as a soluble enzyme. α-L-rhamnosidase purified from C. aurantiacus has a molecular mass of approximately 105 kDa and is predicted to exist as a homodimer with a native enzyme of 200 kDa. The purified enzyme exhibited the highest specific activity for rutin among the reported isoquercitrin producing α-L-rhamnosidases and was applied in the production of isoquercitrin from rutin. Under the optimised conditions of pH 6.0, 50 °C, 0.6 U mL-1 α-L-rhamnosidase, and 30 mM rutin, α-L-rhamnosidase from C. aurantiacus produced 30 mM isoquercitrin after 2 h with a 100% conversion yield and productivity of 15 mM h-1. CONCLUSIONS: We achieved a high productivity of isoquercitrin from rutin. Moreover, these results suggest that α-L-rhamnosidase from C. aurantiacus is an effective isoquercitrin producer.

Discovery of a novel thermostable Zn2+ -dependent alcohol dehydrogenase from Chloroflexus aurantiacus through conserved domains mining.

AIMS: The purpose of the study was to demonstrate feasibility of the Conserved Domains Database (CDD) for identification of novel biocatalysts with desirable properties from a class of well-characterized biocatalysts. METHODS AND RESULTS: The thermostable ADH from Sulfolobus solfataricus with a broad substrate range was applied as a template for the search for novel thermostable ADHs via CDD. From the resulting hits, a putative ADH gene from the thermophilic organism Chloroflexus aurantiacus was cloned and expressed in Escherichia coli. The resulting enzyme was purified and characterized. With a temperature activity optimum of 70°C and a broad substrate spectrum especially for diketones, a versatile new biocatalyst was obtained. CONCLUSIONS: Database-based mining in CDD is a suitable approach to obtain novel biocatalysts with desirable properties. Thereby, the available diversity of similar but not equal enzymes within this class can be increased. SIGNIFICANCE AND IMPACT OF THE STUDY: For industrial applications, there is a demand for larger diversity of similar well-characterized enzymes in order to test them for a given process (biodiversity screening). For fundamental science, the comparison of enzymes with similar function but different sequence can provide insight into structure function relationships or the evolution of enzymes. This study gives a good example on how this demand can be efficiently met.

Production of 3-hydroxypropionic acid via the malonyl-CoA pathway using recombinant fission yeast strains.

3-Hydroxypropionic acid (3-HP) can be converted into derivatives such as acrylic acid, a source for producing super absorbent polymers. Although Escherichia coli has often been used for 3-HP production, it exhibits low tolerance to 3-HP. To circumvent this problem, we selected the fission yeast Schizosaccharomyces pombe as this microorganism has higher tolerance to 3-HP than E. coli. Therefore, we constructed S. pombe transformants overexpressing two genes, one encoding the S. pombe acetyl-CoA carboxylase (Cut6p) and the other encoding the malonyl-CoA reductase derived from Chloroflexus aurantiacus (CaMCR). To prevent the degradation of these expressed proteins, we employed an S. pombe protease-deficient strain. Moreover, to increase the cytosolic concentration of acetyl-CoA, we supplemented acetate to the medium, which improved 3-HP production. To further produce 3-HP by overexpressing Cut6p and CaMCR, we exploited the highly expressing S. pombe hsp9 promoter. Finally, culturing in high-density reached 3-HP production to 7.6 g/L at 31 h.

Engineering and systems-level analysis of Saccharomyces cerevisiae for production of 3-hydroxypropionic acid via malonyl-CoA reductase-dependent pathway.

