If the MDF is sufficiently high, the pathway contains no thermodynamic bottlenecks that would hamper its operation in vivo. The condensation of formaldehyde can be catalyzed by the N-heterocyclic carbine in chemistry 29 , In biology, the thiazolium ring of the cofactor thiamine diphosphate ThDP has similar function, which could activate one aldehyde and then form dimer with another aldehyde In order to find an enzyme to condense two molecules of formaldehyde into one molecule of glycolaldehyde, we referred to the catalytic mechanisms of ThDP-dependent enzymes and constructed a theozyme model, which includes ThDP, glycolaldehyde, and glutamic acid that provides electron for the reaction 32 Fig.
The distance between C2 atom and the product of glycolaldehyde is critical for triggering the catalytic reaction Fig. Thus, we analyzed the distance between C2 atom and glycolaldehyde in each candidate protein.
Theozyme model construction and functional identification of the glycolaldehyde synthase. The glutamate glu is tan; glycolaldehyde is cyan; ThDP is green. The distances between C2 atom in ThDP and glycolaldehyde carbon atoms are represented by d1 and d2.
The green dot is magnesium ion. The amount of product yellow for the tested protein are shown on the right. ND no detection. Error bars represent s.
Source data are provided as a Source Data file. Based on the average distances in each protein using molecular docking Supplementary Data 1 36 , six candidates with short distances and clear functional annotations were defined as candidates Fig.
In addition, three proteins with long distances were randomly chosen as controls. The candidates and controls were expressed and purified to test their ability of producing glycolaldehyde from formaldehyde. Three out of six candidates exhibited the desired activity, while three controls did not have the function, indicating the distance between C2 atom and glycolaldehyde plays a critical role on the condensation of formaldehyde.
After screening, we found that 14 out of 25 positions showed higher activities than M1 mutant Supplementary Fig. N27 was used three times since the variants in this position displayed the highest activity. Using M1 as template, we introduced each group of mutations into M1 and selected the highest active mutant, which was labeled as M2. By performing three rounds of iterative combinatorial mutagenesis among these positions, we totally screened 64, clones and obtained a high activity mutant that contains five novel mutations around the active center.
Finally, the variant with 7 residue mutations was named as glycolaldehyde synthase GALS. Protein engineering and mechanism analysis of the glycolaldehyde synthase. Pink dots represent the volumes of the binding-pockets Supplementary Data 2 , which are In order to figure out how these mutations improve enzyme activity, we proposed to redo crystallization of GALS.
The active pocket locates at the interface of homodimer of GALS The protein structure of GALS may differ from the starting protein after several rounds of mutation. We found that the mutation of LE introduces two additional hydrogen bonds to the hydroxyl group of intermediate analogue IMA , which may contribute in stabilizing transition state and cleaving C—C bond between product and cofactor ThDP Fig. The mutation of LG enlarged the volume of substrate binding pocket by replacing the big isobutyl group with a hydrogen group.
The third mutation of AM may reorient the substrate and then enhance the interaction between ThDP and substrate. The last two mutations HV and QF expanded the pore radius of outside surface and may facilitate access for substrate or product Fig. In nature, no enzyme was reported to achieve the synthesis of AcP from glycolaldehyde.
To confirm our hypothesis, we selected eight candidates Fig. After gene synthesis and protein purification Supplementary Fig. Fortunately, five out of eight PKs displayed significant catalytic activities. Computational analysis and functional identification of the acetyl-phosphate synthase. The details of the selected PKs are present in the supplementary note and supplementary Fig. The activity of each protein was detected by adding 0.
To reveal the mechanism of forming DHEThDP from glycolaldehyde, we proposed to perform theoretical analysis based on the previous computational model of PK Based on computational simulation, we found that the energy barrier is only IM2 is energetically more stable than IM1.
His64, Tyr, and Asn are important for the nucleophilic attack by Pi and they together form the binding site of Pi Site-directed mutagenesis experiments also indicated that these residues are pivotal for enzyme activity Supplementary Fig. Firstly, we measured the yield of glycolaldehyde by GALS under different concentrations of formaldehyde. The results showed that GALS can effectively catalyze the dimerization of formaldehyde, and the yield of glycolaldehyde increased with the concentration of substrate improved Fig.
