Recombinant Marinomonas sp. Acetyl-coenzyme A synthetase (acsA), partial

What is Acetyl-coenzyme A synthetase (acsA) and what reaction does it catalyze?

Acetyl-coenzyme A synthetase (EC 6.2.1.1), also known as acetate--CoA ligase or acyl-activating enzyme, catalyzes the conversion of acetate to acetyl-CoA through an acetyladenylate intermediate . Unlike the alternative pathway involving acetate kinase (Ack) and phosphotransacetylase (Pta), acsA functions as a high-affinity acetate uptake system capable of scavenging extracellular acetate at relatively low concentrations . This enzyme plays a critical role in carbon metabolism by activating acetate for entry into central metabolic pathways.

How does acsA differ from other acyl-CoA synthetases?

While acsA specifically activates acetate, other acyl-CoA synthetases in the same enzyme family have different substrate specificities. For example, acetoacetyl-CoA synthetase (AacS) activates acetoacetate to form acetoacetyl-CoA . These enzymes share the AMP-forming mechanism but differ in substrate preference and can be regulated differently by post-translational modification systems. For instance, in Streptomyces lividans, the protein acetyltransferase SlPatA acetylates SlAacS more efficiently than it does acetyl-CoA synthetase .

What are the recommended storage and reconstitution protocols for recombinant acsA?

For optimal stability and activity of recombinant Marinomonas sp. acsA:

• Store the lyophilized protein at -20°C, or -80°C for extended storage periods

• Briefly centrifuge the vial before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended) and aliquot for long-term storage

• Avoid repeated freeze-thaw cycles; working aliquots can be stored at 4°C for up to one week

• The shelf life is approximately 6 months at -20°C/-80°C for liquid form and 12 months for lyophilized form

What expression systems are suitable for recombinant acsA production?

The commercially available recombinant Marinomonas sp. acsA is produced in yeast expression systems . This suggests that yeast provides an appropriate cellular environment for proper folding and activity of this protein. When designing expression constructs, researchers should consider:

• The presence of appropriate tag sequences for purification (tag type is variable and determined during manufacturing)

• Codon optimization for the chosen expression host

• Expression conditions that minimize protein aggregation and maximize soluble yield

What methods can be used to assess the purity and activity of recombinant acsA?

Purity of recombinant acsA can be assessed using SDS-PAGE, with commercial preparations typically achieving >85% purity . Activity assays for acetyl-CoA synthetase typically measure:

• Formation of acetyl-CoA using coupled enzymatic assays

• Release of pyrophosphate or AMP during the reaction

• Consumption of ATP during the activation step

Researchers should validate enzyme activity under their specific experimental conditions, as factors such as pH, temperature, and buffer composition can significantly affect enzyme performance.

How can researchers optimize recombinant acsA expression and purification?

While specific optimization protocols for Marinomonas sp. acsA are not provided in the search results, general strategies include:

• Testing different expression hosts (yeast systems have proven effective)

• Optimizing induction conditions (temperature, inducer concentration, duration)

• Including appropriate cofactors or stabilizing agents in purification buffers

• Using affinity tags to simplify purification (tag selection should be optimized for each application)

• Implementing quality control measures such as activity assays and stability testing

How is acsA gene expression regulated in bacteria?

In bacteria, acsA expression is controlled by sophisticated regulatory networks. In Vibrio vulnificus, a LuxR-type transcriptional regulator named AcsR positively regulates acsA expression . AcsR directly binds to the upstream region of the acsA open reading frame as demonstrated by in vitro gel-shift assays . The regulatory system also includes a putative histidine kinase gene, acsS, located five ORFs downstream of the acsR gene .

This regulatory circuit is part of a larger network involving the VarS/VarA two-component signal transduction system, which regulates AcsR . Similar regulatory systems exist in other bacteria, such as Shewanella oneidensis, where SO_2742 (sensor kinase) and SO_2648 (response regulator) control acetate metabolism by regulating acetyl-CoA synthetase expression .

What post-translational modifications regulate acsA activity?

Acetyl-CoA synthetase activity is regulated by reversible protein acetylation mechanisms in several bacterial species. Protein acetyltransferases (Pat) can acetylate the epsilon amino group of an active-site lysyl side chain in acetyl-CoA synthetase, leading to enzyme inactivation . This has been demonstrated in Salmonella enterica and Rhodopseudomonas palustris, where Pat enzymes acetylate and inactivate their respective Acs proteins .

Deacetylation of the modified lysine residue by deacetylases can restore enzyme activity . This acetylation/deacetylation cycle provides a rapid post-translational mechanism to control enzyme activity in response to changing metabolic conditions, allowing bacteria to quickly adapt to environmental changes.