BACKGROUND: In the future, oil- and gas-derived polymers may be replaced with bio-based polymers, produced from renewable feedstocks using engineered cell factories. Acrylic acid and acrylic esters with an estimated world annual production of approximately 6 million tons by 2017 can be derived from 3-hydroxypropionic acid (3HP), which can be produced by microbial fermentation. For an economically viable process 3HP must be produced at high titer, rate and yield and preferably at low pH to minimize downstream processing costs. RESULTS: Here we describe the metabolic engineering of baker's yeast Saccharomyces cerevisiae for biosynthesis of 3HP via a malonyl-CoA reductase (MCR)-dependent pathway. Integration of multiple copies of MCR from Chloroflexus aurantiacus and of phosphorylation-deficient acetyl-CoA carboxylase ACC1 genes into the genome of yeast increased 3HP titer fivefold in comparison with single integration. Furthermore we optimized the supply of acetyl-CoA by overexpressing native pyruvate decarboxylase PDC1, aldehyde dehydrogenase ALD6, and acetyl-CoA synthase from Salmonella enterica SEacs (L641P). Finally we engineered the cofactor specificity of the glyceraldehyde-3-phosphate dehydrogenase to increase the intracellular production of NADPH at the expense of NADH and thus improve 3HP production and reduce formation of glycerol as by-product. The final strain produced 9.8 ± 0.4 g L(-1) 3HP with a yield of 13% C-mol C-mol(-1) glucose after 100 h in carbon-limited fed-batch cultivation at pH 5. The 3HP-producing strain was characterized by (13)C metabolic flux analysis and by transcriptome analysis, which revealed some unexpected consequences of the undertaken metabolic engineering strategy, and based on this data, future metabolic engineering directions are proposed. CONCLUSIONS: In this study, S. cerevisiae was engineered for high-level production of 3HP by increasing the copy numbers of biosynthetic genes and improving flux towards precursors and redox cofactors. This strain represents a good platform for further optimization of 3HP production and hence an important step towards potential commercial bio-based production of 3HP.

Interface matters: the stiffness route to stability of a thermophilic tetrameric malate dehydrogenase.

In this work we investigate by computational means the behavior of two orthologous bacterial proteins, a mesophilic and a thermophilic tetrameric malate dehydrogenase (MalDH), at different temperatures. Namely, we quantify how protein mechanical rigidity at different length- and time-scales correlates to protein thermophilicity as commonly believed. In particular by using a clustering analysis strategy to explore the conformational space of the folded proteins, we show that at ambient conditions and at the molecular length-scale the thermophilic variant is indeed more rigid that the mesophilic one. This rigidification is the result of more efficient inter-domain interactions, the strength of which is further quantified via ad hoc free energy calculations. When considered isolated, the thermophilic domain is indeed more flexible than the respective mesophilic one. Upon oligomerization, the induced stiffening of the thermophilic protein propagates from the interface to the active site where the loop, controlling the access to the catalytic pocket, anchors down via an extended network of ion-pairs. On the contrary in the mesophilic tetramer the loop is highly mobile. Simulations at high temperature, could not re-activate the mobility of the loop in the thermophile. This finding opens questions on the similarities of the binding processes for these two homologues at their optimal working temperature and suggests for the thermophilic variant a possible cooperative role of cofactor/substrate.

The crystal structures of the tri-functional Chloroflexus aurantiacus and bi-functional Rhodobacter sphaeroides malyl-CoA lyases and comparison with CitE-like superfamily enzymes and malate synthases.

BACKGROUND: Malyl-CoA lyase (MCL) is a promiscuous carbon-carbon bond lyase that catalyzes the reversible cleavage of structurally related Coenzyme A (CoA) thioesters. This enzyme plays a crucial, multifunctional role in the 3-hydroxypropionate bi-cycle for autotrophic CO2 fixation in Chloroflexus aurantiacus. A second, phylogenetically distinct MCL from Rhodobacter sphaeroides is involved in the ethylmalonyl-CoA pathway for acetate assimilation. Both MCLs belong to the large superfamily of CitE-like enzymes, which includes the name-giving ß-subunit of citrate lyase (CitE), malyl-CoA thioesterases and other enzymes of unknown physiological function. The CitE-like enzyme superfamily also bears sequence and structural resemblance to the malate synthases. All of these different enzymes share highly conserved catalytic residues, although they catalyze distinctly different reactions: C-C bond formation and cleavage, thioester hydrolysis, or both (the malate synthases). RESULTS: Here we report the first crystal structures of MCLs from two different phylogenetic subgroups in apo- and substrate-bound forms. Both the C. aurantiacus and the R. sphaeroides MCL contain elaborations on the canonical ß8/α8 TIM barrel fold and form hexameric assemblies. Upon ligand binding, changes in the C-terminal domains of the MCLs result in closing of the active site, with the C-terminal domain of one monomer forming a lid over and contributing side chains to the active site of the adjacent monomer. The distinctive features of the two MCL subgroups were compared to known structures of other CitE-like superfamily enzymes and to malate synthases, providing insight into the structural subtleties that underlie the functional versatility of these enzymes. CONCLUSIONS: Although the C. aurantiacus and the R. sphaeroides MCLs have divergent primary structures (~37% identical), their tertiary and quaternary structures are very similar. It can be assumed that the C-C bond formation catalyzed by the MCLs occurs as proposed for malate synthases. However, a comparison of the two MCL structures with known malate synthases raised the question why the MCLs are not also able to hydrolyze CoA thioester bonds. Our results suggest the previously proposed reaction mechanism for malate synthases may be incomplete or not entirely correct. Further studies involving site-directed mutagenesis based on these structures may be required to solve this puzzling question.

