Recombinant Escherichia coli O139:H28 Succinyl-CoA ligase [ADP-forming] subunit beta (sucC)

What is the genetic organization of the sucC gene in Escherichia coli?

The sucC gene in E. coli encodes the beta subunit of succinyl-CoA synthetase and is part of a gene cluster encoding several citric acid cycle enzymes arranged as gltA-sdhCDAB-sucABCD. In this cluster, gltA encodes citrate synthase, sdhCDAB encodes succinate dehydrogenase, and sucABCD encode components of the 2-oxoglutarate dehydrogenase complex and succinyl-CoA synthetase. The sucC and sucD genes are separated from sucA and sucB by a 273-base-pair segment containing four palindromic units. These genes are expressed from a sucABCD read-through transcript extending from the suc promoter to a potential rho-independent terminator at the distal end of sucD . The stop codon of sucC overlaps the sucD initiation codon by a single nucleotide, indicating close translational coupling between these genes .

What is the molecular structure of the sucC gene product?

The sucC gene comprises 1161 base pairs (388 codons, excluding the stop codon) and encodes a polypeptide with a molecular weight of approximately 41,390 Da corresponding to the beta subunit of succinyl-CoA synthetase . The gene product forms part of a functional α₂β₂ tetrameric enzyme with the alpha subunit (encoded by sucD). The sucD gene comprises 864 base pairs (288 codons) and encodes a polypeptide of Mr 29,644 . The complete enzyme catalyzes the reversible conversion of succinyl-CoA to succinate, coupled with the synthesis of ATP from ADP and inorganic phosphate, representing an important substrate-level phosphorylation step in the TCA cycle.

What are the key functional residues in the sucC gene product?

Key functional residues in the beta subunit (sucC product) include:

In the alpha subunit (sucD product), His-246α serves as the phosphorylation site and is critical for activity. When this histidine residue is replaced by aspartate (His-246α→Asp), no enzymatic activity is detected, confirming its essential role in the phosphoryl transfer mechanism .

What are the established methods for recombinant expression of sucC?

Recombinant expression of sucC can be achieved through several established methods:

• Subcloning a promoterless sucCD fragment downstream of the lac promoter in vectors such as M13mp10

• Precise splicing of the suc coding regions with an efficient ribosome-binding site (such as atpE) and expression from thermoinducible lambda promoters in vectors like pJLA503

• Co-expression of both subunits to ensure proper assembly of the functional α₂β₂ tetramer

Using thermoinducible lambda promoters, researchers have achieved 40-60-fold amplification of succinyl-CoA synthetase specific activities within 5 hours of thermoinduction, with the alpha and beta subunits accounting for approximately 30% of the protein in supernatant fractions of cell-free extracts . This represents a significant enhancement in expression efficiency compared to native levels.

What purification strategies yield the highest activity for recombinant sucC?

Effective purification strategies for recombinant sucC should address both individual subunit purification and tetrameric enzyme assembly. A comprehensive purification workflow typically includes:

• Expression optimization using thermoinducible promoters or other regulated systems

• Cell lysis under conditions that preserve native protein interactions

• Initial separation through ammonium sulfate fractionation or ion-exchange chromatography

• Affinity chromatography (if tagged constructs are used)

• Size-exclusion chromatography to isolate properly assembled tetramers

Critical considerations include maintaining the proper stoichiometry of alpha and beta subunits, preventing aggregation, and preserving the phosphorylation state of key residues. The purification buffer typically includes components that stabilize the quaternary structure and retain catalytic activity.

How can I verify the structural integrity of purified recombinant sucC?

Verification of structural integrity for purified recombinant sucC should include:

Additionally, thermal shift assays can provide insights into protein stability, while limited proteolysis can reveal structural domains and flexible regions.

What approaches are most effective for site-directed mutagenesis of sucC?

Site-directed mutagenesis of sucC can be performed using several effective approaches:

• Subcloning the gene into suitable vectors (such as M13mp10) for oligonucleotide-directed mutagenesis

• PCR-based methodologies such as QuikChange site-directed mutagenesis

• Complete gene synthesis incorporating desired mutations

• CRISPR-Cas9 systems for precise genomic editing when chromosomal modification is desired

Previous research has successfully employed these techniques to create specific mutations in potential CoA binding-site residues (Trp-43β, His-50β, and Cys-47β) and in the phosphorylation site of the alpha subunit (His-246α) . The effects of these mutations on enzyme activity have provided valuable insights into structure-function relationships.

