Recombinant Methanoplanus petrolearius FAD synthase (ribL)

What is Methanoplanus petrolearius FAD synthase (RibL) and how does it differ from other FAD synthetases?

Methanoplanus petrolearius FAD synthase (RibL) is an enzyme that catalyzes the adenylation of FMN with ATP to produce FAD and pyrophosphate (PP(i)) in the archaeon Methanoplanus petrolearius. As an archaeal FAD synthetase, it differs significantly from both bacterial and eukaryotic counterparts. While eukaryotes possess monofunctional FAD synthetases and bacteria have bifunctional enzymes that catalyze both riboflavin phosphorylation and FMN adenylation, archaeal RibL appears to be monofunctional but with unique characteristics .

The archaeal RibL follows the riboflavin kinase (RibK) step in the archaeal FAD biosynthetic pathway. Unlike bacterial enzymes, archaeal FAD synthetases like RibL do not catalyze the reverse reaction to produce FMN and ATP from FAD and PP(i). Additionally, archaeal RibL is distinctly inhibited by pyrophosphate, whereas other FAD synthetases are not .

What expression systems are most suitable for producing recombinant M. petrolearius RibL?

Based on successful expression of similar archaeal FAD synthetases, Escherichia coli is likely the most suitable heterologous expression system for M. petrolearius RibL. When expressing this protein, researchers should consider:

• Using E. coli strains optimized for expression of proteins containing rare codons, as archaeal genes often contain codons that are rarely used in E. coli

• Employing reducing conditions during purification and storage, as archaeal RibL is oxygen-sensitive and requires reducing conditions for activity

• Including divalent metal ions, particularly Co²⁺, in the purification buffers to maintain enzyme stability

• Considering expression at lower temperatures (16-25°C) to enhance proper protein folding

What are the basic activity assay conditions for M. petrolearius RibL?

The basic activity assay for M. petrolearius RibL should include:

• Buffer conditions: Typically a buffered solution at pH 7.0-8.0

• Substrate requirements: FMN and ATP as primary substrates

• Metal cofactor: Divalent metal ions, with Co²⁺ likely providing optimal activity (approximately 4× greater than with Mg²⁺)

• Reducing agent: A thiol-containing compound (such as DTT or β-mercaptoethanol) to maintain reduced cysteine residues

• Temperature considerations: Likely requires temperatures reflecting the thermophilic nature of the organism (potentially 55-65°C)

• Anaerobic conditions: To prevent oxidation of critical cysteine residues

The activity can be monitored by measuring FAD formation spectrofluorometrically or by HPLC analysis of reaction products .

How stable is recombinant M. petrolearius RibL during purification and storage?

While specific data for M. petrolearius RibL is not provided in the search results, insights from other archaeal FAD synthetases suggest:

• Air sensitivity: The enzyme is likely air-sensitive and requires reducing conditions to maintain activity

• Temperature stability: As an enzyme from an archaeal source, it likely possesses higher thermal stability than mesophilic counterparts

• Storage conditions: Should be stored under reducing conditions, potentially with glycerol as a cryoprotectant

• Metal dependence: The presence of divalent metal ions, particularly Co²⁺, may enhance stability

For optimal storage, researchers should consider maintaining the enzyme under anaerobic conditions at -80°C in the presence of a reducing agent and appropriate metal cofactors.

What is the mechanism of oxygen sensitivity in M. petrolearius RibL and how can it be managed in experimental settings?

The oxygen sensitivity of archaeal RibL enzymes appears to be related to critical cysteine residues in the protein. In the characterized archaeal RibL from Methanocaldococcus jannaschii, alkylation of two conserved cysteines in the C-terminus resulted in complete inactivation of the enzyme . This suggests these cysteines play crucial roles in catalysis and/or structural integrity.

For experimental management of oxygen sensitivity:

• Conduct enzyme purification and assays in an anaerobic chamber or under nitrogen/argon atmosphere

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

• Use oxygen-scavenging enzyme systems in reaction buffers when working outside anaerobic chambers

• Consider rapid work protocols that minimize exposure to air

• Pre-reduce buffers before use by bubbling with nitrogen or adding reducing agents

The relationship between protein oxidation state and activity presents opportunities to study redox regulation mechanisms in archaeal metabolism, potentially revealing unique adaptations to the anaerobic lifestyle of methanogens.

How does the metal cofactor specificity of M. petrolearius RibL affect its catalytic mechanism?