Dissection of malonyl-coenzyme A reductase of Chloroflexus aurantiacus results in enzyme activity improvement.

The formation of fusion protein in biosynthetic pathways usually improves metabolic efficiency either channeling intermediates and/or colocalizing enzymes. In the metabolic engineering of biochemical pathways, generating unnatural protein fusions between sequential biosynthetic enzymes is a useful method to increase system efficiency and product yield. Here, we reported a special case. The malonyl-CoA reductase (MCR) of Chloroflexus aurantiacus catalyzes the conversion of malonyl-CoA to 3-hydroxypropionate (3HP), and is a key enzyme in microbial production of 3HP, an important platform chemical. Functional domain analysis revealed that the N-terminal region of MCR (MCR-N; amino acids 1-549) and the C-terminal region of MCR (MCR-C; amino acids 550-1219) were functionally distinct. The malonyl-CoA was reduced into free intermediate malonate semialdehyde with NADPH by MCR-C fragment, and further reduced to 3HP by MCR-N fragment. In this process, the initial reduction of malonyl-CoA was rate limiting. Site-directed mutagenesis demonstrated that the TGXXXG(A)X(1-2)G and YXXXK motifs were important for enzyme activities of both MCR-N and MCR-C fragments. Moreover, the enzyme activity increased when MCR was separated into two individual fragments. Kinetic analysis showed that MCR-C fragment had higher affinity for malonyl-CoA and 4-time higher K cat/K m value than MCR. Dissecting MCR into MCR-N and MCR-C fragments also had a positive effect on the 3HP production in a recombinant Escherichia coli strain. Our study showed the feasibility of protein dissection as a new strategy in biosynthetic systems.

Functional analysis and expression of the mono-heme containing cytochrome c subunit of Alternative Complex III in Chloroflexus aurantiacus.

The filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus possesses an unusual electron transfer complex called Alternative Complex III instead of the cytochrome bc or bf type complex found in nearly all other known groups of phototrophs. Earlier work has confirmed that Alternative Complex III behaves as a menaquinol:auracyanin oxidoreductase in the photosynthetic electron transfer chain. In this work, we focus on elucidating the contribution of individual subunits to the overall function of Alternative Complex III. The monoheme subunit ActE has been expressed and characterized in Escherichia coli. A partially dissociated Alternative Complex III missing subunit ActE and subunit ActG was obtained by treatment with the chaotropic agent KSCN, and was then reconstituted with the expressed ActE. Enzymatic activity of the partially dissociated Alternative Complex III was greatly reduced and was largely restored in the reconstituted complex. The redox potential of the heme in the recombinant ActE was +385mV vs. NHE, similar to the highest potential heme in the intact complex. The results strongly suggest that the monoheme subunit, ActE, is the terminal electron carrier for Alternative Complex III.

Expression of the sub-pathways of the Chloroflexus aurantiacus 3-hydroxypropionate carbon fixation bicycle in E. coli: Toward horizontal transfer of autotrophic growth.

The 3-hydroxypropionate (3-HPA) bicycle is unique among CO2-fixing systems in that none of its enzymes appear to be affected by oxygen. Moreover, the bicycle includes a number of enzymes that produce novel intermediates of biotechnological interest, and the CO2-fixing steps in this pathway are relatively rapid. We expressed portions of the 3-HPA bicycle in a heterologous organism, E. coli K12. We subdivided the 3-HPA bicycle into four sub-pathways: (1) synthesis of propionyl-CoA from acetyl-CoA, (2) synthesis of succinate from propionyl-CoA, (3) glyoxylate production and regeneration of acetyl-CoA, and (4) assimilation of glyoxylate and propionyl-CoA to form pyruvate and regenerate acetyl-CoA. We expressed the novel enzymes of the 3-HPA bicycle in operon form and used phenotypic tests for activity. Sub-pathway 1 activated a propionate-specific biosensor. Sub-pathway 2, found in non-CO2-fixing bacteria, was reassembled in E. coli using genes from diverse sources. Sub-pathway 3, operating in reverse, generated succinyl-CoA sufficient to rescue a sucAD(-) double mutant of its diaminopimelic acid (DAP) auxotrophy. Sub-pathway 4 was able to reduce the toxicity of propionate and allow propionate to contribute to cell biomass in a prpC(-)(2 methylcitrate synthase) mutant strain. These results indicate that all of the sub-pathways of the 3-HPA bicycle can function to some extent in vivo in a heterologous organism, as indicated by growth tests. Overexpression of certain enzymes was deleterious to cell growth, and, in particular, expression of MMC-CoA lyase caused a mucoid phenotype. These results have implications for metabolic engineering and for bacterial evolution through horizontal gene transfer.