Secondly, we examined the catalytic efficiency from glycolaldehyde to AcP, which was quantified by the content of acetic acid since AcP would be quickly degraded into acetic acid Supplementary Fig. Thus, it is necessary to take a balance for the concentration of formaldehyde in resolving this conflict. The yields of acetic acid or acetyl-CoA were measured at different time points. Samples in all assays were analyzed by HPLC. With increasing the concentration of formaldehyde, the yield of acetic acid in the system initially increased and then decreased Fig.
Interestingly, the final yield of acetic acid is even higher than that in the reaction system from glycolaldehyde to AcP with the same amount of formaldehyde Fig. Finally, the SACA pathway produced 5. However, the yield of acetic acid 7. Our results indicated that it is successful for the biosynthesis of acetyl-CoA from formaldehyde by the SACA pathway in vitro. Firstly, cell lysates were used to verify the biosynthesis of acetyl-CoA by the addition of 13 C-labeled formaldehyde and CoA.
In addition, acetyl-CoA would be converged with oxaloacetate to enter the tricarboxylic acid TCA cycle. However, we did not detect significant differences in higher order mass isotopomers. It would be caused by the low amount of double 13 C-labeled isotopomers in the TCA cycle. Cells were induced in LB and transferred to M9 medium containing 13 C-labeled methanol.
Subsequently, we proposed to test carbon flow within cells. Due to the toxicity of formaldehyde to cells, we introduced the methanol dehydrogenase from Bacillus stearothermophilus BsMDH 41 to maintain a continuously low concentration of formaldehyde in vivo Fig.
The cultured cells were harvested at different time points and were used to detect proteinogenic amino acids and intracellular metabolites Fig. In order to further evaluate the SACA pathway in vivo, we proposed to test the cell growth stepwise by feeding each intermediate in the pathway.
Initially E. By adding different concentrations of glycolaldehyde Supplementary Fig. The contribution of glycolaldehyde to biomass cellular dry weight, CDW was 0. Assessing the SACA pathway by cell growth. Cells initially were cultured in LB medium. IPTG was added to induce protein expression and then the supplemental carbon sources were added. OD was detected at different time points. The strain containing SACA pathway was inhibited initially and then grew up normally under the same condition, which may be caused by its faster rate of formaldehyde consumption than the strain with empty vector Supplementary Fig.
Unfortunately the strain containing SACA pathway did not have more biomass with supplement of formaldehyde than those without formaldehyde. These results indicated that although the SACA pathway is more efficient for removing the toxic formaldehyde, it is not enough to provide biomass. At last, we intended to evaluate the SACA pathway by feeding methanol, which would be continuously converted into formaldehyde.
By comparing with the strain without the SACA pathway, we found that the amount of biomass derived from methanol was 0. Seeking cheaper and more sustainable feedstock for bio-manufacturing is a major challenge in the field of industrial biology.
Organic one-carbon resources, including methane, methanol, formaldehyde and formic acid, would be desired feedstocks, since the annual total output are extremely plentiful and would be sustainably generated from CO 2 26 , 43 , In this study, we successfully constructed the SACA pathway by involving two designed enzymes to convert one-carbon formaldehyde into the central metabolite acetyl-CoA, which is precursor for most of products in bio-manufacturing.
The designed enzyme GALS would produce 1. Therefore, releasing formaldehyde inhibition on ACPS would be a possible strategy improving the efficiency of the SACA pathway in the cell free system in the future. Considering high toxicity of formaldehyde to cell, it is impossible to supply the same amount of formaldehyde in vivo. However we think that there are two strategies to improve performance of the SACA pathway in vivo. It is accessible to increase substrate affinities of both designed enzymes by protein engineering and then facilitate the application of the SACA pathway within cells.
It also should be possible to consider applying other host, like Pichia pastoris 45 , whose peroxisomes allow to maintain formaldehyde at a higher concentration.
In summary, although it is not currently practicable in vivo, the SACA pathway exhibits the following advantages: I it is the shortest pathway from formaldehyde to acetyl-CoA which only contains three enzymes; II it is carbon-conserved and ATP-independent; III it is feasible under both aerobic and anaerobic conditions.
