Recombinant Methanococcus maripaludis FAD synthase (ribL)

What is FAD synthase (ribL) and what is its function in archaea?

FAD synthase (ribL) is an enzyme that catalyzes the transfer of the AMP portion of ATP to FMN (flavin mononucleotide) to produce FAD (flavin adenine dinucleotide) and pyrophosphate (PPi). In archaea, ribL functions as a monofunctional enzyme specifically responsible for the adenylation step in FAD biosynthesis. The enzyme has been designated as RibL to indicate that it follows the riboflavin kinase (RibK) step in the archaeal FAD biosynthetic pathway . Unlike bacterial FAD synthetases, which are bifunctional and catalyze both the phosphorylation of riboflavin and the adenylation of FMN, archaeal RibL is strictly monofunctional and only performs the adenylation reaction .

What are the structural and biochemical characteristics of M. maripaludis FAD synthase?

M. maripaludis FAD synthase (ribL) is a protein classified in the nucleotidyl transferase family. The full-length protein consists of 150 amino acids with the sequence beginning with MEKKIAVTAG and ending with RRWCCKELKV . It has a molecular weight consistent with its amino acid composition. The protein contains conserved cysteine residues in its C-terminus that are critical for enzymatic activity, as demonstrated by complete inactivation when these residues are alkylated . The enzyme requires reducing conditions for activity, making it notably air-sensitive compared to FAD synthetases from other domains of life .

How does archaeal RibL differ from bacterial and eukaryotic FAD synthetases?

Archaeal RibL differs from its counterparts in several significant ways:

• Functionality: While eukaryotes have monofunctional FAD synthetases and bacteria have bifunctional enzymes that catalyze both the phosphorylation of riboflavin and adenylation of FMN, archaeal RibL is uniquely monofunctional but with distinct properties .

• Reaction directionality: Unlike other FAD synthetases, RibL does not catalyze the reverse reaction to produce FMN and ATP from FAD and PPi .

• Inhibition characteristics: In contrast to other FAD synthetases, pyrophosphate (PPi) inhibits the activity of RibL .

• Metal cofactor preference: RibL requires divalent metals for activity, with Co²⁺ providing approximately 4 times greater activity than Mg²⁺ .

• Redox sensitivity: Archaeal RibL is active only under reducing conditions, demonstrating a sensitivity to oxidation not seen in other FAD synthetases .

What is the recommended protocol for storing and handling recombinant M. maripaludis FAD synthase?

Based on manufacturer recommendations, recombinant M. maripaludis FAD synthase should be stored at -20°C, or at -80°C for extended storage periods . Working aliquots can be stored at 4°C for up to one week, but repeated freezing and thawing is not recommended as it may compromise protein integrity and activity .

For reconstitution, it is recommended to briefly centrifuge the vial prior to opening to bring the contents to the bottom. The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, adding glycerol to a final concentration of 5-50% (with 50% being the manufacturer's default) and aliquoting before storing at -20°C/-80°C is recommended .

What experimental conditions are optimal for measuring M. maripaludis FAD synthase activity in vitro?

Based on research with archaeal FAD synthases, the following conditions are recommended for optimal enzymatic activity:

• Reducing environment: Since archaeal RibL is air-sensitive and only active under reducing conditions, assays should include reducing agents such as dithiothreitol (DTT) or β-mercaptoethanol .

• Metal cofactors: Assays should include divalent metal ions, with Co²⁺ being the preferred cofactor as it provides approximately 4 times greater activity compared to Mg²⁺ . A concentration series should be used to determine optimal metal concentrations for the specific preparation.

• Buffer conditions: A buffer system that maintains stable pH and is compatible with the reducing agents and metal cofactors should be used. Phosphate buffers may be problematic due to potential precipitation with some divalent metals.

• Substrate concentrations: Optimal concentrations of FMN and ATP should be determined through enzyme kinetics studies. It's important to note that PPi acts as an inhibitor of RibL activity, so reaction conditions that minimize PPi accumulation may improve enzyme performance .