Biosynthetic pathway for poly(3-hydroxypropionate) in recombinant Escherichia coli.

Poly(3-hydroxypropionate) (P3HP) is a biodegradable and biocompatible thermoplastic. In this study, we engineered a P3HP biosynthetic pathway in recombinant Escherichia coli. The genes for malonyl-CoA reductase (mcr, from Chloroflexus aurantiacus), propionyl-CoA synthetase (prpE, from E. coli), and polyhydroxyalkanoate synthase (phaC1, from Ralstonia eutropha) were cloned and expressed in E. coli. The E. coli genes accABCD encoding acetyl-CoA carboxylase were used to channel the carbon into the P3HP pathway. Using glucose as a sole carbon source, the cell yield and P3HP content were 1.32 g/L and 0.98% (wt/wt [cell dry weight]), respectively. Although the yield is relatively low, our study shows the feasibility of engineering a P3HP biosynthetic pathway using a structurally unrelated carbon source in bacteria.

SANS investigation of the photosynthetic machinery of Chloroflexus aurantiacus.

Green photosynthetic bacteria harvest light and perform photosynthesis in low-light environments, and contain specialized antenna complexes to adapt to this condition. We performed small-angle neutron scattering (SANS) studies to obtain structural information about the photosynthetic apparatus, including the peripheral light-harvesting chlorosome complex, the integral membrane light-harvesting B808-866 complex, and the reaction center (RC) in the thermophilic green phototrophic bacterium Chloroflexus aurantiacus. Using contrast variation in SANS measurements, we found that the B808-866 complex is wrapped around the RC in Cfx. aurantiacus, and the overall size and conformation of the B808-866 complex of Cfx. aurantiacus is roughly comparable to the LH1 antenna complex of the purple bacteria. A similar size of the isolated B808-866 complex was suggested by dynamic light scattering measurements, and a smaller size of the RC of Cfx. aurantiacus compared to the RC of the purple bacteria was observed. Further, our SANS measurements indicate that the chlorosome is a lipid body with a rod-like shape, and that the self-assembly of bacteriochlorophylls, the major component of the chlorosome, is lipid-like. Finally, two populations of chlorosome particles are suggested in our SANS measurements.

Gradual adaptive changes of a protein facing high salt concentrations.

Several experimental techniques were applied to unravel fine molecular details of protein adaptation to high salinity. We compared four homologous enzymes, which suggested a new halo-adaptive state in the process of molecular adaptation to high-salt conditions. Together with comparative functional studies, the structure of malate dehydrogenase from the eubacterium Salinibacter ruber shows that the enzyme shares characteristics of a halo-adapted archaea-bacterial enzyme and of non-halo-adapted enzymes from other eubacterial species. The S. ruber enzyme is active at the high physiological concentrations of KCl but, unlike typical halo-adapted enzymes, remains folded and active at low salt concentrations. Structural aspects of the protein, including acidic residues at the surface, solvent-exposed hydrophobic surface, and buried hydrophobic surface, place it between the typical halo-adapted and non-halo-adapted proteins. The enzyme lacks inter-subunit ion-binding sites often seen in halo-adapted enzymes. These observations permit us to suggest an evolutionary pathway that is highlighted by subtle trade-offs to achieve an optimal compromise among solubility, stability, and catalytic activity.

Enzymatic activity of the alternative complex III as a menaquinol:auracyanin oxidoreductase in the electron transfer chain of Chloroflexus aurantiacus.