How do mutations in acsA affect bacterial phenotypes?

Loss of function mutations in acsA produce specific phenotypic changes:

• Reduced growth when acetate is the sole carbon source, as seen in Vibrio vulnificus ΔacsA mutants

• Increased resistance to organic acids that are otherwise toxic, including acrylate, 3-hydroxypropionate, and propionate

This altered sensitivity to organic acids forms the basis for using acsA as a counter-selection marker in cyanobacteria like Synechococcus sp. PCC 7002 . The mechanism of resistance likely involves prevention of toxic metabolite formation, as these organic acids typically require activation by CoA ligases to exert their inhibitory effects.

How can acsA be used as a counter-selection marker in cyanobacteria?

The acsA gene provides an elegant counter-selection system for cyanobacteria based on organic acid toxicity. In Synechococcus sp. PCC 7002, loss of AcsA function confers resistance to acrylate, which is otherwise toxic at low concentrations . This counter-selection method involves:

• Designing constructs with homologous regions flanking the acsA gene

• Transforming the constructs into wild-type cells

• Selecting transformants on media containing acrylate

• Validating insertions or deletions by PCR and phenotypic testing

This approach enables markerless gene modifications and has been successfully applied to both the acsA locus and other sites like the glpK pseudogene . The system is potentially applicable to other cyanobacterial species where AcsA activity confers acrylate sensitivity, such as Synechocystis sp. PCC 6803 .

What experimental approaches can be used to study acsA regulation?

Based on published research methods, several approaches have proven effective for studying acsA regulation:

• Transcriptional reporter fusions (e.g., acsA::luxAB) to monitor gene expression levels under various conditions

• Gel-shift assays using recombinant regulatory proteins (e.g., AcsR) and DNA fragments containing the acsA promoter region to study direct binding interactions

• Comparative transcriptome analyses to identify genes co-regulated with acsA

• Mutational analysis of regulatory genes (e.g., acsR, acsS) to assess their impact on acsA expression

• In vitro acetylation/deacetylation assays to study post-translational regulation of enzyme activity

How can researchers generate and validate acsA mutants?

To generate and validate acsA mutants:

• Design constructs with homologous regions (approximately 600 bp) flanking the acsA gene or target region

• Include appropriate selection markers (e.g., streptomycin resistance marker aadA)

• Transform the linear DNA fragment into target cells

• Select transformants on appropriate media (antibiotics for insertion, acrylate for deletion)

• Verify mutations by:

  • Colony PCR to confirm integration and complete segregation of all chromosomes

  • Growth tests on acetate minimal medium (mutants should show growth defects)

  • Resistance tests with organic acids like acrylate (mutants should show increased resistance)

  • Complementation studies to confirm phenotype specificity

What factors affect acsA enzyme stability and activity?

Several factors can influence the stability and activity of recombinant acsA:

• Storage conditions: The enzyme should be stored at -20°C or -80°C with 5-50% glycerol as a cryoprotectant

• Buffer composition: Optimal pH, ionic strength, and presence of cofactors (ATP, CoA)

• Presence of inhibitors: Organic acids like acrylate may inhibit enzyme activity

• Post-translational modifications: Acetylation of key lysine residues can inactivate the enzyme

• Protein concentration: Appropriate working concentrations range from 0.1-1.0 mg/mL

What are the common challenges in acsA functional studies?

When working with acsA in functional studies, researchers should be aware of:

• Potential cross-reactivity with other CoA ligases with similar substrate specificity

• The need for complete segregation of all chromosomes when creating mutants in polyploid organisms like cyanobacteria

• Growth medium considerations, as acsA mutants may show differential growth depending on carbon sources

• Regulatory complexities involving two-component systems and transcriptional regulators

• Effects of post-translational modifications on enzyme activity

How can researchers design experiments to study acsA in different bacterial species?

When investigating acsA in diverse bacterial species, consider:

• Comparative genomic approaches to identify orthologs (e.g., acsA homologs in Vibrio vulnificus, Synechococcus, Shewanella oneidensis)

• Designing species-specific primers for gene amplification and expression analysis

• Testing sensitivity to organic acids like acrylate as a functional assay for AcsA activity

• Investigating regulatory elements by analyzing promoter regions and potential regulatory protein binding sites

• Employing heterologous expression to study enzyme properties in well-characterized systems

Table 1: Properties of Recombinant Marinomonas sp. Acetyl-coenzyme A synthetase (acsA)

Table 2: Regulatory Elements Controlling acsA Expression in Bacterial Species

Table 3: Phenotypic Effects of acsA Mutations in Various Bacterial Species