• Temperature and pH: As M. maripaludis is a mesophilic archaeon, activity assays typically perform well at moderate temperatures (30-37°C), but specific optimal conditions should be determined empirically.

How can I design a gene silencing experiment to study FAD synthase function in archaeal systems?

Designing gene silencing experiments for archaeal systems, particularly for FAD synthase, can draw inspiration from successful approaches used in related organisms. Based on research with Entamoeba histolytica, which utilizes an archaeal-type FADS acquired through lateral gene transfer, the following methodological approach can be adapted :

• Target identification: Identify the specific ribL gene sequence in your archaeal system using genomic or transcriptomic data. Ensure the target region is unique to avoid off-target effects.

• Silencing construct design: For archaea, consider using a markerless mutagenesis approach as demonstrated with M. maripaludis . This technique uses negative selection strategies, such as the sensitivity conferred by the hpt gene (encoding hypoxanthine phosphoribosyltransferase) to base analogs like 8-azahypoxanthine .

• Transformation method: Use established transformation protocols for your specific archaeal species. For M. maripaludis, transformation with plasmid DNA followed by selection on appropriate media has been demonstrated .

• Verification of silencing: Confirm successful gene silencing through:

  • RT-qPCR to measure transcript levels

  • Western blotting to assess protein levels

  • Enzymatic assays to measure FAD synthase activity

  • Measurement of cellular FAD levels using HPLC or fluorescence-based methods

• Phenotypic analysis: Evaluate the impact of silencing on:

  • Growth rate and viability

  • Activity of FAD-dependent enzymes

  • Metabolic profiles

  • Stress responses, particularly to oxidative stress

What are the kinetic parameters of archaeal RibL compared to FAD synthetases from other domains of life?

Comparison of kinetic parameters between archaeal RibL and FAD synthetases from other domains reveals significant differences that reflect their evolutionary divergence and functional adaptations. Based on available data, the following table summarizes key kinetic parameters:

These kinetic differences highlight the unique evolutionary adaptation of archaeal RibL and suggest potential selective pressures that shaped its biochemical properties .

How does the sensitivity to oxidation affect experimental design when working with archaeal RibL?

The air sensitivity of archaeal RibL presents significant challenges for experimental design that must be carefully addressed to obtain reliable results:

• Purification strategies: When isolating recombinant archaeal RibL, all steps should be performed under anaerobic conditions or with the inclusion of strong reducing agents. Consider using anaerobic chambers or glove boxes for protein handling .

• Buffer formulation: All buffers should contain appropriate reducing agents (such as DTT, β-mercaptoethanol, or TCEP) at sufficient concentrations to maintain the enzyme in its reduced, active state. The reducing agents should be freshly prepared to ensure efficacy .

• Assay design: Enzymatic assays should be conducted in sealed reaction vessels with minimal headspace to reduce oxygen exposure. Consider using oxygen scavenging systems in assay buffers.

• Storage considerations: For long-term storage, protein samples should be flash-frozen in the presence of reducing agents and stored at -80°C. Aliquoting prevents repeated freeze-thaw cycles that could expose the protein to oxygen .

• Activity verification: Include positive controls with known activity to verify that assay conditions are sufficiently reducing. Consider measuring activity immediately after purification as a baseline and then at later time points to assess stability.

• Spectroscopic monitoring: The redox state of critical cysteine residues can potentially be monitored using thiol-reactive probes or spectroscopic techniques to correlate with enzymatic activity.

• Protective mutations: For research purposes, consider exploring whether strategic mutations could enhance oxidative stability while maintaining catalytic function.

What is the evolutionary significance of the archaeal FAD synthase and its potential acquisition through lateral gene transfer?

The archaeal FAD synthase represents a fascinating case study in molecular evolution and the role of lateral gene transfer in metabolic adaptation. Research suggests several important evolutionary aspects:

Understanding these evolutionary relationships not only illuminates the history of essential metabolic pathways but may also provide insights into the adaptability and metabolic versatility of microbial life across diverse environments .

What are recommended approaches for expressing and purifying recombinant M. maripaludis FAD synthase?