The surprising lack of the cytochrome bc1 complex in the filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus suggests that a functional replacement exists to link the cyclic electron transfer chain. Earlier work identified the alternative complex III (ACIII) as a substitute of cytochrome bc1 complex. Herein, the enzymatic activity of ACIII is studied. The results strongly support the view that the ACIII functions as menaquinol:auracyanin oxidoreductase in the C. aurantiacus electron transfer chain. Among all the substrates tested, auracyanin is the most efficient electron acceptor of ACIII, suggesting that ACIII directly transfers the electron to auracyanin instead of cytochrome c-554. The lack of sensitivity to common inhibitors of the cytochrome bc1 complex indicates a different catalytic mechanism for the ACIII complex.

Iron binding effects on the kinetic stability and unfolding energetics of a thermophilic phenylalanine hydroxylase from Chloroflexus aurantiacus.

The effects of non-heme iron binding on the function, structure, and stability of a monomeric phenylalanine hydroxylase from the thermophile Chloroflexus aurantiacus (caPAH) were investigated. Comparative studies on holo (iron-bound) and apo (iron-depleted) caPAH indicated that iron(II) binding does not significantly affect the overall structure of the enzyme. Thermal denaturation studies performed using differential scanning calorimetry showed that the unfolding reaction was kinetically controlled and that holo-caPAH displayed a large increase in thermal stability (approximately 15 degrees C upshift in the T (m) value) compared with the apoenzyme. Analysis using a simple irreversible two-state model also showed a higher kinetic stability for holo-caPAH at optimal growth temperature (denaturing approximately 8 times more slowly than the apo form at 55 degrees C). Experiments performed in the presence of urea in combination with structure-energetics calculations suggest that iron binding reduces the change in accessible surface area exposed in the unfolding transition state (from approximately 36% to approximately 5% of the total change in accessible surface area) and also the surface involved in water-unsatisfied broken internal contacts (solvation barriers). Additional comparative analyses using phenylalanine hydroxylase from mesophilic and psychrophilic organisms suggest that, in addition to its catalytic role, the non-heme iron serves to enhance the kinetic stability of phenylalanine hydroxylase at the optimal growth temperature of the organism.

Purification, characterization and crystallization of menaquinol:fumarate oxidoreductase from the green filamentous photosynthetic bacterium Chloroflexus aurantiacus.

The integral membrane protein complex, menaquinol:fumarate oxidoreductase (mQFR) has been purified, identified and characterized from the thermophilic green filamentous anoxygenic photosynthetic bacterium Chloroflexus aurantiacus. The complex is composed of three subunits: a 74 kDa flavoprotein that contains a covalently bound flavin adenine dinucleotide, a 28 kDa iron-sulfur cluster-containing polypeptide, and a 27 kDa transmembrane polypeptide, which is also the binding site of two b-type hemes and two menaquinones. The purified complex has an apparent molecular mass of 260 kDa by blue-native PAGE, which is indicative of a native homodimeric form. The isolated complex is active in vitro in both fumarate reduction and succinate oxidation. It has been analyzed by visible absorption, redox titration, chemical analysis and EPR spectroscopy. In addition, phylogenetic analysis shows that the QFR of both C. aurantiacus and Chlorobium tepidum are most closely related to those found in the delta-proteobacteria. The purified enzyme was crystallized and X-ray diffraction data obtained up to 3.2 A resolution.

Properties of R-citramalyl-coenzyme A lyase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus.

The autotrophic CO(2) fixation pathway (3-hydroxypropionate cycle) in Chloroflexus aurantiacus results in the fixation of two molecules of bicarbonate into one molecule of glyoxylate. Glyoxylate conversion to the CO(2) acceptor molecule acetyl-coenzyme A (CoA) requires condensation with propionyl-CoA (derived from one molecule of acetyl-CoA and one molecule of CO(2)) to beta-methylmalyl-CoA, which is converted to citramalyl-CoA. Extracts of autotrophically grown cells contained both S- and R-citramalyl-CoA lyase activities, which formed acetyl-CoA and pyruvate. Pyruvate is taken out of the cycle and used for cellular carbon biosynthesis. Both the S- and R-citramalyl-CoA lyases were up-regulated severalfold during autotrophic growth. S-Citramalyl-CoA lyase activity was found to be due to l-malyl-CoA lyase/beta-methylmalyl-CoA lyase. This promiscuous enzyme is involved in the CO(2) fixation pathway, forms acetyl-CoA and glyoxylate from l-malyl-CoA, and condenses glyoxylate with propionyl-CoA to beta-methylmalyl-CoA. R-Citramalyl-CoA lyase was further studied. Its putative gene was expressed and the recombinant protein was purified. This new enzyme belongs to the 3-hydroxy-3-methylglutaryl-CoA lyase family and is a homodimer with 34-kDa subunits that was 10-fold stimulated by adding Mg(2) or Mn(2+) ions and dithioerythritol. The up-regulation under autotrophic conditions suggests that the enzyme functions in the ultimate step of the acetyl-CoA regeneration route in C. aurantiacus. Genes similar to those involved in CO(2) fixation in C. aurantiacus, including an R-citramalyl-CoA lyase gene, were found in Roseiflexus sp., suggesting the operation of the 3-hydroxypropionate cycle in this bacterium. Incomplete sets of genes were found in aerobic phototrophic bacteria and in the gamma-proteobacterium Congregibacter litoralis. This may indicate that part of the reactions may be involved in a different metabolic process.

