Recombinant Ashbya gossypii Lipoyl synthase, mitochondrial (AGR231C)

What is the function of Ashbya gossypii Lipoyl synthase in mitochondrial metabolism?

Ashbya gossypii Lipoyl synthase (LIAS) is a mitochondrial enzyme that catalyzes the insertion of sulfur atoms into octanoyl substrates to form lipoyl moieties. This enzyme is critical for the lipoylation pathway, which is essential for the function of key metabolic enzymes including pyruvate dehydrogenase complex (PDC), α-ketoglutarate dehydrogenase complex (OGDC), and the glycine cleavage system (GCS) .

The reaction involves the insertion of two sulfur atoms at the C6 and C8 positions of an octanoyl chain that is bound to a specific lysine residue of the lipoyl domain (LD) of these enzyme complexes. This modification is crucial for the proper functioning of these metabolic enzymes in the mitochondria .

How does the structure of A. gossypii Lipoyl synthase relate to its function?

A. gossypii Lipoyl synthase contains two distinct iron-sulfur cluster binding motifs that are critical for its catalytic function:

• A C-X₂-C iron-sulfur cluster binding motif that coordinates a reducing [4Fe-4S] cluster, which acts as an electron source to generate the radical form of S-adenosylmethionine (SAM)

• A C-X₄-C-X₅-C motif that is exclusive to lipoyl synthases and coordinates an auxiliary [4Fe-4S] cluster, which serves as the source of sulfur atoms inserted into the octanoyl substrate

These structural features enable LIAS to perform the complex radical-based chemistry required for sulfur insertion. The enzyme uses two equivalents of SAM to generate radicals at the sixth and eighth carbons of the octanoyl chain, facilitating sulfur insertion from the auxiliary [4Fe-4S] cluster .

What expression systems are optimal for producing recombinant A. gossypii Lipoyl synthase?

Based on current research methodologies for similar proteins, E. coli expression systems have proven effective for recombinant production of iron-sulfur proteins like LIAS . For A. gossypii LIAS specifically, consider the following optimization strategies:

• Expression vector selection: Use vectors with strong, inducible promoters (e.g., T7) that can be tightly regulated to prevent premature expression that might be toxic

• Host strain selection: Choose E. coli strains optimized for iron-sulfur protein expression (e.g., BL21(DE3) derivatives supplemented with plasmids encoding iron-sulfur cluster assembly proteins)

• Culture conditions:

  • Low-temperature induction (16-18°C)

  • Supplementation with iron (ferrous ammonium sulfate, 100-200 μM)

  • Addition of L-cysteine (200 μM) to support Fe-S cluster assembly

  • Anaerobic or microaerobic conditions during expression and purification

• Purification strategy:

  • Include affinity tags that don't interfere with Fe-S cluster assembly (His₆ tag is commonly used)

  • Maintain anaerobic conditions during all purification steps

  • Include reducing agents (DTT or β-mercaptoethanol) in all buffers

For heterologous expression in A. gossypii itself, the newly developed promoter systems described in search result provide valuable tools, with the dual luciferase reporter system enabling quantitative evaluation of promoter strength.

How can the activity of recombinant A. gossypii Lipoyl synthase be accurately measured?

An effective assay system for A. gossypii LIAS activity should include:

• Substrate preparation: Use a truncated version of the lipoyl carrier protein (H-protein) where the octanoyl side chain is attached to the lysine through an amide bond, similar to methods described for human LIAS

• Reaction components:

  • Reconstituted LIAS enzyme with both [4Fe-4S] clusters

  • S-adenosylmethionine (SAM)

  • Octanoylated substrate protein

  • Reducing system (typically sodium dithionite or a physiological electron donor)

  • Buffer system (typically HEPES or Tris at pH 7.5-8.0)

• Detection method:

  • LC-MS analysis for detection of lipoylated product (m/z shift from octanoylated substrate)

  • The doubly sulfurated product shows a characteristic m/z signature (e.g., m/z = 1010 for the human system)

• Controls:

  • Reactions without enzyme

  • Reactions without SAM

  • Reactions with partially reconstituted enzyme (only auxiliary or only reducing cluster)

Sample data from a typical activity assay might resemble:

This approach provides quantitative data on LIAS activity and can be used to evaluate the effects of mutations or different reconstitution methods .