For successful expression and purification of recombinant M. maripaludis FAD synthase, consider the following methodological approaches:

• Expression system selection: While the commercial recombinant protein is produced in yeast , heterologous expression in E. coli has been successfully demonstrated for archaeal FAD synthetases . Consider using specialized E. coli strains designed for expression of proteins with rare codons or those requiring a reducing environment.

• Expression construct design:

  • Include appropriate affinity tags (His-tag, GST, etc.) for purification

  • Consider the placement of tags (N- or C-terminal) based on the critical C-terminal cysteines

  • Include TEV or similar protease cleavage sites if tag removal is desired

  • Optimize codon usage for the expression host

• Induction conditions:

  • Test various induction temperatures (16-30°C), with lower temperatures often favoring proper folding

  • Optimize inducer concentration and induction duration

  • Consider auto-induction media for archaeal proteins

• Purification strategy:

  • Perform all steps under reducing conditions with freshly prepared reducing agents

  • Include protease inhibitors during initial lysis

  • Consider purification under anaerobic conditions if possible

  • Employ a multi-step purification approach (e.g., affinity chromatography followed by size exclusion)

• Quality control:

  • Verify purity using SDS-PAGE (>85% purity is recommended)

  • Confirm identity using mass spectrometry

  • Assess enzyme activity immediately after purification

  • Monitor protein stability over time under various storage conditions

How can I troubleshoot low activity issues when working with recombinant archaeal RibL?

When encountering low activity with recombinant archaeal RibL, consider the following troubleshooting approaches:

• Oxidation issues:

  • Ensure all buffers contain fresh reducing agents (DTT, β-mercaptoethanol, or TCEP)

  • Consider increasing the concentration of reducing agents

  • Purge buffers with inert gas to remove dissolved oxygen

  • Minimize exposure to air during handling

• Metal cofactor optimization:

  • Test various divalent metal ions, with particular emphasis on Co²⁺, which provides significantly higher activity than Mg²⁺

  • Optimize metal concentration through a concentration gradient

  • Ensure the absence of chelating agents in buffers

• Substrate quality:

  • Use fresh, high-quality FMN and ATP

  • Verify substrate integrity using spectroscopic methods or HPLC

  • Test different substrate concentrations to identify optimal conditions

• Enzyme integrity:

  • Verify protein folding using circular dichroism or fluorescence spectroscopy

  • Check for potential proteolytic degradation using Western blotting or mass spectrometry

  • Consider the impact of fusion tags on enzyme activity

• Assay conditions:

  • Optimize temperature and pH for enzyme activity

  • Ensure that PPi, which inhibits RibL activity, is kept at minimal levels

  • Consider adding PPi-scavenging systems to the reaction

• Alternate activity assays:

  • If the standard assay yields low activity, consider alternative methods for measuring FAD production

  • Test the enzyme's ability to use CTP for FCD production as an alternative activity measure

What analytical methods are most appropriate for studying the products of archaeal RibL reactions?

Several analytical methods are suitable for studying the products of archaeal RibL reactions, each with specific advantages:

• HPLC analysis:

  • Reverse-phase HPLC with UV-Vis detection at 260 nm (nucleotide moiety) and 450 nm (flavin moiety)

  • Ion-pair HPLC for improved separation of nucleotides and flavin compounds

  • HPLC coupled with fluorescence detection (ex. 450 nm, em. 520 nm) for enhanced sensitivity for flavin compounds

• Spectrophotometric assays:

  • Continuous monitoring of the reaction by tracking the spectral differences between FMN and FAD

  • Coupled enzyme assays that utilize the produced FAD for a secondary reaction with spectrophotometric output

• Mass spectrometry:

  • LC-MS/MS for precise identification and quantification of reaction products

  • High-resolution MS for distinguishing between closely related flavin compounds

  • Analysis of reaction intermediates or alternate products like FCD

• Radioactive assays:

  • Using radiolabeled ATP (³²P or ³H) to track the formation of FAD

  • Thin-layer chromatography coupled with autoradiography or phosphorimaging

• Enzymatic coupled assays:

  • Using FAD-dependent enzymes to indirectly measure FAD production

  • Monitoring NADH oxidation in coupled enzyme systems

• Fluorescence-based approaches:

  • Taking advantage of the different fluorescence properties of FMN and FAD

  • Developing FRET-based assays for real-time monitoring

Each method has specific sample preparation requirements and sensitivity limits, so the choice should be based on the specific research question and available equipment.