Thus, continuing to improve enzyme activities in this pathway or to increase formaldehyde tolerance would enable carbon-conserving and ATP-independent conversion to produce chemicals and fuels from plentiful one-carbon resources, which could alleviate the pressure of the resource supply for bio-manufacturing and also greatly reduce production costs.
Furthermore, combining with the electrochemical conversion from CO 2 to formic acid 43 and biotransformation from formic acid to formaldehyde 26 , the SACA pathway would also be applied for industrial chemicals manufacture from CO 2 in the future. For detailed information, please see Supplementary information. Plasmids used in the study are listed in Supplementary Table 4. All plasmids were constructed by Gibson DNA assembly.
Acetate kinase and hexokinase were purchased from Sigma-Aldrich. All genes were transformed into E. Proteins were purified by His-Spin protein mini-prep columns Zymo Research. First, a single-point saturation mutation method was used to construct the clone library.
The activity of the mutants were determined by measuring the amount of glycolaldehyde produced by the whole cell catalytic systems. Amino acids with highly active mutants were selected for subsequent iterative saturation mutations.
The procedures of various control experiments were consistent with the above. Cells from different culture times were collected and used to detect intracellular metabolites and proteinogenic amino acids. Samples were concentrated and tested with LC-MS. Engineered E. Cells were induced using IPTG with 0. And then, OD was measured at each specified time point.
Similarly, bacterial parasites like Rickettsia were implied to depend on import of external CoA Daugherty et al. Transporter-mediated uptake of CoA has been demonstrated for isolated mitochondria from plants Neuburger, Day, and Douce and animals Tahiliani , raising the possibility that mitochondria and some intracellular parasites share a conserved CoA transporter.
The phylogenetic pattern of CoA genes in Plasmodium falciparum , the causal agent of human malaria, is similar to that found in animals, indicating that P.
This P. PPAT was previously demonstrated to be essential in E. The genomes from the eukaryotic crown group fungi, plants, animals all contain highly conserved homologues to the five human enzymes required for conversion of pantothenate into CoA.
Archaea lack highly conserved homologues to E. The possibility that archaeal genomes might contain more distantly related homologues to these enzymes was investigated by using iterative Psi-Blast searches against the archaeal subset of the NCBI peptide sequence database. Psi-Blast detected weak similarity between E. In summary, Blast or Psi-Blast searches cannot identify any of the missing archaeal genes involved in the synthesis of phosphopantothenate.
More distantly related archaeal members of COG, which are not identified in figure 2 , can be detected in the second and third Psi-Blast iterations using the human PPAT domain as a query sequence. COG dephospho-CoA kinase includes the conserved bacterial and eukaryotic DPCKs and also includes a family of nucleotide kinases conserved in archaea. While archaeal COG members were not detected by Blast fig.
Thus, at least a subset of archaeal species should be able to convert pantoate into phosphopantothenate. They are present in all archaea except in Thermoplasma , but they are absent from bacteria or eukaryotes.
COG members are distantly related to mevalonate and homoserine kinases of the GHMP-kinase family, and searching the Pfam protein families database Bateman et al. Genschel, unpublished data.
The following picture of archaeal CoA biosynthesis emerges from the above results. Enzymes for the synthesis of pantoate in methanogens could not be identified using homology or non-homology methods.
This confirms that the set of enzymes for synthesis of phosphopantothenate in methanogens is unrelated to the corresponding bacterial enzymes. Thermoplasma lack bacterial or archaeal enzymes for the synthesis of pantothenate and may be dependent on exogenous pantothenate. Phylogenetic analysis was carried out for individual enzymes of pantothenate and CoA synthesis to shed light on the origin of eukaryotic CoA genes and to reveal cases of horizontal gene transfer that are not apparent from the mere presence or absence of these genes in the genome.
The scope of ancient horizontal gene transfers and the criteria for the detection of such events were recently reviewed Brown However, the pairwise similarity scores between Thermotoga and archaeal KPHMTs are within the range of the scores obtained within the archaeal group, and some archaeal KPHMTs are more similar to the enzyme from Thermotoga than to homologues in other archaea. The assumption that KPHMT was present in the common ancestor of bacteria and archaea would also require that the gene was subsequently lost in many archaeal species.