How does the A. gossypii lipoylation pathway compare to that of other organisms?

The A. gossypii lipoylation pathway shares core components with other eukaryotes but may have organism-specific features. Based on comparative studies of lipoylation pathways:

• Core pathway components:

  • LIPT2: Transfers octanoyl groups from ACP to lipoyl domains (primarily GCSH)

  • LIAS: Inserts sulfur atoms into octanoyl moieties

  • LIPT1: Transfers lipoyl groups between proteins

• Pathway organization in different organisms:

• Unique features of A. gossypii system:

  • May have adaptations related to high riboflavin production

  • Could have differences in regulation due to filamentous fungal metabolism

The efficient functioning of this pathway is essential for A. gossypii metabolism and potentially impacts its biotechnological applications in riboflavin production .

How do iron-sulfur cluster assembly pathways impact A. gossypii Lipoyl synthase function?

Iron-sulfur cluster assembly and delivery pathways are critical for LIAS function, as the enzyme requires two distinct [4Fe-4S] clusters. Research on human LIAS provides insights applicable to A. gossypii:

• Cluster assembly proteins:

  • ISCU functions as a primary scaffold for [2Fe-2S] cluster assembly

  • ISCA proteins (ISCA1, ISCA2) are involved in [4Fe-4S] cluster formation

• Cluster transfer preferences:

  • ISCU has been shown to be the most effective donor for LIAS reconstitution

  • Full-length ISCA2 is also effective (95 ± 10% relative to ISCU-promoted activity)

• Impact on LIAS activity:

  • Native LIAS as isolated is typically inactive due to incomplete cluster assembly

  • Full reconstitution requires delivery of clusters to both the auxiliary and reducing sites

• Experimental approach for A. gossypii:

  • Identify A. gossypii homologs of key Fe-S cluster assembly proteins

  • Test their ability to reconstitute A. gossypii LIAS in vitro

  • Use spectroscopic methods (UV-Vis, EPR) to monitor cluster assembly

Understanding these interactions is crucial for optimizing recombinant LIAS production and activity .

What are common challenges in purifying active A. gossypii Lipoyl synthase and how can they be addressed?

Several challenges can arise when working with A. gossypii LIAS:

• Iron-sulfur cluster instability:

  • Problem: Fe-S clusters are oxygen-sensitive and can degrade during purification

  • Solution: Perform all steps under anaerobic conditions; use glove box or Schlenk techniques; include reducing agents in all buffers; work quickly and keep samples cold

• Low yield of active enzyme:

  • Problem: Expression of Fe-S proteins often results in low yields of properly folded protein

  • Solution: Optimize expression conditions (temperature, induction time); co-express with Fe-S cluster assembly proteins; test multiple purification strategies

• Insufficient cluster reconstitution:

  • Problem: Recombinant LIAS often lacks complete Fe-S clusters

  • Solution: Use effective reconstitution methods with appropriate Fe-S cluster donors; monitor reconstitution spectroscopically

• Activity assessment challenges:

  • Problem: Complex assay systems with multiple components can be difficult to optimize

  • Solution: Develop robust controls; ensure adequate substrate preparation; optimize detection methods

• Storage stability:

  • Problem: LIAS activity can degrade during storage

  • Solution: Store under anaerobic conditions at -80°C; test addition of glycerol (typically 50%) as a cryoprotectant ; avoid repeated freeze-thaw cycles

Addressing these challenges requires careful experimental design and appropriate controls to ensure reproducible results with active enzyme.

How can site-directed mutagenesis be used to study A. gossypii Lipoyl synthase mechanism?

