Recombinant Acanthamoeba polyphaga mimivirus Probable FAD-linked sulfhydryl oxidase R368 (MIMI_R368)

What is the structural organization of mimivirus sulfhydryl oxidases?

Mimivirus sulfhydryl oxidases belong to the Erv (Essential for respiration and viability) family of enzymes and possess a characteristic domain organization. These proteins contain a canonical Erv domain with a FAD-binding region and, in many cases, an additional ORFan domain with a novel fold. The Erv domain houses a FAD-proximal CxxC disulfide that forms the active site, while the ORFan domain may contribute to substrate recognition or enzyme localization .

How are conserved cysteine residues distributed in mimivirus sulfhydryl oxidases?

Mimivirus sulfhydryl oxidases contain several strategically positioned cysteine residues that serve distinct functional roles. Using R596 as an example model:

The active-site CxxC motif is essential for catalysis, directly interacting with the FAD cofactor. Additional cysteines, such as the CX₉C motif identified in R596, may function as shuttle disulfides that facilitate electron transfer during substrate oxidation. Mutation of these conserved cysteines significantly impacts enzymatic activity, demonstrating their importance in the catalytic mechanism .

What expression systems are suitable for producing recombinant mimivirus sulfhydryl oxidases?

Recombinant mimivirus sulfhydryl oxidases can be expressed in bacterial systems, though several challenges must be addressed:

• Standard E. coli strains: Production in common laboratory strains often leads to aggregation and precipitation due to improper disulfide formation .

• OrigamiTM B E. coli: This thiol reductase-deficient strain facilitates disulfide bond formation in the cytoplasm but may still yield aggregated protein .

• Strategic mutagenesis: To overcome aggregation issues, construct design should consider removing non-essential cysteines while preserving those critical for structure and function. For example:

  • Full-length constructs (all cysteines) → protein aggregation

  • Selected conserved cysteines only → stable, functional protein

For purification, a typical protocol includes:

• Nickel affinity chromatography (using His₆-tag)

• Thrombin cleavage to remove affinity tags

• Secondary affinity chromatography

• Size exclusion chromatography in 20 mM Tris, 300 mM NaCl (pH 7.7)

Protein concentration can be determined spectroscopically at 446 nm using the FAD absorbance (ε = 11,300 M⁻¹ cm⁻¹) in 6 M guanidine-HCl containing 20 mM sodium phosphate buffer (pH 6.5) .

What spectroscopic methods can be used to characterize mimivirus sulfhydryl oxidases?

Several spectroscopic approaches are valuable for characterizing mimivirus sulfhydryl oxidases:

• UV-visible spectroscopy:

  • FAD absorption (λₘₐₓ ~450 nm) provides information about cofactor binding

  • Spectral changes during reduction/oxidation reveal enzyme redox state

  • Concentration determination using ε = 11,300 M⁻¹ cm⁻¹ at 446 nm

• Fluorescence spectroscopy:

  • Monitoring FAD fluorescence quenching during protein binding

  • Following conformational changes that affect FAD environment

• Circular dichroism (CD):

  • Secondary structure analysis

  • Thermal stability assessment (melting temperature determination)

• NMR spectroscopy:

  • TROSY-HSQC 2D NMR for conformational dynamics analysis

  • Detection of structural perturbations upon mutation

These techniques provide complementary information about protein structure, stability, and dynamics, enabling comprehensive characterization of wild-type and mutant enzymes.

What is the mechanism of electron transfer in mimivirus sulfhydryl oxidases?

Mimivirus sulfhydryl oxidases utilize a sophisticated electron transfer mechanism involving multiple redox-active disulfide centers:

• Substrate oxidation: The substrate protein's dithiol motif is oxidized to a disulfide with the concurrent reduction of a disulfide within the sulfhydryl oxidase.

• Shuttle disulfide mechanism: Mimivirus R596 demonstrates evidence for an intermolecular dithiol/disulfide relay within the dimer—the first such extended, intersubunit redox-active site identified in a viral sulfhydryl oxidase. The CX₉C motif located in one subunit functions as a shuttle disulfide that can accept electrons from substrates and transfer them to the active-site disulfide of the second subunit .

• FAD reduction/oxidation: The active-site disulfide (CxxC), proximal to the FAD, transfers electrons to the flavin cofactor, which in turn passes them to molecular oxygen, producing hydrogen peroxide.

Experimental evidence for this mechanism comes from activity comparisons between wild-type enzyme and variants lacking the putative shuttle cysteines. The R596-4C variant (retaining four conserved cysteines) showed considerably higher activity in DTT oxidation than the R596-2C variant (retaining only the active-site cysteines), supporting the functional importance of the additional disulfide in the catalytic mechanism .

How does the ORFan domain contribute to substrate specificity and enzyme function?