How can I design a comprehensive mutational analysis strategy for sucC?

A comprehensive mutational analysis strategy for sucC should include:

• Evolutionary analysis to identify conserved residues across species

• Structural mapping of residues involved in:

  • Substrate binding (succinate, CoA, ATP)

  • Catalysis (phosphoryl transfer)

  • Subunit-subunit interactions

  • Allosteric regulation

• Systematic replacement of key residues with:

  • Alanine (to remove side chain functionality)

  • Conservative substitutions (to test specific physicochemical properties)

  • Non-conservative substitutions (to significantly alter properties)

• Analysis of mutant proteins using:

  • Steady-state kinetics (Km, kcat, kcat/Km for each substrate)

  • Pre-steady-state kinetics (to identify rate-limiting steps)

  • Structural analysis (to assess conformational effects)

  • Stability assays (to measure effects on protein folding and assembly)

Previous research has shown the critical importance of residues such as Trp-43β and His-50β for enzyme activity, while Cys-47β appears less essential since it can be replaced by serine without inactivating the enzyme .

What assay systems provide the most reliable data for analyzing sucC variants?

The most reliable assay systems for analyzing sucC variants include:

• Spectrophotometric coupled assays:

  • Forward reaction: Monitoring ADP formation coupled to NADH oxidation via pyruvate kinase and lactate dehydrogenase

  • Reverse reaction: Monitoring ATP formation coupled to NADH formation via hexokinase and glucose-6-phosphate dehydrogenase

• Direct assays:

  • Radioactive assays using labeled substrates

  • HPLC-based quantification of substrates and products

  • Mass spectrometry-based analyses for precise quantification

• Biophysical methods for structural analysis:

  • Circular dichroism to assess secondary structure integrity

  • Fluorescence spectroscopy to monitor tertiary structure changes

  • Thermal shift assays to determine stability effects

When interpreting activity data, it's essential to consider the potential effects of mutations on protein stability and oligomerization, not just catalytic function. Therefore, a comprehensive analysis should include both activity measurements and structural characterization.

How does the catalytic mechanism of succinyl-CoA synthetase operate at the molecular level?

The catalytic mechanism of succinyl-CoA synthetase involves:

• Formation of a phosphorylated enzyme intermediate at His-246α in the alpha subunit

• Sequential binding and reaction of substrates in an ordered mechanism

• Phosphoryl transfer between the enzyme and substrates

• Conformational changes coordinating catalysis across the tetramer

What structural elements are responsible for subunit assembly in the succinyl-CoA synthetase tetramer?

The succinyl-CoA synthetase functions as an α₂β₂ tetramer with specific structural elements mediating subunit assembly:

• The interface between alpha and beta subunits involves:

  • Complementary surface charge distributions

  • Hydrophobic interactions

  • Hydrogen bonding networks

  • Potential domain-swapping arrangements

• The alpha-alpha and beta-beta interfaces contribute to dimer formation and tetramer stabilization

• The close translational coupling of the sucC and sucD genes (with a single-nucleotide overlap between the sucC stop codon and the sucD initiation codon) suggests co-translational assembly may play a role in proper complex formation

Research approaches to study these interfaces include systematic mutagenesis of interface residues, cross-linking studies, and structural analysis through X-ray crystallography or cryo-electron microscopy.

How do post-translational modifications affect sucC activity and regulation?

The most significant post-translational modification in succinyl-CoA synthetase is the phosphorylation of His-246α in the alpha subunit, which forms an essential part of the catalytic mechanism rather than serving a regulatory role . This phosphorylation creates a high-energy intermediate during the reaction cycle.

Additional potential modifications that might affect enzyme activity include:

• Redox modifications of cysteine residues (although Cys-47β can be replaced by serine without inactivating the enzyme)

• Potential phosphorylation of serine, threonine, or tyrosine residues

• Acetylation or other modifications that might respond to metabolic state

Research to identify and characterize such modifications requires techniques such as mass spectrometry, specific antibodies against modified forms, and mutation of candidate modification sites followed by functional analysis.