Site-directed mutagenesis is a powerful approach for investigating LIAS mechanism:

• Key targets for mutagenesis:

  • Cluster-binding cysteines: Based on human LIAS studies, mutations like C106A (auxiliary cluster) and C137A/C141A (reducing cluster) can selectively eliminate binding at each site

  • Conserved residues in substrate binding pocket: Identify potential substrate-interacting residues through structural modeling

  • Residues involved in SAM binding: Target conserved motifs that interact with SAM

• Expected effects:

  • Mutations in auxiliary cluster binding site (e.g., C106A) typically result in complete loss of Fe-S clusters and activity

  • Mutations in reducing cluster site may retain the auxiliary cluster but lose activity

  • Mutations affecting substrate binding may show altered kinetics without complete loss of activity

• Characterization methods:

  • Iron quantitation to determine cluster content

  • EPR spectroscopy to assess cluster type and environment

  • Activity assays to measure functional impact

  • LC-MS to detect intermediate formation

• Experimental design example:

This approach has provided valuable insights into human LIAS mechanism and could be adapted for A. gossypii LIAS .

How does Lipoyl synthase activity impact the biotechnological applications of A. gossypii?

LIAS activity has significant implications for A. gossypii biotechnology applications:

• Riboflavin production:

  • Lipoylated proteins (PDC, OGDC) are essential for central carbon metabolism that generates precursors for riboflavin biosynthesis

  • Optimization of LIAS activity could potentially enhance riboflavin yields by improving metabolic efficiency

• Biolipid production:

  • A. gossypii has been engineered for single cell oil (SCO) production

  • Lipoylated enzymes influence acetyl-CoA and NADPH availability, which are critical for lipid biosynthesis

  • Engineering strains with optimized lipoylation could increase lipid yields beyond current levels (up to 70% of cell dry weight)

• Recombinant protein production:

  • A. gossypii is emerging as a host for recombinant protein production

  • Energy metabolism dependent on lipoylated enzymes supports protein synthesis

  • Ensuring optimal LIAS function could enhance the metabolic capacity for protein production

• Other metabolites:

  • A. gossypii has been engineered to produce nucleosides like inosine

  • Central metabolism dependent on lipoylated enzymes provides precursors for these pathways

Understanding and optimizing LIAS function could therefore be a valuable target for enhancing various biotechnological applications of A. gossypii.

How might altering A. gossypii Lipoyl synthase expression affect cellular metabolism?

Modifying LIAS expression can have complex effects on cellular metabolism:

• Effects of increased LIAS expression:

  • May increase lipoylation of target enzymes if other pathway components (LIPT1, LIPT2) and iron-sulfur cluster assembly machinery are not limiting

  • Could enhance activity of PDC, OGDC, BCKDC, and GCS, potentially increasing flux through these pathways

  • Might lead to higher acetyl-CoA production, benefiting pathways like lipid synthesis

  • In humans, overexpression of LIP1 has shown varied effects depending on the genetic background, suggesting complex regulatory interactions

• Effects of decreased LIAS expression:

  • Would likely reduce lipoylation of target enzymes

  • Could create bottlenecks in central carbon metabolism

  • Might lead to accumulation of pathway intermediates (pyruvate, 2-OG, BCAAs)

  • In humans, LIAS deficiency leads to severe metabolic dysfunction affecting multiple pathways

• Competitive iron-sulfur cluster utilization:

  • LIAS competes with other Fe-S proteins for limited Fe-S clusters

  • In contexts where Fe-S cluster assembly is limiting, overexpression of abundant Fe-S proteins like aconitase (ACO) can reduce LIAS activity

  • This has been demonstrated in Arabidopsis, where deletion of ACO3 suppressed phenotypes of GRXS15 mutation by redirecting Fe-S clusters to LIAS

• Potential metabolic engineering strategy:

These engineering approaches would need to be carefully tested as the outcomes may be difficult to predict due to the complex regulatory networks involved .

What new promoter systems can enhance expression control for A. gossypii Lipoyl synthase research?

