Recombinant Anopheles gambiae Molybdopterin synthase catalytic subunit (AGAP004290)

What is the molecular identity of Recombinant Anopheles gambiae Molybdopterin synthase catalytic subunit?

The Recombinant Anopheles gambiae Molybdopterin synthase catalytic subunit (AGAP004290) is a protein with UniProt accession number Q7QAD7. It represents the large subunit (MoaE) of the molybdopterin synthase enzyme complex derived from Anopheles gambiae (African malaria mosquito). The protein is also known as Molybdenum cofactor synthesis protein 2 large subunit or MOCS2B. The full-length protein consists of 154 amino acids with a sequence beginning with MNYLKLTFDK and ending with NHIL .

What is the structural organization of molybdopterin synthase?

Molybdopterin synthase is a heterotetrameric enzyme composed of two types of subunits: a large subunit (MoaE) and a small subunit (MoaD). Crystal structure analysis has revealed that the C-terminus of each MoaD subunit inserts deeply into a MoaE subunit to form the active site. In the activated form of the enzyme, the MoaD C-terminus is present as a thiocarboxylate, which is critical for its catalytic function . The structure also includes a binding pocket for the terminal phosphate of molybdopterin and a proposed binding site for the pterin moiety present in the substrate (precursor Z) and the product (molybdopterin) .

What are optimal storage conditions for the recombinant AGAP004290 protein?

The shelf life of recombinant AGAP004290 is influenced by multiple factors including storage state, buffer ingredients, storage temperature, and the inherent stability of the protein. For optimal preservation:

• Liquid form has a shelf life of approximately 6 months at -20°C/-80°C

• Lyophilized form demonstrates extended stability with a shelf life of 12 months at -20°C/-80°C

• Repeated freezing and thawing cycles should be avoided

• Working aliquots can be stored at 4°C for up to one week

How should AGAP004290 protein be reconstituted for experimental use?

For proper reconstitution:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation)

• Aliquot for long-term storage at -20°C/-80°C

What is the biochemical function of molybdopterin synthase in Anopheles gambiae?

Molybdopterin synthase catalyzes a critical step in the molybdenum cofactor biosynthesis pathway. Specifically, it generates the dithiolene group of molybdopterin, which is responsible for molybdenum ligation in the final cofactor. The enzyme converts precursor Z to molybdopterin by introducing sulfur atoms to form the dithiolene group . This reaction is essential for producing functional molybdenum cofactors that serve as active sites in various enzymes including those involved in nitrogen, sulfur, and carbon metabolism in A. gambiae and other organisms .

How can the enzymatic activity of recombinant AGAP004290 be measured?

A methodological approach to measuring AGAP004290 activity involves:

• In vitro reconstitution assay: Combine purified recombinant AGAP004290 (MoaE) with its partner subunit MoaD (which should be present as a thiocarboxylate at its C-terminus)

• Substrate preparation: Generate precursor Z either through chemical synthesis or isolation from bacteria lacking the molybdopterin synthase

• Reaction conditions: Incubate the enzyme complex with precursor Z under anaerobic conditions with appropriate buffer systems (typically at pH 7.0-7.5)

• Activity detection: Monitor the conversion of precursor Z to molybdopterin using:

  • HPLC analysis with fluorescence detection

  • Mass spectrometry to detect the formation of the dithiolene group

  • Coupled enzyme assays that depend on active molybdopterin formation

What is the relationship between molybdopterin synthase and molybdenum cofactor biosynthesis?

Molybdopterin synthase catalyzes the second major step in the four-step molybdenum cofactor biosynthesis pathway in organisms like E. coli, which serves as a model for understanding this process. The complete pathway includes:

• Formation of precursor Z

• Conversion of precursor Z to molybdopterin (MPT) by molybdopterin synthase

• Insertion of molybdenum to form Moco via an MPT-AMP intermediate

• Potential additional modifications, such as the covalent addition of GMP or CMP to form MGD or MCD cofactors

In A. gambiae, the AGAP004290 protein (MoaE) is specifically involved in the second step of this pathway, working in conjunction with its partner subunit to generate the dithiolene group essential for molybdenum coordination .