The preference for Co²⁺ as a metal cofactor in archaeal RibL represents a significant divergence from typical FAD synthetases, which generally prefer Mg²⁺. For M. petrolearius RibL, based on archaeal homologs:

• Co²⁺ likely provides approximately 4× greater activity than Mg²⁺

• The metal ion presumably coordinates the phosphate groups of ATP to facilitate nucleophilic attack by the phosphate group of FMN

• The unique preference for Co²⁺ may reflect adaptation to the specific cellular environment of M. petrolearius

To investigate metal specificity:

• Conduct activity assays with various divalent metals (Mg²⁺, Co²⁺, Mn²⁺, Ni²⁺, Zn²⁺) at equivalent concentrations

• Determine metal binding constants using isothermal titration calorimetry or fluorescence quenching

• Perform site-directed mutagenesis of predicted metal-coordinating residues to identify the metal-binding site

Understanding this unique metal preference could provide insights into the evolution of metalloproteins in archaeal organisms and potential biotechnological applications requiring Co²⁺-dependent enzymes.

What structural features distinguish M. petrolearius RibL from bacterial and eukaryotic FAD synthetases?

While the specific structure of M. petrolearius RibL is not described in the search results, archaeal RibL proteins likely possess several distinguishing structural features:

• Nucleotidyl transferase domain: M. petrolearius RibL is expected to belong to the nucleotidyl transferase protein family

• Absence of riboflavin kinase domain: Unlike bacterial bifunctional enzymes, archaeal RibL likely lacks the domain for riboflavin phosphorylation

• Critical C-terminal cysteines: Contains conserved cysteine residues in the C-terminus that are essential for activity and sensitive to oxidation

• Metal binding site: Likely possesses a unique metal-binding pocket optimized for Co²⁺ coordination rather than Mg²⁺

In contrast, human FAD synthase (hFADS2) contains both a 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase domain for FAD formation and a molybdopterin-binding (MPTb) domain involved in FAD hydrolysis . Bacterial enzymes typically combine riboflavin kinase and FAD synthetase activities in a bifunctional enzyme.

What is the substrate specificity of M. petrolearius RibL beyond its primary FMN and ATP substrates?

Archaeal RibL enzymes demonstrate interesting substrate flexibility. Based on studies of the M. jannaschii homolog:

• Alternative nucleotide donor: M. petrolearius RibL likely can use CTP as an alternative to ATP, producing flavin cytidine dinucleotide (FCD) instead of FAD

• Substrate recognition: The enzyme likely recognizes the isoalloxazine ring and ribityl chain of FMN with high specificity

• Reverse reaction: Unlike other FAD synthetases, archaeal RibL does not appear to catalyze the reverse reaction (FAD + PP(i) → FMN + ATP)

To investigate substrate specificity:

• Test various nucleotide donors (ATP, CTP, GTP, UTP) under standard assay conditions

• Examine FMN analogs with modifications to the isoalloxazine ring or ribityl chain

• Determine kinetic parameters (K₍ₘ₎, k(cat), k(cat)/K₍ₘ₎) for each viable substrate

• Use isothermal titration calorimetry to measure binding affinities for various substrates

The unique substrate preferences of archaeal RibL may provide insights into the evolutionary divergence of flavin metabolism in archaea.

How can researchers distinguish between FAD synthetase and FAD hydrolase activities in recombinant preparations?

Some FAD synthetases, such as human FADS2, exhibit both synthetic and hydrolytic activities . To distinguish between these functions in M. petrolearius RibL:

• Directional assays:

  • Synthetase activity: Measure FAD formation from FMN and ATP

  • Hydrolase activity: Measure FMN formation from FAD

• Inhibitor-based approach:

  • Pyrophosphate inhibition: Since pyrophosphate inhibits archaeal RibL synthetase activity , its presence would selectively affect the forward reaction

  • Metal dependence: Evaluate activity with different metals, as synthetase and hydrolase activities may have different metal preferences

• Time-course analysis:

  • Monitor the reaction over extended periods to detect any reversal or hydrolysis of products

  • In plant mitochondrial preparations, prolonged incubation leads to FAD decrease and riboflavin increase due to hydrolytic activity

Based on archaeal RibL characteristics, M. petrolearius RibL likely lacks significant hydrolytic activity, as the M. jannaschii homolog does not catalyze the reverse reaction .

What analytical techniques are most effective for studying the redox state of critical cysteines in M. petrolearius RibL?

Given the importance of cysteine residues in archaeal RibL function , several techniques can effectively analyze their redox states:

• Alkylation-based methods:

  • Differential alkylation with iodoacetamide or N-ethylmaleimide followed by mass spectrometry

  • Fluorescent labeling of free thiols using maleimide dyes

• Mass spectrometry approaches:

  • Liquid chromatography-mass spectrometry (LC-MS) of peptide digests to identify modified cysteines

  • Hydrogen-deuterium exchange mass spectrometry to examine structural changes upon oxidation

• Activity correlation:

  • Parallel analysis of enzyme activity and thiol content using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB)

  • Site-directed mutagenesis of individual cysteines to determine their specific roles

• Protein crystallography:

  • X-ray crystallography under reducing and oxidizing conditions to visualize structural changes

  • Anomalous scattering to precisely locate sulfur atoms in the protein structure

These techniques can reveal how the redox state of specific cysteines impacts enzyme structure and function, providing insights into the mechanism of oxygen sensitivity.