Recent advances in A. gossypii promoter characterization provide valuable tools for LIAS expression control:

• Newly characterized promoters:

  • A dual luciferase reporter system has been developed for A. gossypii, enabling quantitative evaluation of promoter strength

  • Ten new promoters with diverse expression characteristics have been identified

• Promoter strength comparison:

• Implementation strategy:

  • For basic research: Use constitutive promoters of appropriate strength to achieve desired LIAS expression levels

  • For metabolic engineering: Consider regulatable promoters that respond to carbon source to coordinate LIAS expression with metabolic state

  • Use genomic integrative cassettes rather than episomic vectors for stable expression

• Future directions:

  • Development of more finely tunable expression systems

  • Identification of promoters responsive to specific metabolic conditions

  • Engineering synthetic promoters with multiple regulatory elements

These advances in promoter characterization provide opportunities for more precise control of LIAS expression for both research and biotechnological applications .

How might cryo-EM and advanced structural techniques advance our understanding of A. gossypii Lipoyl synthase?

Advanced structural biology techniques offer promising avenues for deeper insights into A. gossypii LIAS:

These advanced structural studies could provide unprecedented insights into the catalytic mechanism of LIAS and guide rational engineering approaches for biotechnological applications.

How does A. gossypii Lipoyl synthase differ from bacterial and human homologs?

Comparing A. gossypii LIAS with homologs from other organisms reveals important similarities and differences:

• Sequence and structural conservation:

  • All LIAS enzymes contain conserved motifs for binding two [4Fe-4S] clusters

  • The core radical SAM domain is highly conserved across species

  • Variations in N- and C-terminal regions may reflect organism-specific interactions

• Functional comparison:

• Catalytic mechanism:

  • The fundamental radical SAM mechanism is conserved across species

  • Species-specific differences may exist in substrate recognition and protein-protein interactions

  • The requirement for two SAM molecules and the auxiliary cluster as the sulfur source is universal

• Biotechnological implications:

  • Knowledge from bacterial and human systems can inform A. gossypii research

  • Species-specific features must be considered when engineering A. gossypii LIAS

  • Comparative studies could reveal optimizations for biotechnological applications

Understanding these similarities and differences provides context for A. gossypii LIAS research and can guide experimental approaches.

What can we learn from iron-sulfur cluster transfer studies in other organisms for A. gossypii research?

Studies on iron-sulfur cluster transfer in other organisms provide valuable insights for A. gossypii research:

• Lessons from Arabidopsis studies:

  • Competitive utilization of iron-sulfur clusters occurs between different proteins

  • Deletion of abundant Fe-S proteins (like ACO3) can redirect clusters to less abundant ones (like LIAS)

  • GRXS15 is critical for [2Fe-2S] cluster transfer, affecting downstream [4Fe-4S] cluster assembly

  • LIAS activity directly impacts metabolite profiles, with deficiencies causing accumulation of α-keto acids

• Insights from human studies:

  • ISCU is the most effective [2Fe-2S] cluster donor for LIAS reconstitution

  • ISCA1/2 are involved in [4Fe-4S] cluster formation and delivery

  • Post-translational modifications may affect LIAS activity

  • Both clusters must be present for activity, with the auxiliary cluster typically assembled first

• Bacterial system insights:

  • The bacterial Fe-S cluster assembly machinery (ISC and SUF systems) shows functional parallels with eukaryotic systems

  • Bacterial studies have revealed detailed mechanistic insights into radical SAM enzymes like LIAS

• Application to A. gossypii research:

  • Identify and characterize A. gossypii homologs of key Fe-S cluster assembly proteins

  • Investigate competitive Fe-S cluster utilization in A. gossypii

  • Consider co-expression of Fe-S cluster assembly proteins when producing recombinant LIAS

  • Study the impact of LIAS activity on A. gossypii metabolite profiles

This cross-organism knowledge can accelerate A. gossypii LIAS research by providing established frameworks and experimental approaches .