A similar situation is found in the case of KPR, which catalyzes the reduction of ketopantoate to pantoate. Archaeal KPRs are clustered with the homologue from Aquifex aeolicus in the tree shown in figure 3 b. Previous analyses of the genomes of T. The fact that the distributions and phylogenetic positions of archaeal KPHMT and KPR genes are more parsimoniously accounted for by horizontal gene transfer than by vertical inheritance suggests that a subset of archaea received the genes for the synthesis of pantoate from bacterial thermophiles after the separation of the bacterial and archaeal lineages.
Homologues to KPR are present in fungi but absent from plants and several bacterial species. Both S. The high similarity between S.
The remaining eukaryotic KPHMTs in figure 3 a are monophyletic, indicating that the gene might have been present in the common ancestor of plants and fungi. In contrast, plant and fungal sequences for PS are polyphyletic fig. This suggests that unrelated forms of PANK were recruited independently after the separation of the domains. However, these species would be expected to contain some form of PANK because the remaining CoA pathway is conserved in them fig.
Psi-Blast searches identify distant homologues to E. However, no homologues can be detected in the other species mentioned above. While the identity of PANK in archaea and in some bacteria remains to be demonstrated experimentally, these observations indicate clearly that there are at least three and possibly more unrelated forms of PANK. Conservation of these enzymes across all three domains can be established by Blast or Psi-Blast searches.
The E. Indeed, using the M. PPCDC is well conserved in all three domains of life, which directly supports monophyly of this enzyme. Phe and Asn correspond to invariant or highly conserved sites, respectively, while Tyr is conserved in eukaryotes but is changed to isoleucine or valine in prokaryotes, including bacterial species with monofunctional PPCS. Asn is conserved in eukaryotes and in most bacteria but is changed to His in archaea and actinobacteria. Cys is part of the proposed substrate recognition clamp of E.
This motif is largely conserved in bacteria and eukaryotes, except in actinobacteria where Cys is changed to Gly. One possible explanation is that these exchanges, affecting both Asn and Cys, are compensatory and restore activity. The position of streptococcal and enterococcal taxa between the archaeal and eukaryotic domains in the PPCS tree fig. Eukaryotes appear as a sister domain to archaea in the case of PPCS fig.
PPAT from E. Monophyly of this group was inferred by sequence and structure comparisons Bork et al. Analysis of the phyletic patterns of NTases and structurally related proteins suggested that at least four members of this superfamily were present in the universal ancestor and that diversification of NTases predated the universal ancestor Aravind, Anantharaman, and Koonin Bacterial and archaeal PPATs may, therefore, originate from distinct ancestral NTases, which would explain the observed sequence divergence between them.
Phylogenetic tree analysis of the conserved archaeal and eukaryotic PPATs supports monophyly of these domains data not shown , indicating that the eukaryotic ancestor inherited PPAT from archaea. This indicates that the two metazoan forms of DPCK are not derived from gene duplication in the ancestor of metazoa but were inherited independently from bacteria. Phylogenetic profiling revealed a mosaic of orthologous relationships of CoA biosynthetic genes in bacteria, archaea, and eukaryotes.
The set of CoA pathway enzymes from E. Similarly, the human CoA enzymes are well conserved within the eukaryotic domain. However, based on homology, only the four ultimate CoA enzymes can be identified in archaea.
This suggests that archaea have unrelated enzymes with PS and PANK activities and that methanogens additionally have unrelated enzymes for the synthesis of pantoate. Archaeal PS and PANK were predicted by chromosomal proximity and found to be unrelated to the bacterial or eukaryotic functional analogues, indicating that convergent evolution acted in the bacterial and archaeal lineages.
Therefore, the enzymes required for the synthesis of phosphopantothenate were recruited independently in bacteria and archaea. Eukaryotes inherited their genes for pantothenate synthesis from bacteria, whereas eukaryotic genes for CoA biosynthesis were partly derived from bacteria and partly from archaea. This explanation is based on the view that eukaryotes arose through some fusion of an archaeon with a bacterium, a view that figures in several models aiming to explain the transition from prokaryotes to eukaryotes Martin et al.
