Active acetyl-CoA synthase from
 Clostridium thermoaceticum
 obtained by cloning and heterologous expression of
 acsAB
 in
 Escherichia coli

Loke, Huay-Keng; Bennett, George N.; Lindahl, Paul A.

doi:10.1073/pnas.220404397

Cited by 35 publications

(44 citation statements)

References 64 publications

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“…The mutant lacked significant amounts of Ni and ~ 30% less Fe relative to WT recombinant protein ( Table 3). Note that the A-cluster is largely devoid of Ni in these recombinant proteins (10) and so the observed Ni should reflect that present in the C-cluster.…”

Section: Resultsmentioning

confidence: 96%

Evidence for a Proton Transfer Network and a Required Persulfide-Bond-Forming Cysteine Residue in Ni-Containing Carbon Monoxide Dehydrogenases

et al. 2004

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Carbon monoxide dehydrogenase from Moorella thermoacetica catalyzes the reversible oxidation of CO to CO 2 at a nickel-iron-sulfur active-site called the C-cluster. Mutants of a proposed proton transfer pathway and of a cysteine residue recently found to form a persulfide bond with the C-cluster were characterized. Four semi-conserved histidine residues were individually mutated to alanine. His116 and His122 were essential to catalysis, while His113 and His119 attenuated catalysis but were not essential. Significant activity was "rescued" by a double mutant where His116 was replaced by Ala and His was also introduced at position 115. Activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or 123. Activity was also "rescued" by replacing His with Cys at position 116. Mutation of conserved Lys587 near the C-cluster attenuated activity but did not eliminate it. Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala. Mutations of conserved Asn284 also attenuated activity. These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway. The Ser mutant of the persulfide-forming Cys316 was essentially inactive and displayed no EPR signals originating from the C-cluster.Electronic absorption and metal analysis suggests that the C-cluster is absent in this mutant. The persulfide bond appears to be essential for either the assembly or stability of the C-cluster, and/or for eliciting the redox chemistry of the C-cluster required for catalytic activity. Ni-containing carbon monoxide dehydrogenases (CODH's) are found in methanogenic archaea, acetogenic bacteria, and CO-utilizing bacteria, where they play critical roles in C 1 - In this study, we tested these hypotheses using site-directed mutagenesis, enzyme activity assays and EPR spectroscopy. We also examined the effect of mutating the cysteine residue involved in persulfide bond formation. Our results indicate that the persulfide-associated cysteine residue is required for catalysis and possibly for C-cluster assembly or stability. Our evidence also suggests that the 4 His residues as well as Lys587 and Asn284 are indeed used as a catalytic base and a proton relay network. Experimental ProceduresOligonucleotides used to construct mutants were synthesized in the Gene TechnologiesLaboratory at Texas A&M University. Mutants were constructed using the QuikChange sitedirected mutagenesis method from Stratagene, using plasmid pTM02, which contains the genes acsA and acsB, as the template (10). Double mutants were constructed using one oligonucleotide containing both mutations, except for Asn284Ala:His119Ala and Lys587Ala:His113Ala, for which two different oligonucleotides were used in two different PCR reactions. All mutant plasmids were produced using an MJ Research Minicycler PCR machine. Mutants were transformed, expressed, harvested, and purified as described (10). ...

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Section: Resultsmentioning

confidence: 96%

Evidence for a Proton Transfer Network and a Required Persulfide-Bond-Forming Cysteine Residue in Ni-Containing Carbon Monoxide Dehydrogenases

et al. 2004

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“…In these organisms, a reduced anaerobic environment provides the conditions for CO 2 fixation (43). The CODH/ACS enzyme has been expressed in a nonnative host (E. coli) before; however, the enzyme was shown to be active only in the direction of CO oxidation in vitro (18). Never before has this enzyme been shown to function in vivo in a nonnative host.…”

Section: Discussionmentioning

confidence: 99%

Heterologous Expression of the Clostridium carboxidivorans CO Dehydrogenase Alone or Together with the Acetyl Coenzyme A Synthase Enables both Reduction of CO ₂ and Oxidation of CO by Clostridium acetobutylicum