What are the optimal conditions for measuring kinetic parameters of M. petrolearius RibL?

For accurate determination of kinetic parameters of M. petrolearius RibL:

• Basic assay conditions:

  • Buffer: 50 mM HEPES or Tris-HCl, pH 7.5-8.0

  • Temperature: Likely 55-65°C (reflecting the thermophilic nature of the organism)

  • Metal: 5-10 mM Co²⁺ (which provides optimal activity)

  • Reducing agent: 1-5 mM DTT or β-mercaptoethanol

  • Anaerobic environment

• For K₍ₘ₎ determination for FMN:

  • Vary FMN concentration (0.1-100 μM) while maintaining excess ATP (1-5 mM)

  • Consider potential substrate inhibition at high FMN concentrations

• For K₍ₘ₎ determination for ATP:

  • Vary ATP concentration (1 μM-5 mM) while maintaining excess FMN (20-50 μM)

• For alternative substrate kinetics:

  • Use similar approaches with CTP to characterize the formation of FCD

• Data analysis:

  • Initial velocity measurements should be taken within the linear range of the reaction

  • Fit data to appropriate enzyme kinetic models (Michaelis-Menten, allosteric models if cooperative behavior is observed)

Note that pyrophosphate inhibition should be considered when designing experiments, as product accumulation may affect reaction rates.

How does M. petrolearius RibL compare with FAD synthetases from other archaeal species?

The archaeal FAD synthetases represent a distinct evolutionary group with unique properties:

• Shared characteristics likely include:

  • Oxygen sensitivity due to critical cysteine residues

  • Preference for Co²⁺ as a metal cofactor over Mg²⁺

  • Ability to use CTP as an alternative substrate

  • Inhibition by pyrophosphate

  • Monofunctional nature (lacking riboflavin kinase activity)

• Phylogenetic positioning:

  • Archaeal RibL enzymes like those from M. petrolearius and M. jannaschii belong to the nucleotidyl transferase protein family

  • They are evolutionarily distinct from both bacterial bifunctional enzymes and eukaryotic monofunctional FAD synthetases

• Functional adaptation:

  • The unique properties likely reflect adaptation to the anaerobic, often thermophilic lifestyle of methanogenic archaea

  • The inability to catalyze the reverse reaction distinguishes archaeal enzymes from other FAD synthetases

Comparative analysis of archaeal RibL proteins can provide insights into the evolution of flavin metabolism across different domains of life.

What is the evolutionary relationship between archaeal RibL and the bifunctional enzymes of bacteria?

Archaeal RibL represents a distinct evolutionary solution to FAD synthesis compared to bacterial systems:

• Domain architecture differences:

  • Archaeal FAD synthesis involves separate monofunctional enzymes: RibK (riboflavin kinase) and RibL (FAD synthetase)

  • Bacterial systems typically employ bifunctional enzymes that catalyze both steps

• Nucleotide specificity divergence:

  • Archaeal RibK is CTP-dependent, whereas bacterial riboflavin kinases use ATP

  • Archaeal RibL can use both ATP and CTP as substrates

• Evolutionary implications:

  • The archaeal system may represent either an ancestral state or a specialized adaptation

  • The separation of functions in archaea versus fusion in bacteria suggests different evolutionary pressures on flavin metabolism

This divergence in enzyme architecture provides a fascinating case study in the evolution of metabolic pathways and enzyme specificity across domains of life.

How do the dual catalytic capabilities of human FAD synthetase compare with the properties of M. petrolearius RibL?

Human FAD synthetase and archaeal RibL show significant functional and structural differences:

This comparison highlights the diverse evolutionary strategies for regulating cellular FAD homeostasis across different domains of life.

What potential biotechnological applications exist for recombinant M. petrolearius RibL?

The unique properties of archaeal RibL enzymes suggest several potential biotechnological applications:

• Biocatalysis:

  • Production of FAD and flavin derivatives under anaerobic conditions

  • Synthesis of flavin cytidine dinucleotide (FCD) and other non-standard flavin cofactors

• Biosensors:

  • Development of redox-sensitive biosensors based on the oxygen sensitivity of the enzyme

  • Creation of metal-responsive systems utilizing the Co²⁺ preference

• Structural biology tools:

  • Model system for studying metal-nucleotide interactions

  • Platform for investigating redox control mechanisms in enzymes

• Thermostable enzyme applications:

  • Heat-resistant flavin production systems

  • Robust components for cell-free biosynthetic pathways

The ability to function under reducing conditions and at elevated temperatures makes archaeal RibL enzymes potentially valuable for specialized biocatalytic applications.