There are many CoA-dependent enzymes with universal phyletic distribution, for example, citrate synthase EC 2. Hence, CoA must have been available already in the RNA world or at a very early stage of the universal ancestor. Experimental evidence implied pantothenate and pantetheine, but not dephospho-CoA or CoA, as potential prebiotic compounds Miller and Schlesinger ; Keefe, Newton, and Miller Alternatively, the most ancient step of the CoA pathway could have been the synthesis of phosphopantetheine from prebiotic pantothenate.
It may be possible to discriminate among these alternatives if comparative genome analysis can reveal whether phosphopantetheine-dependent or CoA-dependent enzymes are the more ancient. Geoffrey McFadden, Associate Editor. Bacterial biosynthesis of coenzyme A. See text for abbreviations of enzyme names.
Phylogenetic profile of E. The putative homologues retrieved in this way were scored according to pairwise amino acid identity in the best local Blast alignment. The boxes indicate groups of species in which these bifunctional enzymes are conserved. In the genomes of Arabidopsis and Drosophila , a uridine kinase domain was detected with the E. The accession numbers of the E. Neighbor-joining trees were generated from maximum likelihood distances using the JTT-F matrix.
The neighbor-joining trees were then refined by local rearrangement searches using the ProtML algorithm. The scale bar indicates substitutions for each tree. For clarity, some sequences from groups of highly related species were excluded from the trees. The trees were constructed as described in figure 3. Monophyly of the archaeal, bacterial, and eukaryotic domains is supported in both trees, except for the position of PPCS from Streptococcus and Enterococcus.
Abiko, Y. Investigations on pantothenic acid and its related compounds. Biochemical studies. Separation and substrate specificity of pantothenate kinase and phosphopantothenoylcysteine synthetase. Tokyo 61 : Metabolism of coenzyme A. Greenberg, ed. Metabolism of sulphur compounds. Academic Press, New York. Adachi, J. Computer science monographs, No.
Institute of Statistical Mathematics, Tokyo. Adam, R. Biology of Giardia lamblia. Altschul, S. Gish, W. Miller, E. Myers, and D. Basic local alignment search tool. Madden, A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. Nucleic Acids Res. Aravind, L.
Anantharaman, and E. Proteins 48 : 1 Tatusov, Y. Wolf, D. Walker, and E. Evidence for massive gene exchange between archaeal and bacterial hyperthermophiles. Trends Genet. Bateman, A. Birney, L. Cerruti, R.
Durbin, L. Etwiller, S. Eddy, S. Steps 3 and 4. CoA binds the succinyl group to form succinyl CoA. Step 5. A phosphate group is substituted for coenzyme A, and a high- energy bond is formed. This energy is used in substrate-level phosphorylation during the conversion of the succinyl group to succinate to form either guanine triphosphate GTP or ATP. There are two forms of the enzyme, called isoenzymes, for this step, depending upon the type of animal tissue in which they are found.
One form is found in tissues that use large amounts of ATP, such as heart and skeletal muscle. This form produces ATP. The second form of the enzyme is found in tissues that have a high number of anabolic pathways, such as liver. This form produces GTP. In particular, protein synthesis primarily uses GTP. Step 6. Step six is a dehydration process that converts succinate into fumarate. Unlike NADH, this carrier remains attached to the enzyme and transfers the electrons to the electron transport chain directly.
This process is made possible by the localization of the enzyme catalyzing this step inside the inner membrane of the mitochondrion. Step 7. Water is added to fumarate during step seven, and malate is produced. The last step in the citric acid cycle regenerates oxaloacetate by oxidizing malate. Another molecule of NADH is produced. Two carbon atoms come into the citric acid cycle from each acetyl group, representing four out of the six carbons of one glucose molecule.
Two carbon dioxide molecules are released on each turn of the cycle; however, these do not necessarily contain the most recently-added carbon atoms. The two acetyl carbon atoms will eventually be released on later turns of the cycle; thus, all six carbon atoms from the original glucose molecule are eventually incorporated into carbon dioxide.
These carriers will connect with the last portion of aerobic respiration to produce ATP molecules. Several of the intermediate compounds in the citric acid cycle can be used in synthesizing non-essential amino acids; therefore, the cycle is amphibolic both catabolic and anabolic.
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