What expression systems are most effective for producing recombinant AGAP004290?

Based on the available data, yeast has been used successfully as an expression system for recombinant AGAP004290 . When designing an expression strategy:

• Yeast expression system:

  • Offers eukaryotic protein processing capabilities

  • Provides post-translational modifications

  • Typically yields properly folded protein

• Optimization considerations:

  • Codon optimization for the host organism

  • Selection of appropriate promoters (constitutive vs. inducible)

  • Fusion tags for purification and detection

  • Growth conditions (temperature, media, induction time)

• Purification strategy:

  • Affinity chromatography using appropriate tags

  • Size exclusion chromatography for oligomeric state confirmation

  • Yield assessment using SDS-PAGE with expected purity >85%

How can researchers assess the structural integrity of purified AGAP004290?

Multiple complementary techniques can be employed to verify structural integrity:

• SDS-PAGE and Western blotting:

  • Confirm protein size and purity (>85% as reported)

  • Detect specific epitopes using antibodies against AGAP004290

• Circular Dichroism (CD) spectroscopy:

  • Assess secondary structure elements

  • Monitor thermal stability and folding

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS):

  • Determine oligomeric state in solution

  • Verify heterotetramer formation when combined with MoaD

• Limited proteolysis:

  • Probe domain organization and folding

  • Identify stable domains for structural studies

• Differential Scanning Fluorimetry (DSF):

  • Measure thermal stability under various buffer conditions

  • Optimize storage conditions based on stability data

What are the key considerations for designing interaction studies between AGAP004290 and its partner proteins?

When investigating protein-protein interactions involving AGAP004290:

• Partner identification:

  • The MoaD subunit (small subunit) is the primary interaction partner

  • Ensure the MoaD C-terminus is properly processed for thiocarboxylate formation

• Interaction analysis methods:

  • Pull-down assays using tagged AGAP004290

  • Surface Plasmon Resonance (SPR) for binding kinetics

  • Isothermal Titration Calorimetry (ITC) for thermodynamic parameters

  • Native mass spectrometry to confirm heterotetramer formation

  • Yeast two-hybrid or bacterial two-hybrid screens for novel partners

• Functional validation:

  • In vitro reconstitution of enzymatic activity with purified components

  • Mutational analysis targeting the interaction interface

  • Cross-linking studies to capture transient interactions

How can structural information about molybdopterin synthase inform inhibitor design for pest control strategies?

The crystal structure analysis of molybdopterin synthase provides crucial insights for rational inhibitor design:

• Target site identification:

  • The deep insertion of the MoaD C-terminus into MoaE creates a unique interface

  • The binding pocket for the terminal phosphate of molybdopterin

  • The proposed binding site for the pterin moiety

• Structure-based design strategy:

  • Develop compounds that mimic the thiocarboxylate group but resist catalysis

  • Target species-specific differences in the active site between human and insect enzymes

  • Design allosteric inhibitors that prevent MoaE-MoaD complex formation

• Validation approaches:

  • In vitro enzyme inhibition assays

  • Structural studies of enzyme-inhibitor complexes

  • Cellular assays in insect cell lines

  • Specificity testing against human molybdopterin synthase

• Potential applications:

  • Development of selective insecticides targeting A. gambiae

  • Creation of molecular tools to study molybdenum cofactor metabolism in mosquitoes

What methods can be used to investigate the role of AGAP004290 in the physiology of Anopheles gambiae?

Several complementary approaches can elucidate the physiological significance:

• Gene silencing techniques:

  • RNA interference (RNAi) to knockdown AGAP004290 expression

  • CRISPR-Cas9 gene editing to create defined mutations

  • Analysis of resulting phenotypes in development, survival, and reproduction

• Tissue-specific expression analysis:

  • RT-qPCR to quantify transcripts across tissues and developmental stages

  • Immunohistochemistry to localize protein expression

  • Single-cell transcriptomics to identify cell types expressing AGAP004290

• Functional assays:

  • Measure activities of molybdoenzymes in normal and AGAP004290-depleted mosquitoes

  • Metabolomic profiling to identify altered biochemical pathways

  • Supplementation studies with molybdenum compounds

• Ecological and behavioral studies:

  • Assess impact on blood-feeding behavior

  • Evaluate effects on susceptibility to insecticides

  • Investigate potential role in malaria parasite interactions

How do mutations in the AGAP004290 gene affect enzyme function and mosquito physiology?