How should experiments be designed to assess the impact of Lipoyl synthase on A. gossypii metabolism?

Robust experimental design is critical for understanding LIAS impacts on metabolism:

• Key experimental approaches:

  • Genetic manipulation: Gene deletion, overexpression, and site-directed mutagenesis

  • Metabolomics: Targeted and untargeted analysis of metabolite profiles

  • Protein analysis: Assessment of lipoylation status of target proteins

  • Flux analysis: Measurement of metabolic flux through key pathways

• Experimental design principles:

  • Controls: Include appropriate wild-type and negative controls

  • Replication: Minimum of three biological replicates per condition

  • Variables: Control for culture conditions, growth phase, and media composition

  • Statistical analysis: Apply appropriate statistical methods (e.g., Student's t-test for comparing two conditions, ANOVA for multiple conditions)

• Sample experimental design for metabolic impact study:

• Key metabolites to measure:

  • Pyruvate and other α-keto acids (accumulate with LIAS deficiency)

  • TCA cycle intermediates (affected by PDC and OGDC activity)

  • Glycine and serine (reflect GCS activity)

  • Acetyl-CoA and other acyl-CoAs (central to many pathways)

  • Riboflavin and precursors (industrially relevant)

• Data analysis considerations:

  • Normalize metabolite data appropriately (e.g., to cell dry weight)

  • Consider both absolute concentrations and relative changes

  • Apply multivariate analysis for untargeted metabolomics

  • Validate key findings with targeted analyses

This systematic approach will provide robust insights into LIAS's impact on A. gossypii metabolism.

What are the most effective protocols for analyzing the lipoylation status of target proteins?

Assessing protein lipoylation status is crucial for LIAS research:

• Western blot analysis:

  • Use anti-lipoic acid antibodies to detect lipoylated proteins

  • Sample preparation is critical—use protease inhibitors and maintain reducing conditions

  • Separate mitochondrial proteins by SDS-PAGE

  • Transfer to PVDF or nitrocellulose membranes

  • Probe with anti-lipoic acid antibody followed by appropriate secondary antibody

  • Compare band intensities across samples to quantify relative lipoylation levels

• Mass spectrometry approaches:

  • Bottom-up proteomics: Digest proteins, identify lipoylated peptides by MS/MS

  • Top-down proteomics: Analyze intact proteins to determine modification stoichiometry

  • Targeted MRM/PRM: Focus on specific lipoylated peptides for quantitative analysis

  • Look for mass shifts characteristic of lipoylation (+188 Da) or octanoylation (+126 Da)

• Enzyme activity assays:

  • Measure activities of lipoylation-dependent enzymes (PDC, OGDC, BCKDC, GCS)

  • Compare activities with total protein levels to infer lipoylation status

  • Use specific assays for each enzyme complex:

    • PDC: NADH production from pyruvate

    • OGDC: NADH production from α-ketoglutarate

    • GCS: CO₂ release from glycine or serine formation

• Example protocol workflow:

  a. Sample preparation:

  • Isolate mitochondria from A. gossypii cultures

  • Lyse mitochondria in buffer containing protease inhibitors

  • Determine protein concentration

  b. Western blot analysis:

  • Separate 20-50 μg protein per lane by SDS-PAGE

  • Transfer to membrane

  • Block with 5% non-fat milk or BSA

  • Incubate with anti-lipoic acid antibody (1:1000 dilution)

  • Wash and incubate with HRP-conjugated secondary antibody

  • Develop using chemiluminescence and image

  c. Quantification:

  • Normalize lipoylated protein bands to loading control

  • Compare across samples to determine relative lipoylation levels

• Expected results interpretation:

  • Wild-type A. gossypii should show strong lipoylation of E2 subunits and H-protein

  • LIAS deficiency would result in decreased lipoylation of all targets

  • Partial LIAS activity might show preferential lipoylation of certain targets

  • Changes in LIAS expression should correlate with lipoylation levels