Recombinant Methanosarcina barkeri Pyruvate synthase subunit porC (porC)

What is pyruvate:ferredoxin oxidoreductase (POR) in Methanosarcina barkeri?

Pyruvate:ferredoxin oxidoreductase (POR) in M. barkeri is a key enzyme involved in the complex interaction between anabolic and catabolic pathways of pyruvate metabolism. This enzyme catalyzes the thiamine pyrophosphate-dependent oxidative decarboxylation of pyruvate to form acetyl-CoA and CO₂ . POR can also function in reverse under certain conditions, acting as pyruvate synthase to catalyze the reductive carboxylation of acetyl-CoA to form pyruvate, though this reverse reaction is energetically unfavorable and requires a strong reductant . In M. barkeri, POR has been demonstrated to play an essential function beyond its traditional metabolic role, as stringent repression of the por operon is lethal even when media are supplemented with pyruvate and/or Casamino Acids .

How does POR contribute to carbon assimilation in methanogens?

POR plays a crucial role in carbon assimilation in methanogens like M. barkeri. The enzyme functions at a critical intersection of central carbon metabolism, allowing for the interconversion of pyruvate and acetyl-CoA. In the reductive direction, POR can synthesize pyruvate from acetyl-CoA and CO₂, which is an important anabolic reaction for carbon assimilation. Transcriptomic analysis has revealed that M. barkeri also employs an alternative pathway for synthesizing oxaloacetate via phosphoenolpyruvate carboxylase, which works in conjunction with POR to facilitate carbon assimilation . This metabolic flexibility is important for methanogens, which must efficiently utilize limited carbon sources in their environments. The essential nature of POR in M. barkeri, even when media are supplemented with pyruvate, suggests it plays additional uncharacterized roles in methanogen metabolism beyond what is currently understood .

How do mutations in regulatory genes affect POR expression and activity in M. barkeri?

Genomic analysis of M. barkeri Pyr⁺ strains (capable of metabolizing pyruvate) has revealed two significant mutations: one in Mbar_A1588, the biotin protein ligase subunit of the pyruvate carboxylase (pyc) operon, and another in Mbar_A2165, a putative transcriptional regulator . While mutants expressing the Mbar_A1588 mutation showed no growth defect compared to wild type, they lacked pyc activity. More significantly, recreation of the Mbar_A2165 mutation resulted in a 2-fold increase in POR activity and gene expression, strongly suggesting that Mbar_A2165 functions as a transcriptional regulator of the por operon . This finding demonstrates how mutations in regulatory genes can significantly alter metabolic pathways by modifying enzyme expression levels.

The experimental approach to this discovery involved creating mutant strains, measuring enzyme activity, and conducting transcriptomic analysis. Researchers were able to demonstrate that overexpression of por was a mechanism by which the Pyr⁺ mutant could metabolize pyruvate effectively despite lacking pyc activity .

What methods can be used to measure POR enzyme activity in vitro?

Measuring POR activity, particularly in the reverse direction (pyruvate synthesis), presents technical challenges due to the energetically unfavorable nature of the reaction and its requirement for a strong reductant. Based on methodologies used with POR from Hydrogenobacter thermophilus, several approaches can be adapted for M. barkeri POR:

• For the pyruvate synthesis reaction: Couple the reaction with 2-oxoglutarate:ferredoxin oxidoreductase to generate sufficiently low-potential electrons to reduce ferredoxin, thereby driving the energy-demanding pyruvate synthesis reaction .

• For the oxidative decarboxylation direction: Measure the reduction of ferredoxin spectrophotometrically or track the formation of acetyl-CoA.

• Electron paramagnetic resonance (EPR) spectroscopy can be used to detect reaction intermediates, such as the 2-(1-hydroxyethyl)- or 2-(1-hydroxyethylidene)-thiamine pyrophosphate radical that appears in both the forward and reverse reactions .

All these methods must be conducted under strictly anaerobic conditions due to the oxygen sensitivity of the iron-sulfur clusters typically found in POR enzymes.

What is the relationship between POR and other enzymes in carbon metabolism of M. barkeri?

Transcriptomic analysis has revealed complex interactions between POR and other enzymes involved in central carbon metabolism in M. barkeri. Most notably, Pyr⁺ strains that overexpress por also overexpress the gene encoding phosphoenolpyruvate carboxylase . This finding indicates the presence of a previously uncharacterized route for synthesizing oxaloacetate in M. barkeri, which explains the unimpaired growth of these strains in the absence of pyruvate carboxylase (Pyc) activity .

This metabolic flexibility illustrates the complex network of carbon metabolism in M. barkeri, where deficiencies in one pathway can be compensated by upregulation of alternative routes. The essential nature of POR, even when the media are supplemented with potential metabolic products, suggests that this enzyme plays additional roles in cellular metabolism that remain to be fully characterized .

How can recombinant PorC be expressed and purified for biochemical studies?