The ORFan domain of mimivirus sulfhydryl oxidases presents a fascinating case of structural innovation in viral proteins. This domain:

• Forms a novel fold: The ORFan domain of R596 assumes a compact, folded structure unlike any previously characterized protein fold, representing potential "protein structural innovation" in large double-stranded DNA viruses .

• Creates a substrate-binding interface: In the R596 dimer, the ORFan domain contributes to forming a broad cleft enriched with exposed aromatic groups and basic side chains—features consistent with binding target proteins or nucleic acids .

• Influences enzyme localization: The unique surface properties may facilitate localization within viral factories or virions, positioning the enzyme where its activity is most needed .

• Shows evolutionary diversity: Comparison of related viral sulfhydryl oxidase sequences indicates variable conservation of the ORFan domain structure. While some orthologs (e.g., megavirus mg174) likely share the complete fold, others show significant truncation or sequence divergence in this region .

Experimental approaches to investigate ORFan domain function include:

• Truncation constructs to assess the domain's contribution to activity

• Chimeric proteins to evaluate domain-specific functions

• Site-directed mutagenesis of surface residues in the putative binding cleft

• Protein-protein interaction studies using pulldown assays or crosslinking

What strategies can overcome challenges in crystallizing mimivirus sulfhydryl oxidases?

Crystallization of mimivirus sulfhydryl oxidases presents several challenges, particularly related to protein stability and homogeneity. Successful strategies include:

• Construct optimization:

  • Identify and remove non-essential cysteines that promote aggregation

  • Design constructs based on sequence conservation analysis

  • For R596, a four-cysteine variant (R596-4C) maintaining only the most conserved cysteines (Cys80, Cys83, Cys146, Cys156) yielded diffraction-quality crystals

• Protein stabilization:

  • Buffer optimization to prevent oxidation/aggregation

  • Addition of reducing agents at appropriate concentrations

  • Inclusion of ligands or substrates to stabilize conformation

• Selenomethionine incorporation:

  • Preparation of selenomethionine variants following published protocols

  • Facilitates phase determination in crystallographic analysis

• Crystal optimization:

  • Microseeding to improve crystal quality

  • Additive screening to enhance crystallization

  • Crystal cryoprotection optimization

For proteins resistant to crystallization (such as mutant variants), alternative structural approaches like NMR spectroscopy can provide valuable conformational information. TROSY-HSQC 2D NMR has been successfully employed to detect increased conformational flexibility in mutant sulfhydryl oxidases compared to wild-type proteins .

How can enzyme kinetics be used to assess the functional importance of specific residues?

Comprehensive kinetic analysis provides insights into the catalytic mechanism and the contribution of specific residues in mimivirus sulfhydryl oxidases:

• Steady-state kinetics:

  • Determine kcat and KM using various substrates (DTT, reduced proteins)

  • Compare parameters between wild-type and mutant enzymes

  • Assess substrate specificity through analysis of kcat/KM values

• Stopped-flow spectroscopy:

  • Monitor rapid reactions using FAD absorbance changes

  • Determine pre-steady-state kinetic parameters

  • Measure FAD association/dissociation rates

  • Quantify the effect of mutations on cofactor binding

• Redox potential measurements:

  • Determine the redox potential of active-site and shuttle disulfides

  • Evaluate thermodynamic parameters for electron transfer

• Structure-function analysis:

  • Correlate kinetic parameters with structural features

  • Design rational mutations based on crystal structures

  • Analyze the effect of mutations on protein stability and catalytic efficiency

Case study: Comparison of R596-4C (containing the active-site disulfide and a potential shuttle disulfide) with R596-2C (containing only the active-site disulfide) revealed substantially higher DTT oxidation activity in the former, supporting the functional importance of the shuttle disulfide in the catalytic mechanism .

What methods can be used to study the stability and assembly of mimivirus sulfhydryl oxidase dimers?

The dimeric structure of mimivirus sulfhydryl oxidases is crucial for their function, and several approaches can assess dimer stability and assembly:

• Thermal stability assessment:

  • Differential scanning calorimetry to determine melting temperature

  • Circular dichroism with temperature ramping

  • Thermal shift assays using fluorescent dyes

• Chemical stability:

  • Resistance to denaturants (guanidine hydrochloride, urea)

  • Susceptibility to partial proteolysis

  • Stability in the presence of reducing agents like glutathione

• Cofactor binding studies:

  • Measure FAD dissociation rates using stopped-flow spectroscopy

  • Determine apoprotein preparation conditions

  • Compare wild-type and mutant proteins for cofactor retention

• Size exclusion chromatography:

  • Monitor oligomeric state under various conditions

  • Detect changes in assembly due to mutations or conditions

• Analytical ultracentrifugation:

  • Determine equilibrium constants for dimer formation

  • Assess heterogeneity in oligomeric states

The interplay between FAD binding, disulfide bridge formation, and dimer stability appears crucial for enzyme function. Studies with ALR (another sulfhydryl oxidase) demonstrated that mutations affecting FAD binding also impact dimer stability and intersubunit disulfide reduction susceptibility .