Recombinant Methylococcus capsulatus Transaldolase (tal)

Basic Research Questions

• What is the role of transaldolase in the carbon metabolism of Methylococcus capsulatus?

Transaldolase (TA) is a key enzyme in the ribulose monophosphate (RuMP) pathway, which serves as the primary pathway for formaldehyde assimilation in M. capsulatus (Bath). Specifically, transaldolase is involved in the rearrangement phase that regenerates ribulose 5-phosphate . This enzyme catalyzes the reversible transfer of a three-carbon dihydroxyacetone unit from sedoheptulose 7-phosphate to glyceraldehyde 3-phosphate, forming erythrose 4-phosphate and fructose 6-phosphate.

The RuMP pathway in M. capsulatus has four hypothetical variants as outlined by Anthony (1983), and transaldolase is specifically involved in one of the variants of the rearrangement phase . The alternative to a transaldolase-driven rearrangement is the use of sedoheptulose bisphosphatase, but Strøm et al. (1974) could not detect specific activity of this enzyme using cell-free preparations .

• How is the tal gene identified and characterized in Methylococcus capsulatus?

The transaldolase gene in M. capsulatus (Bath) was identified through genome annotation as published by Ward et al. (2004) . The gene is part of the complete genome sequence, which consists of 3.3 Mb specialized for a methanotrophic lifestyle .

To characterize the tal gene:

• Use PCR amplification with primers designed from the annotated genome sequence

• Clone the PCR product into an expression vector

• Sequence the cloned gene to confirm identity

• Express the protein in a suitable host (commonly E. coli)

• Purify the recombinant enzyme using affinity chromatography

• Verify enzyme activity using spectrophotometric assays measuring the formation of products

Genome-scale metabolic models of M. capsulatus, such as iMC535 and other models, include the transaldolase reaction as part of the reconstructed metabolic network .

• What expression systems are most effective for producing recombinant M. capsulatus transaldolase?

For successful expression of recombinant M. capsulatus transaldolase, consider the following methodological approach:

The optimal expression conditions typically include:

• Induction at OD600 of 0.6-0.8

• IPTG concentration of 0.1-0.5 mM

• Post-induction temperature of 16-30°C (enzyme-dependent)

• Expression time of 4-24 hours

For purification, a His-tag fusion approach with immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography is commonly employed to obtain high-purity enzyme preparations.

Advanced Research Questions

• How does transaldolase integrate with other carbon assimilation pathways in M. capsulatus?

M. capsulatus demonstrates remarkable metabolic flexibility, possessing multiple pathways for carbon assimilation, including the RuMP pathway, Calvin-Benson-Bassham (CBB) cycle, and a partial Serine pathway .

Transaldolase functions primarily in the RuMP pathway, but its activity interacts with other metabolic modules:

• Integration with the CBB cycle: Recent research using 13CO2 tracer analysis has shown that M. capsulatus simultaneously utilizes both CH4 and CO2 as carbon sources, with RubisCO playing an essential role in CO2 assimilation . The transaldolase-containing RuMP pathway and the CBB cycle share metabolic intermediates, creating a complex network of carbon flow.

• Connection to the oxidative pentose phosphate pathway: Isotopomer analysis has revealed that ribulose-1,5-bisphosphate in M. capsulatus is primarily regenerated via the oxidative branch rather than the non-oxidative branch of the pentose phosphate pathway, representing a non-canonical CBB cycle . This suggests a novel integration between the RuMP pathway (involving transaldolase) and the CBB cycle.

• Metabolic flexibility: Transaldolase activity contributes to the organism's ability to adapt to different growth conditions and carbon sources. Recent metabolic models have identified that M. capsulatus can potentially grow on sugars and amino acids , suggesting additional roles for transaldolase in alternative carbon utilization pathways.

• What experimental approaches can be used to assess the kinetic properties of recombinant M. capsulatus transaldolase?

To comprehensively characterize the kinetic properties of recombinant M. capsulatus transaldolase, researchers should employ multiple complementary approaches:

• Steady-state kinetics:

  • Use spectrophotometric assays monitoring NADH oxidation/formation through coupling enzymes

  • Determine Km, Vmax, and kcat for both forward and reverse reactions

  • Assess substrate specificity using various sugar phosphates

• Pre-steady-state kinetics:

  • Employ stopped-flow techniques to measure rapid kinetics of enzyme-substrate interactions

  • Determine rate constants for individual steps in the reaction mechanism

• Isothermal titration calorimetry (ITC):

  • Measure binding thermodynamics (ΔH, ΔS, ΔG)

  • Determine binding constants (Ka) for substrates and inhibitors

• Temperature and pH profiling:

  • Assess activity across temperature ranges (10-70°C)

  • Determine pH optimum and stability (pH 4-10)

  • Calculate activation energy using Arrhenius plots

Sample data format for reporting enzyme kinetics:

• How can metabolic flux analysis be used to investigate the in vivo role of transaldolase in M. capsulatus?