Carlson

Papoutsakis

2017

Appl Environ Microbiol

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With recent advances in synthetic biology, CO 2 could be utilized as a carbon feedstock by native or engineered organisms, assuming the availability of electrons. Two key enzymes used in autotrophic CO 2 fixation are the CO dehydrogenase (CODH) and acetyl coenzyme A (acetyl-CoA) synthase (ACS), which form a bifunctional heterotetrameric complex. The CODH/ACS complex can reversibly catalyze CO 2 to CO, effectively enabling a biological water-gas shift reaction at ambient temperatures and pressures. The CODH/ACS complex is part of the Wood-Ljungdahl pathway (WLP) used by acetogens to fix CO 2 , and it has been well characterized in native hosts. So far, only a few recombinant CODH/ACS complexes have been expressed in heterologous hosts, none of which demonstrated in vivo CO 2 reduction. Here, functional expression of the Clostridium carboxidivorans CODH/ACS complex is demonstrated in the solventogen Clostridium acetobutylicum, which was engineered to express CODH alone or together with the ACS. Both strains exhibited CO 2 reduction and CO oxidation activities. The CODH reactions were interrogated using isotopic labeling, thus verifying that CO was a direct product of CO 2 reduction, and vice versa. CODH apparently uses a native C. acetobutylicum ferredoxin as an electron carrier for CO 2 reduction. Heterologous CODH activity depended on actively growing cells and required the addition of nickel, which is inserted into CODH without the need to express the native Ni insertase protein. Increasing CO concentrations in the gas phase inhibited CODH activity and altered the metabolite profile of the CODHexpressing cells. This work provides the foundation for engineering a complete and functional WLP in nonnative host organisms. IMPORTANCE Functional expression of CO dehydrogenase (CODH) fromClostridium carboxidivorans was demonstrated in C. acetobutylicum, which is natively incapable of CO 2 fixation. The expression of CODH, alone or together with the C. carboxidivorans acetyl-CoA synthase (ACS), enabled C. acetobutylicum to catalyze both CO 2 reduction and CO oxidation. Importantly, CODH exhibited activity in both the presence and absence of ACS. 13 C-tracer studies confirmed that the engineered C. acetobutylicum strains can reduce CO 2 to CO and oxidize CO during growth on glucose.KEYWORDS CO 2 fixation, CO 2 reduction, CO oxidation, Wood-Ljungdahl pathway, acetogenesis, carbon dioxide fixation, carbon dioxide reduction, metabolic engineering, synthetic biology, acetogens

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“…However, it remains to be shown if this regulation is dose-dependent for the whole Methanosarcina population, as is the case for tetO/TetR systems in bacteria and eukaryotes, or an autocatalytic induction of expression due to active uptake of the inducer, as is the case for Plac-and Para-dependent gene expression (Novick andWeiner 1957, Morgan-Kiss et al 2002). With the Pmcr(tetO)/TetR system established for Methanosarcina it seems feasible now to overproduce enzymes in a catalytically active form where other host/overexpresion systems have resulted in partially inactive protein (Roberts et al 1989, Sauer et al 1997, Sauer and Thauer Rudolf 1998, Loke et al 2000. Furthermore, even toxic genes can probably be overproduced because of the tight repression of the hybrid promoter in the absence of tetracycline.…”

Section: Discussionmentioning

confidence: 99%

New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

Guss

Rother

Zhang

et al. 2008

Archaea

119

179

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Summary A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage φC31. Host strains expressing the φC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri PmcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline in strains that express the tetR gene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR-regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion.

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Active acetyl-CoA synthase from Clostridium thermoaceticum obtained by cloning and heterologous expression of acsAB in Escherichia coli

Cited by 35 publications

References 64 publications

Evidence for a Proton Transfer Network and a Required Persulfide-Bond-Forming Cysteine Residue in Ni-Containing Carbon Monoxide Dehydrogenases

Evidence for a Proton Transfer Network and a Required Persulfide-Bond-Forming Cysteine Residue in Ni-Containing Carbon Monoxide Dehydrogenases

Heterologous Expression of the Clostridium carboxidivorans CO Dehydrogenase Alone or Together with the Acetyl Coenzyme A Synthase Enables both Reduction of CO ₂ and Oxidation of CO by Clostridium acetobutylicum

New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

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Active acetyl-CoA synthase from Clostridium thermoaceticum obtained by cloning and heterologous expression of acsAB in Escherichia coli

Cited by 35 publications

References 64 publications

Evidence for a Proton Transfer Network and a Required Persulfide-Bond-Forming Cysteine Residue in Ni-Containing Carbon Monoxide Dehydrogenases

Evidence for a Proton Transfer Network and a Required Persulfide-Bond-Forming Cysteine Residue in Ni-Containing Carbon Monoxide Dehydrogenases

Heterologous Expression of the Clostridium carboxidivorans CO Dehydrogenase Alone or Together with the Acetyl Coenzyme A Synthase Enables both Reduction of CO 2 and Oxidation of CO by Clostridium acetobutylicum

New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

Contact Info

Product

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Heterologous Expression of the Clostridium carboxidivorans CO Dehydrogenase Alone or Together with the Acetyl Coenzyme A Synthase Enables both Reduction of CO ₂ and Oxidation of CO by Clostridium acetobutylicum