How can computational enzyme design be applied to engineer improved sucC variants?

Computational enzyme design for sucC improvement can utilize several sophisticated approaches:

• Transition state stabilization modeling:

  • Identify residues that can better stabilize the reaction transition state

  • Design mutations that optimize electrostatic and hydrogen-bonding interactions

• Machine learning algorithms:

  • Train models on enzyme datasets to predict beneficial mutations

  • Apply deep learning approaches that can model protein structure-function relationships

• Molecular dynamics simulations:

  • Analyze protein flexibility and dynamics

  • Identify regions for engineering improved substrate binding or product release

• Sequence-based approaches:

  • Analyze multiple sequence alignments to identify co-evolving residues

  • Design consensus sequences based on evolutionary information

• Combined approaches:

  • Integrate computational predictions with experimental validation

  • Use iterative cycles of design and testing

Recent advances in computational enzyme design have achieved remarkable successes, with engineered enzymes reaching catalytic efficiencies (kcat/KM) up to 106 M-1s-1 through proper active site design and transition state stabilization .

What methods are available for incorporating engineered sucC into metabolic engineering strategies?

Incorporating engineered sucC into metabolic engineering strategies involves:

• Integration approaches:

  • Chromosomal integration using homologous recombination

  • Plasmid-based expression with appropriate copy number control

  • CRISPR-Cas9 genome editing for precise modifications

• Expression optimization:

  • Promoter selection for appropriate expression levels

  • Ribosome binding site engineering for translation efficiency

  • Codon optimization for the production host

• Pathway engineering:

  • Balancing enzyme activities throughout connected pathways

  • Eliminating competing pathways

  • Coordinating with upstream and downstream steps

  • Building recombinant enzyme cascades that avoid purification of intermediates

• Systems-level analysis:

  • Metabolic flux analysis to identify bottlenecks

  • Omics approaches to understand system-wide effects

  • Kinetic modeling to predict optimal enzyme parameters

Recombinant DNA technology enables customization of the enzyme for specific applications, providing advantages in terms of reproducibility, defined biological properties, and the ability to generate variants with novel properties .

How can cross-disciplinary approaches enhance our understanding of sucC function in cellular metabolism?

Cross-disciplinary approaches that enhance our understanding of sucC function include:

• Systems biology:

  • Integration of transcriptomics, proteomics, and metabolomics data

  • Construction of comprehensive metabolic models

  • Flux balance analysis to predict metabolic flows

• Synthetic biology:

  • Design of minimal synthetic pathways incorporating sucC

  • Creation of orthogonal metabolic modules

  • Development of biosensors to monitor sucC activity in vivo

• Structural biology combined with computational approaches:

  • Cryo-EM analysis of sucC in different functional states

  • Molecular dynamics simulations of enzyme dynamics

  • Quantum mechanics/molecular mechanics for reaction mechanism studies

• Evolutionary biochemistry:

  • Ancestral sequence reconstruction to understand evolutionary trajectories

  • Phylogenetic analysis across diverse species

  • Experimental evolution to identify adaptive mutations

These interdisciplinary approaches provide complementary insights that can reveal new aspects of sucC function, regulation, and potential applications in biotechnology.

What are common problems in recombinant sucC expression and how can they be resolved?

Common problems in recombinant sucC expression and their solutions include:

Based on previous research, thermoinducible lambda promoters in vectors like pJLA503 have been particularly effective for sucC expression, achieving 40-60-fold amplification of enzyme activity .

How can I address inconsistencies in kinetic data between different sucC preparations?

Addressing inconsistencies in kinetic data requires systematic investigation of several factors:

• Expression system variability:

  • Standardize expression conditions

  • Use the same host strain and growth protocol

  • Validate protein sequence by mass spectrometry

• Purification considerations:

  • Develop reproducible purification protocols

  • Verify oligomeric state (tetramer formation)

  • Confirm purity by SDS-PAGE and other methods

• Assay standardization:

  • Use multiple assay methods to cross-validate results

  • Standardize buffer conditions, pH, and temperature

  • Include appropriate controls in each experiment

• Data analysis:

  • Apply rigorous statistical analysis

  • Use consistent kinetic models for data fitting

  • Report all experimental conditions in detail

• Sample handling:

  • Standardize protein storage conditions

  • Test for activity loss during storage

  • Validate freeze-thaw stability

By systematically addressing these factors, researchers can identify the sources of inconsistency and establish reliable protocols for obtaining reproducible kinetic data.