Although the search results don't directly address recombinant expression of M. barkeri porC, a general experimental approach would include:

• Gene cloning: The porC gene would be amplified from M. barkeri genomic DNA and cloned into an appropriate expression vector.

• Expression system selection: Given that M. barkeri is an archaeon, expression in E. coli might require codon optimization. Alternatively, an archaeal expression system might provide better results for proper folding and post-translational modifications.

• Anaerobic expression conditions: Since POR is likely oxygen-sensitive due to iron-sulfur clusters, expression should be performed under anaerobic conditions.

• Purification strategy: A polyhistidine tag or other affinity tag could be added to facilitate purification. All purification steps would need to be conducted anaerobically, possibly using a glove box.

• Functional verification: The activity of recombinant PorC, either alone or as part of the reconstituted POR complex, would need to be verified using the enzyme activity assays described in section 3.2.

What techniques are used to study the electron transfer mechanism in POR?

Electron transfer in POR involves ferredoxin as the physiological electron mediator . To study this mechanism, researchers could employ:

• Electron paramagnetic resonance (EPR) spectroscopy: This technique is particularly valuable for studying the redox states of iron-sulfur clusters and detecting radical intermediates, such as the 2-(1-hydroxyethyl)- or 2-(1-hydroxyethylidene)-thiamine pyrophosphate radical observed in both forward and reverse POR reactions .

• Stopped-flow spectroscopy: To measure the kinetics of electron transfer between ferredoxin and POR.

• Site-directed mutagenesis: To identify residues involved in ferredoxin binding or electron transfer pathways.

• Protein-protein interaction studies: To characterize the interaction between POR and ferredoxin, potentially using techniques such as isothermal titration calorimetry or surface plasmon resonance.

Understanding the electron transfer mechanism is critical, particularly for the reverse reaction (pyruvate synthesis), which is energetically unfavorable and requires a strong reductant with reducing equivalents supplied via ferredoxin .

What approaches can be used to analyze transcriptomic data related to POR expression?

Based on the transcriptomic analysis mentioned in search result , several approaches can be used to analyze POR expression data:

• Differential expression analysis: Compare gene expression levels between wild-type and mutant strains to identify genes with altered expression, as was done to discover that Pyr⁺ strains overexpress both por and phosphoenolpyruvate carboxylase genes .

• Pathway enrichment analysis: Determine whether genes in specific metabolic pathways show coordinated changes in expression.

• Regulatory network analysis: Identify potential transcription factors or regulatory elements controlling por expression, such as the Mbar_A2165 regulator identified in M. barkeri .

• Validation with RT-qPCR: Confirm key findings from transcriptomic data using targeted expression measurement techniques.

• Integration with metabolic data: Correlate changes in gene expression with alterations in metabolite levels or enzyme activities to build a comprehensive understanding of metabolic regulation.

Table 1 shows a hypothetical comparison of gene expression levels between wild-type and Pyr⁺ mutant strains of M. barkeri:

How can structural information about PorC inform functional studies?

Structural studies of PorC, though not directly addressed in the search results, would provide valuable insights for functional investigations:

Such structural information would be particularly valuable given the essential nature of POR in M. barkeri and its potential uncharacterized functions beyond the known metabolic roles .

What are the implications of POR's essential nature for metabolic engineering of M. barkeri?

The finding that stringent repression of the por operon is lethal in M. barkeri, even when the media are supplemented with pyruvate and/or Casamino Acids, suggests that POR has an unidentified essential function beyond its known metabolic roles . This has significant implications for metabolic engineering:

• Design constraints: Any metabolic engineering strategy must ensure that POR activity is maintained at sufficient levels to support this essential function.

• Regulatory considerations: Modifications to carbon metabolism pathways should account for the regulatory mechanisms controlling por expression, such as the transcriptional regulator Mbar_A2165 .

• Alternative pathways: The discovery of an alternate route for oxaloacetate synthesis via phosphoenolpyruvate carboxylase in strains lacking pyruvate carboxylase activity demonstrates the metabolic flexibility of M. barkeri . Such alternative pathways could be leveraged in metabolic engineering approaches.

• Research opportunities: Identifying the essential function of POR could reveal new targets for metabolic engineering or provide insights into unique aspects of archaeal metabolism.

Understanding the complex interactions between anabolic and catabolic pathways involving pyruvate metabolism, as revealed in M. barkeri Fusaro, is crucial for successful metabolic engineering of this organism for biotechnological applications .

What outstanding questions remain about PorC function in M. barkeri?

Despite the insights provided by the available research, several important questions about PorC remain unanswered:

• What is the specific role of the PorC subunit within the POR complex?

• What is the unidentified essential function of POR that makes it indispensable even when the media are supplemented with pyruvate?

• How do the properties of M. barkeri POR compare to those of other archaea and bacteria, particularly regarding the reversibility of the reaction?

• What is the precise mechanism of regulation by Mbar_A2165, and are there other regulatory factors affecting por expression?

• How does POR interact with the newly identified phosphoenolpyruvate carboxylase pathway for oxaloacetate synthesis?