How can archaeal RibL be used as a tool for studying flavin metabolism in different systems?

Archaeal RibL offers several unique applications as a research tool for studying flavin metabolism:

• Selective FAD synthesis: Since archaeal RibL catalyzes the forward reaction (FMN to FAD) but not the reverse, it can be used for the selective production of FAD in complex reaction mixtures .

• Novel flavin compound generation: The ability of archaeal RibL to use CTP to produce flavin cytidine dinucleotide (FCD) opens possibilities for generating novel flavin compounds with potential biotechnological applications .

• Redox-responsive systems: The sensitivity of archaeal RibL to oxidation makes it a potential component in redox-responsive biosensors or synthetic biology circuits that respond to cellular redox status.

• Metabolic engineering: Archaeal RibL could be incorporated into engineered metabolic pathways in heterologous hosts to create redox-dependent regulation of flavin cofactor availability.

• Comparative enzymology: The distinct properties of archaeal RibL provide valuable contrast for comparative studies with bacterial and eukaryotic FAD synthetases, potentially revealing fundamental principles of enzyme evolution.

• Structural biology: The unique characteristics of archaeal RibL make it an interesting target for structural studies to understand the molecular basis of its distinctive properties.

• Teaching tool: The clear differences between archaeal, bacterial, and eukaryotic FAD synthetases make this enzyme family valuable for teaching concepts in comparative biochemistry and molecular evolution.

What are the implications of archaeal RibL's unique properties for drug development against pathogens with archaeal-type FAD synthases?

The unique properties of archaeal-type FAD synthases present promising opportunities for selective drug development against pathogens that utilize these enzymes:

• Selective targeting: The significant differences between archaeal-type FAD synthases and human FADS allow for the development of inhibitors that selectively target the pathogen enzyme without affecting the host .

• Essential metabolic function: As demonstrated in E. histolytica, archaeal-type FADS is essential for parasite growth and survival, making it a promising drug target . Inhibition of this enzyme would disrupt FAD-dependent processes critical for pathogen viability.

• Unique catalytic mechanisms: The distinctive properties of archaeal RibL, including its metal preferences, redox sensitivity, and inhibition characteristics, provide multiple biochemical vulnerabilities that could be exploited for inhibitor design .

• Structure-based drug design: Structural information about archaeal-type FAD synthases can guide rational design of selective inhibitors targeting unique features of these enzymes.

• Resistance considerations: The distinctive evolutionary origin of archaeal-type FAD synthases suggests that resistance mechanisms developed against inhibitors of conventional FAD synthetases might not protect against drugs targeting the archaeal-type enzyme.

• Broader applications: Insights gained from targeting archaeal-type FADS in one pathogen may be applicable to other pathogens that have acquired similar enzymes through lateral gene transfer .

• Potentiation of existing therapies: In E. histolytica, silencing of archaeal-type FADS made the parasite more susceptible to metronidazole, suggesting potential synergistic drug combinations .

What advances in protein engineering might improve the stability and utility of archaeal RibL for biotechnological applications?

Several protein engineering approaches could enhance the stability and utility of archaeal RibL for biotechnological applications:

• Oxidative stability engineering:

  • Strategic mutation of non-catalytic cysteines to serine or alanine

  • Introduction of stabilizing disulfide bonds in non-critical regions

  • Computational design to identify mutations that improve stability while maintaining activity

  • Directed evolution under oxidizing conditions to select for more stable variants

• Metal binding optimization:

  • Engineering the metal binding site to improve affinity for specific metals

  • Modifying the enzyme to function efficiently with more readily available metals (e.g., Mg²⁺ instead of Co²⁺)

  • Creating variants with altered metal specificity for specialized applications

• Substrate specificity modification:

  • Engineering the active site to accept modified flavin substrates

  • Enhancing the enzyme's ability to use alternative nucleotide donors beyond ATP and CTP

  • Creating variants with altered product specificity

• Immobilization strategies:

  • Introducing specific attachment sites for controlled immobilization

  • Designing fusion proteins with self-assembling domains for defined spatial organization

  • Creating enzyme arrays with optimized activity retention upon immobilization

• Thermostability enhancement:

  • Rigidifying the enzyme structure through introduction of proline residues

  • Increasing surface salt bridges and hydrogen bonding networks

  • Applying consensus design approaches based on multiple archaeal FAD synthases

• Fusion protein development:

  • Creating bifunctional enzymes by fusing RibL with riboflavin kinase

  • Developing chimeric proteins combining beneficial properties from different FAD synthases

  • Introducing protein domains that enhance solubility or stability

These engineering approaches could significantly expand the utility of archaeal RibL in various biotechnological applications, from biocatalysis to biosensor development and synthetic biology.

What are the most significant open questions regarding archaeal FAD synthases?

Despite progress in understanding archaeal FAD synthases, several significant questions remain unresolved:

• Structural basis of unique properties: What structural features account for the distinct properties of archaeal RibL, including its air sensitivity, metal preference, and inability to catalyze the reverse reaction?

• Evolutionary origin: What is the precise evolutionary history of archaeal RibL, and what selective pressures drove its divergence from bacterial and eukaryotic FAD synthetases?

• Regulatory mechanisms: How is the expression and activity of archaeal RibL regulated in response to changing metabolic needs and environmental conditions?

• Protein-protein interactions: Does archaeal RibL form complexes with other proteins involved in flavin metabolism, and how do these interactions influence its function?

• Physiological role of FCD synthesis: What is the biological significance of archaeal RibL's ability to synthesize flavin cytidine dinucleotide (FCD), and how is this alternative cofactor utilized in archaeal metabolism?

• Distribution across archaeal lineages: How widespread are these unique FAD synthases across different archaeal lineages, and what patterns of conservation or variation exist?

• Lateral gene transfer dynamics: What factors facilitated the lateral transfer of archaeal FADS genes to organisms like E. histolytica, and how frequently has this occurred in evolution?

Addressing these questions will require interdisciplinary approaches combining structural biology, biochemistry, genetics, and evolutionary analysis. The answers will not only enhance our understanding of fundamental aspects of flavin metabolism but may also reveal new targets for antimicrobial development and expand the biotechnological utility of these unique enzymes.

What emerging technologies might advance research on archaeal RibL in the coming decade?

Several emerging technologies are poised to significantly advance research on archaeal RibL in the coming years:

• Cryo-electron microscopy: Advanced cryo-EM techniques will enable determination of high-resolution structures of archaeal RibL, potentially capturing different conformational states during catalysis.

• AI-driven protein structure prediction: Tools like AlphaFold and RoseTTAFold will provide increasingly accurate structural models of archaeal RibL variants from diverse species, facilitating comparative structural analysis.

• Archaeal genetic engineering tools: Improved CRISPR-Cas and other genome editing technologies for archaea will enable more sophisticated in vivo studies of RibL function.

• Single-molecule enzymology: Advanced techniques to study individual enzyme molecules will reveal the dynamics and heterogeneity of archaeal RibL catalysis at unprecedented resolution.

• Microfluidic enzyme assays: High-throughput microfluidic platforms will accelerate screening of conditions, variants, and potential inhibitors of archaeal RibL.

• Synthetic biology frameworks: Expanding toolkits for incorporating archaeal enzymes into synthetic biological systems will reveal new applications and insights.

• Metagenomic mining: Advanced metagenomic techniques will uncover novel archaeal RibL variants from extreme environments, potentially with unique properties.

• Quantum biochemistry simulations: Improved computational methods will enable detailed modeling of the reaction mechanism and electronic structure of archaeal RibL.

• Artificial enzyme evolution: Directed evolution platforms coupled with high-throughput screening will generate archaeal RibL variants with enhanced stability and novel functions.