A systematic approach to mutation analysis would include:

• Mutation identification and characterization:

  • Natural variants in wild mosquito populations

  • Engineered mutations based on structural information

  • Classification of mutations (active site, dimerization interface, etc.)

• Functional analysis of mutant proteins:

  • Expression and purification of recombinant mutant proteins

  • Enzymatic activity assays compared to wild-type

  • Structural studies to determine molecular effects of mutations

  • Protein stability and partner binding studies

• Physiological impact assessment:

  • Generation of transgenic mosquitoes expressing mutant variants

  • Phenotypic characterization across developmental stages

  • Stress response analysis (oxidative, nutritional, etc.)

  • Fitness measurements in laboratory and simulated field conditions

• Comparative analysis with human disease mutations:

  • Parallels with human molybdenum cofactor deficiency

  • Conservation of critical residues across species

  • Potential for mosquito models of human disease

Comparative Analysis with Related Systems

Evolutionary analysis provides multiple insights:

• Sequence conservation patterns:

  • Highly conserved catalytic residues across all domains of life

  • Variable regions that may confer species-specific properties

  • Correlation between sequence conservation and functional importance

• Phylogenetic relationships:

  • Construction of evolutionary trees based on MoaE sequences

  • Identification of insect-specific sequence features

  • Correlation with species divergence patterns

• Selective pressures:

  • Analysis of positive and negative selection on different protein regions

  • Identification of rapidly evolving sites that may indicate functional adaptation

  • Correlation with environmental factors (e.g., molybdenum availability)

• Applications:

  • Prediction of functionally important residues

  • Identification of insect-specific features for targeted intervention

  • Understanding of evolutionary constraints on enzyme function

How does the nucleotide specificity mechanism in molybdopterin dinucleotide formation relate to AGAP004290 function?

While not directly related to the core function of AGAP004290, understanding the downstream processing of molybdopterin provides context:

• Dinucleotide formation process:

  • After molybdopterin formation by molybdopterin synthase (including AGAP004290)

  • Further modification can occur with the addition of nucleotides

  • In bacteria, GTP:molybdopterin guanylyltransferase (MobA) or CTP:molybdopterin cytidylyltransferase (MocA) catalyze the addition of GMP or CMP

• Structural basis for nucleotide specificity:

  • The N-terminal domain determines nucleotide recognition and binding

  • Specific amino acid motifs in the N-terminal domain control GTP vs. CTP specificity

  • Exchange of five key amino acids can alter nucleotide preference

• Evolutionary implications:

  • MobA and MocA share 22% sequence identity despite different nucleotide preferences

  • Paralogue evolution from gene duplication and specialization

  • Potential for similar specialization in other components of the pathway

• Research applications:

  • Engineering altered nucleotide specificity through targeted mutations

  • Understanding how downstream modifications affect molybdenum cofactor function

  • Potential for bioengineering novel cofactor variants

What are common challenges in expressing and purifying active AGAP004290?

Researchers may encounter several technical issues:

• Expression challenges:

  • Insoluble protein formation or inclusion bodies

  • Low expression yield

  • Improper folding in heterologous systems

• Purification difficulties:

  • Co-purification of host proteins

  • Aggregation during concentration

  • Loss of activity during purification steps

• Solutions and optimization strategies:

  • Adjusting expression temperature (typically lower temperatures improve folding)

  • Co-expression with chaperones or partner proteins

  • Testing different fusion tags (His, GST, MBP) for improved solubility

  • Optimizing buffer conditions with stabilizing additives

  • Using mild detergents to prevent aggregation

  • Employing refolding protocols if necessary

How can researchers troubleshoot inactive recombinant AGAP004290 preparations?