What are the most significant unanswered questions regarding the structure and function of archaeal FAD synthetases?

Several important questions remain regarding archaeal FAD synthetases:

• Structural basis of function:

  • What is the three-dimensional structure of archaeal RibL?

  • How does the active site accommodate both ATP and CTP as substrates?

  • What is the structural basis for Co²⁺ preference over Mg²⁺?

• Redox regulation:

  • What is the precise role of the conserved cysteines in catalysis?

  • Is there a physiological redox regulation mechanism in archaea?

  • How does oxygen sensitivity relate to the anaerobic lifestyle of the organism?

• Evolutionary questions:

  • Did the monofunctional archaeal system precede the bifunctional bacterial system?

  • What selective pressures led to the unique properties of archaeal FAD metabolism?

  • Are there archaeal species with alternative FAD synthesis pathways?

• Physiological roles:

  • How is FAD synthesis regulated in archaeal cells?

  • Does the ability to synthesize FCD have physiological significance?

  • What is the biological significance of pyrophosphate inhibition?

Addressing these questions would significantly advance our understanding of flavin metabolism across domains of life.

What technical challenges must be overcome to obtain structural information about M. petrolearius RibL?

Obtaining structural information about archaeal RibL presents several technical challenges:

• Protein expression and purification:

  • Maintaining reducing conditions throughout purification to preserve activity

  • Obtaining sufficient quantities of soluble protein from a thermophilic source

  • Preventing oxidative damage during handling

• Crystallization challenges:

  • Identifying appropriate crystallization conditions for an oxygen-sensitive protein

  • Maintaining protein stability during crystal growth

  • Incorporating appropriate metal cofactors into crystals

• Data collection considerations:

  • Collecting X-ray diffraction data under anaerobic conditions

  • Potential need for specialized equipment for anaerobic crystal handling

  • Optimizing cryoprotection for thermophilic proteins

• Alternative structural approaches:

  • Consider electron microscopy as an alternative approach

  • Use NMR for structural studies of specific domains

  • Apply hydrogen-deuterium exchange mass spectrometry for conformational studies

Overcoming these challenges requires specialized equipment and techniques for handling oxygen-sensitive proteins, but would provide valuable insights into the unique structural features of archaeal RibL.

What are common problems in heterologous expression of M. petrolearius RibL and how can they be addressed?

Heterologous expression of archaeal proteins like M. petrolearius RibL often encounters several challenges:

• Insoluble protein expression:

  • Solution: Lower expression temperature (16-20°C), use solubility-enhancing fusion tags (SUMO, MBP), add compatible solutes (trehalose, betaine)

  • Screen different E. coli expression strains optimized for difficult proteins

• Loss of activity during purification:

  • Solution: Maintain strict reducing conditions using higher concentrations of reducing agents

  • Conduct all purification steps in an anaerobic chamber if possible

  • Include Co²⁺ in purification buffers to stabilize the protein

• Codon bias issues:

  • Solution: Use codon-optimized synthetic genes for expression in E. coli

  • Alternatively, use E. coli strains supplemented with rare tRNAs

• Protein instability:

  • Solution: Include stabilizing agents (glycerol, trehalose) in storage buffers

  • Store under anaerobic conditions at -80°C

  • Avoid freeze-thaw cycles by preparing single-use aliquots

• Low expression yield:

  • Solution: Optimize media composition and induction conditions

  • Consider archaeal expression hosts for difficult cases

  • Explore cell-free protein synthesis systems

Each of these approaches should be systematically tested to identify optimal conditions for expressing active M. petrolearius RibL.

How can researchers accurately measure enzyme activity when dealing with an oxygen-sensitive enzyme like M. petrolearius RibL?

Measuring activity of oxygen-sensitive enzymes presents methodological challenges that can be addressed by:

• Anaerobic techniques:

  • Conduct assays in an anaerobic chamber with controlled atmosphere

  • Use sealed cuvettes or microplates with oxygen-impermeable films

  • Include oxygen-scavenging systems (glucose oxidase/catalase) in reaction mixtures

• Redox potential control:

  • Monitor and maintain specific redox potentials using redox buffers

  • Use redox indicators to verify anaerobic conditions

  • Pre-reduce all assay components before mixing

• Rapid analysis methods:

  • Develop quick-quench approaches to minimize oxygen exposure

  • Use stopped-flow spectrophotometry for faster kinetic measurements

  • Implement automated sampling systems within anaerobic environments

• Data analysis considerations:

  • Account for potential activity loss during measurement

  • Include appropriate controls to assess oxygen effects

  • Consider mathematical modeling to correct for oxygen inactivation

• Alternative activity assays:

  • Develop endpoint assays with immediate sample denaturation

  • Use HPLC-based product analysis under anaerobic sample preparation

These approaches can be combined to develop robust activity assays for oxygen-sensitive enzymes like M. petrolearius RibL.