What are the best strategies for studying sucC in the context of the complete TCA cycle?

Studying sucC in the context of the complete TCA cycle requires integrative approaches:

These approaches collectively provide a comprehensive understanding of how sucC functions within the broader context of cellular metabolism.

What emerging technologies might revolutionize sucC research in the next decade?

Emerging technologies with potential to revolutionize sucC research include:

• Advanced structural biology approaches:

  • Time-resolved cryo-EM to capture catalytic intermediates

  • Micro-electron diffraction for structural analysis with minimal material

  • Integrative structural biology combining multiple data types

• Single-molecule techniques:

  • Single-molecule FRET to monitor conformational changes

  • Force spectroscopy to investigate mechanical properties

  • Single-molecule activity measurements

• Advanced computational methods:

  • AI-powered protein design platforms

  • Quantum computing for more accurate reaction simulations

  • Automated laboratory systems for high-throughput testing

• Genome engineering advances:

  • Base editing for precise genetic modifications

  • In vivo directed evolution using continuous evolution systems

  • Synthetic genomics approaches for completely redesigned pathways

• Cellular and subcellular analysis:

  • Super-resolution microscopy for enzyme localization

  • Metabolic sensors for real-time activity monitoring

  • Single-cell metabolomics for heterogeneity analysis

These technologies will provide unprecedented insights into sucC structure, function, and integration with cellular metabolism.

How might engineered sucC variants contribute to sustainable bioproduction processes?

Engineered sucC variants could contribute to sustainable bioproduction through:

• Enhanced biofuel and biochemical production:

  • Optimized succinate production for bio-based polymers

  • Engineered pathways incorporating succinyl-CoA as a key intermediate

  • Creation of novel TCA cycle variants for increased carbon efficiency

• CO2 fixation and carbon capture:

  • Integration into synthetic carbon fixation pathways

  • Enhanced CO2 incorporation into central metabolism

  • Development of carbon-negative production processes

• Bioremediation applications:

  • Degradation of recalcitrant environmental pollutants

  • Conversion of waste streams into valuable products

  • Detoxification of industrial byproducts

• Process improvements:

  • Thermostable variants for high-temperature processes

  • pH-tolerant enzymes for operation under diverse conditions

  • Solvent-resistant variants for non-aqueous biocatalysis

Recombinant DNA technology enables these applications by providing tools for enzyme customization, enhancing reproducibility, and allowing for large-scale production of optimized variants .

What interdisciplinary collaborations would most advance our understanding of sucC biology?

Interdisciplinary collaborations with the greatest potential to advance sucC biology include:

• Structural biology and computational chemistry:

  • High-resolution structures of reaction intermediates

  • Quantum mechanics/molecular mechanics studies of catalytic mechanism

  • Molecular dynamics simulations of conformational changes

• Systems biology and mathematical modeling:

  • Comprehensive metabolic models integrating experimental data

  • Multi-scale modeling from molecular to cellular levels

  • Prediction of system-wide effects of sucC modifications

• Synthetic biology and bioengineering:

  • Development of novel pathways incorporating sucC

  • Creation of minimal synthetic cells with redesigned TCA cycles

  • Engineering of protein scaffolds for pathway optimization

• Evolutionary biology and bioinformatics:

  • Phylogenetic analysis across diverse organisms

  • Ancestral sequence reconstruction and resurrection

  • Identification of evolutionary constraints and opportunities

• Chemical biology and organic chemistry:

  • Design of mechanism-based inhibitors or activators

  • Development of chemical probes for studying enzyme dynamics

  • Creation of artificial cofactors for expanded catalytic capabilities

Effective collaboration across these disciplines would integrate diverse expertise and methodologies to address complex questions about sucC biology and applications.