Properties of succinyl-coenzyme A:D-citramalate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus.

The phototrophic bacterium Chloroflexus aurantiacus uses the 3-hydroxypropionate cycle for autotrophic CO(2) fixation. This cycle starts with acetyl-coenzyme A (CoA) and produces glyoxylate. Glyoxylate is an unconventional cell carbon precursor that needs special enzymes for assimilation. Glyoxylate is combined with propionyl-CoA to beta-methylmalyl-CoA, which is converted to citramalate. Cell extracts catalyzed the succinyl-CoA-dependent conversion of citramalate to acetyl-CoA and pyruvate, the central cell carbon precursor. This reaction is due to the combined action of enzymes that were upregulated during autotrophic growth, a coenzyme A transferase with the use of succinyl-CoA as the CoA donor and a lyase cleaving citramalyl-CoA to acetyl-CoA and pyruvate. Genomic analysis identified a gene coding for a putative coenzyme A transferase. The gene was heterologously expressed in Escherichia coli and shown to code for succinyl-CoA:d-citramalate coenzyme A transferase. This enzyme, which catalyzes the reaction d-citramalate + succinyl-CoA --> d-citramalyl-CoA + succinate, was purified and studied. It belongs to class III of the coenzyme A transferase enzyme family, with an aspartate residue in the active site. The homodimeric enzyme composed of 44-kDa subunits was specific for succinyl-CoA as a CoA donor but also accepted d-malate and itaconate instead of d-citramalate. The CoA transferase gene is part of a cluster of genes which are cotranscribed, including the gene for d-citramalyl-CoA lyase. It is proposed that the CoA transferase and the lyase catalyze the last two steps in the glyoxylate assimilation route.

Properties of succinyl-coenzyme A:L-malate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus.

The 3-hydroxypropionate cycle has been proposed to operate as the autotrophic CO2 fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. In this pathway, acetyl coenzyme A (acetyl-CoA) and two bicarbonate molecules are converted to malate. Acetyl-CoA is regenerated from malyl-CoA by L-malyl-CoA lyase. The enzyme forming malyl-CoA, succinyl-CoA:L-malate coenzyme A transferase, was purified. Based on the N-terminal amino acid sequence of its two subunits, the corresponding genes were identified on a gene cluster which also contains the gene for L-malyl-CoA lyase, the subsequent enzyme in the pathway. Both enzymes were severalfold up-regulated under autotrophic conditions, which is in line with their proposed function in CO2 fixation. The two CoA transferase genes were cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Succinyl-CoA:L-malate CoA transferase forms a large (alphabeta)n complex consisting of 46- and 44-kDa subunits and catalyzes the reversible reaction succinyl-CoA + L-malate --> succinate + L-malyl-CoA. It is specific for succinyl-CoA as the CoA donor but accepts L-citramalate instead of L-malate as the CoA acceptor; the corresponding d-stereoisomers are not accepted. The enzyme is a member of the class III of the CoA transferase family. The demonstration of the missing CoA transferase closes the last gap in the proposed 3-hydroxypropionate cycle.

New class of bacterial membrane oxidoreductases.

A new class of bacterial multisubunit membrane-bound electron-transfer complexes has been identified based on biochemical and bioinformatic data. It contains subunits homologous to the three-subunit molybdopterin oxidoreductases and four additional subunits, two of which are c-type cytochromes. The complex was purified from the filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus, and putative operons for similar complexes were identified in a wide range of bacteria. In most cases, the presence of the new complex is anticorrelated with the cytochrome bc or bf electron-transfer complex, suggesting that it replaces it functionally. This appears to be a widespread yet previously unrecognized protein complex involved in energy metabolism in bacteria.