When facing activity issues, consider:

• Protein integrity verification:

  • Confirm full-length protein by mass spectrometry

  • Verify correct disulfide bond formation

  • Check for post-translational modifications

• Partner protein requirements:

  • Ensure availability of properly activated MoaD (with thiocarboxylate)

  • Verify complex formation between MoaE and MoaD

  • Test different ratios of subunits

• Assay conditions optimization:

  • Vary buffer composition, pH, and ionic strength

  • Test different metal ion requirements

  • Optimize substrate concentration

  • Ensure anaerobic conditions if required

• Stability considerations:

  • Minimize freeze-thaw cycles

  • Add stabilizing agents (glycerol, reducing agents)

  • Consider freshly prepared enzyme for critical experiments

What analytical methods are most informative for characterizing AGAP004290 interactions with substrates and inhibitors?

Multiple complementary techniques provide comprehensive characterization:

• Binding affinity determination:

  • Isothermal Titration Calorimetry (ITC) for thermodynamic parameters

  • Surface Plasmon Resonance (SPR) for association/dissociation kinetics

  • Microscale Thermophoresis (MST) for interactions in solution

  • Fluorescence-based assays with labeled substrates or inhibitors

• Structural characterization:

  • X-ray crystallography of enzyme-substrate or enzyme-inhibitor complexes

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational changes

  • NMR for dynamic interactions in solution

  • Cryo-EM for larger complexes

• Functional analysis:

  • Enzyme kinetics (Michaelis-Menten parameters)

  • Inhibition kinetics (Ki determination, mode of inhibition)

  • Competition assays with natural substrates

  • Activity assays under varying conditions

How might AGAP004290 be targeted for developing novel mosquito control strategies?

Several promising approaches emerge:

• Inhibitor development strategy:

  • Structure-based design of specific inhibitors targeting the active site

  • Allosteric inhibitors disrupting protein-protein interactions

  • Peptidomimetics based on the MoaD C-terminal region

  • Small molecule screening against the purified enzyme

• Delivery methods:

  • Development of inhibitor-impregnated bed nets

  • Baited traps containing enzyme inhibitors

  • Transgenic approaches affecting AGAP004290 expression

• Resistance management:

  • Target conserved regions resistant to mutation

  • Develop combination approaches targeting multiple pathway steps

  • Monitor for resistance development in field populations

• Validation approach:

  • Laboratory testing on mosquito populations

  • Semi-field trials in controlled environments

  • Assessment of effects on non-target organisms

What fundamental questions remain unanswered about molybdopterin synthase function in Anopheles gambiae?

Critical knowledge gaps include:

• Regulatory mechanisms:

  • How is AGAP004290 expression regulated during different life stages?

  • What controls the balance between MoaE and MoaD production?

  • Are there post-translational modifications affecting activity?

• Structural details:

  • Complete structure of A. gambiae molybdopterin synthase with bound substrate

  • Conformational changes during catalysis

  • Species-specific structural features

• Metabolic integration:

  • Connection between molybdoenzymes and mosquito metabolism

  • Role in response to environmental stressors

  • Potential involvement in insecticide resistance mechanisms

• Population variation:

  • Natural variation in AGAP004290 sequence across mosquito populations

  • Correlation with ecological factors or resistance phenotypes

  • Selective pressures acting on the gene

How might systems biology approaches enhance our understanding of AGAP004290 in the context of mosquito physiology?

Integrative approaches offer comprehensive insights:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Map AGAP004290 function within broader metabolic networks

  • Identify compensatory mechanisms when the enzyme is inhibited

• Computational modeling:

  • Develop metabolic flux models incorporating molybdoenzymes

  • Simulate effects of AGAP004290 inhibition on cellular metabolism

  • Predict system-level consequences of targeted interventions

• Ecological context:

  • Correlate molybdenum availability in different habitats with enzyme activity

  • Model population-level effects of enzyme inhibition

  • Predict evolutionary responses to selective pressure

• Translational applications:

  • Identify synergistic targets for combination approaches

  • Predict potential off-target effects of inhibitors

  • Design intervention strategies with maximal impact and minimal resistance potential