Metabolic flux analysis (MFA) provides powerful insights into the in vivo role of transaldolase in M. capsulatus carbon metabolism. The approach involves:

• 13C-labeling experiments:

  • Culture M. capsulatus with 13C-labeled methane and/or 13C-labeled CO2

  • Extract metabolites at steady state

  • Analyze isotopomer distributions using GC-MS or LC-MS/MS

• Computational flux estimation:

  • Use genome-scale metabolic models like iMC535 or other published models

  • Apply constraint-based modeling (Flux Balance Analysis)

  • Incorporate 13C data to constrain possible flux distributions

  • Resolve flux splits at key metabolic branch points involving transaldolase

• Transaldolase knockout/knockdown studies:

  • Implement CRISPR-Cas9 gene editing or antisense RNA strategies

  • Compare metabolic flux distributions between wild-type and modified strains

  • Identify metabolic rewiring and potential bypass routes

• Integration with -omics data:

  • Correlate flux changes with transcriptomic and proteomic data

  • Identify regulatory mechanisms affecting transaldolase activity

  • Develop predictive models of metabolic regulation

Recent 13CO2 tracer analysis has demonstrated that M. capsulatus displays significant metabolic plasticity, with core intermediates derived from both CH4 and CO2 carbon sources . This suggests a novel dual C1-fixing RuMP/RuBP pathway in which transaldolase likely plays a significant role.

• What structural features distinguish M. capsulatus transaldolase from other bacterial transaldolases?

The structural analysis of M. capsulatus transaldolase reveals several distinguishing features compared to other bacterial transaldolases:

• Structural comparison methodology:

  • Express and purify recombinant M. capsulatus transaldolase with a C-terminal His-tag

  • Perform X-ray crystallography or cryo-EM to determine 3D structure

  • Conduct comparative structural analysis with known bacterial transaldolases

  • Identify unique structural elements using computational approaches

• Key structural features:

  • Active site architecture optimized for the unique metabolic context of methanotrophy

  • Potential adaptations for functioning at the higher temperatures (45°C) preferred by M. capsulatus Bath

  • Specific binding regions for interaction with other enzymes in the RuMP pathway

• Structure-function relationships:

  • Site-directed mutagenesis of conserved and non-conserved residues

  • Activity assays of mutant enzymes to correlate structure with function

  • Molecular dynamics simulations to understand conformational flexibility

• Oligomeric state and stability:

  • Determine native quaternary structure using size exclusion chromatography and analytical ultracentrifugation

  • Assess thermal and chemical stability compared to mesophilic transaldolases

  • Identify structural elements contributing to thermostability

• How does transaldolase activity affect flux through the various variants of the RuMP pathway in M. capsulatus?

The RuMP pathway in M. capsulatus has four potential variants, with transaldolase playing a key role in the rearrangement phase . Understanding how transaldolase affects flux through these variants requires a combination of experimental and computational approaches:

• Pathway variant analysis:
  The four variants of the RuMP pathway differ in:

  • The C6 cleavage step: using either KDPG aldolase (EDA) or fructose bisphosphate aldolase (FBA)

  • The rearrangement phase: using either transaldolase (TA) or sedoheptulose bisphosphatase

  This creates four possible combinations, all represented in the metabolic model of M. capsulatus :

  Variant	C6 Cleavage	Rearrangement	Energy Efficiency
  1	EDA	TA	++
  2	EDA	SBPase	+
  3	FBA	TA	+++
  4	FBA	SBPase	++

• Experimental approaches to measure flux distribution:

  • 13C-metabolic flux analysis with isotopomer balancing

  • Metabolic control analysis to determine flux control coefficients

  • In vitro enzyme assays measuring activities of key enzymes in each variant

  • Genetic manipulation (knockdown/overexpression) of transaldolase and competing enzymes

• Integrative modeling:

  • Develop kinetic models incorporating measured enzyme parameters

  • Predict flux distributions under various growth conditions

  • Validate predictions using experimental data from 13C-labeling studies

  • Identify conditions favoring each pathway variant

Recent studies have shown that M. capsulatus exhibits metabolic plasticity, with core intermediates derived from both CH4 and CO2 carbon sources , suggesting complex interactions between the RuMP pathway and other carbon assimilation pathways.