Recombinant Drosophila yakuba Adenylyltransferase and sulfurtransferase MOCS3 (GE18783)

What is the domain organization of MOCS3 in Drosophila yakuba?

Drosophila yakuba MOCS3 (GE18783, UniProt ID: B4NXF7) exhibits a two-domain structure similar to MOCS3 proteins in other organisms. The protein contains an N-terminal domain that resembles the Escherichia coli MoeB protein with adenylyltransferase activity and a C-terminal segment displaying similarities to sulfurtransferase rhodanese . This dual-domain architecture is essential for its bifunctional role in cellular metabolism. The N-terminal domain is responsible for ATP-dependent adenylation reactions, while the C-terminal rhodanese-like domain (MOCS3-RLD) is involved in sulfur transfer reactions. The complete protein features conserved cysteine residues, particularly the catalytic cysteine in the rhodanese-like domain that forms the critical persulfide intermediate during sulfur transfer reactions.

What are the primary biochemical functions of MOCS3 in cellular metabolism?

MOCS3 serves dual critical roles in cellular metabolism:

• Molybdenum cofactor biosynthesis: MOCS3 catalyzes both the adenylation and subsequent generation of a thiocarboxylate group at the C-terminus of MOCS2A, the smaller subunit of molybdopterin (MPT) synthase, during Moco biosynthesis . This modification is essential for MPT synthase to catalyze the conversion of precursor Z to molybdopterin.

• tRNA thiolation: MOCS3 activates the URM1 protein through adenylation and sulfur transfer to form a thiocarboxylate group at its C-terminus . This modified URM1 participates in the thiolation of specific tRNAs, which is crucial for proper translation.

Both pathways involve the transfer of persulfide sulfur, with MOCS3 serving as the common factor between these essential cellular processes. The adenylation activity of the N-terminal domain and the sulfurtransferase activity of the C-terminal domain work in concert to perform these functions.

What are the recommended protocols for recombinant expression and purification of D. yakuba MOCS3?

For optimal recombinant expression and purification of Drosophila yakuba MOCS3:

Expression System:

• E. coli BL21(DE3) cells transformed with a pET-based vector containing the D. yakuba MOCS3 gene

• Include a His6-tag at either N- or C-terminus for purification

• Express at 18-20°C overnight after IPTG induction (0.2-0.5 mM) to improve protein solubility

Purification Protocol:

• Harvest cells and lyse in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, and protease inhibitors

• Purify using Ni-NTA affinity chromatography with an imidazole gradient (20-250 mM)

• Further purify by size exclusion chromatography using a Superdex 200 column

• Verify protein purity by SDS-PAGE and identity by ESI-MS

Important Considerations:

• Be aware of potential N-terminal modifications such as gluconoylation that may occur during E. coli expression, which creates protein heterogeneity but doesn't affect sulfurtransferase activity

• For functional studies of the separate domains, express them individually, as demonstrated with successful expression of the isolated MOCS3-RLD

How can the adenylyltransferase and sulfurtransferase activities of MOCS3 be measured in vitro?

Adenylyltransferase Activity Assay:

• Incubate purified MOCS3 with its substrate protein (MOCS2A or URM1), ATP, and MgCl2

• Monitor ATP consumption using coupled enzyme assays (pyruvate kinase/lactate dehydrogenase system)

• Detect the adenylated intermediate by:

  • SDS-PAGE mobility shift analysis

  • Mass spectrometry to observe the mass increment of adenylation (+329 Da)

Sulfurtransferase Activity Assays:

• Rhodanese Activity Assay (Spectrophotometric):

  • Measure the formation of thiocyanate from cyanide and thiosulfate

  • Detect thiocyanate formation by reaction with Fe3+ to form Fe(SCN)3 (red complex)

  • Monitor absorbance at 460 nm

• Persulfide Formation Assay:

  • Treat MOCS3-RLD with thiosulfate as sulfur donor

  • Identify persulfide formation on the catalytic cysteine residue using ESI-MS/MS

  • Observe mass increment of +32 Da corresponding to persulfide addition

• Functional MPT Synthesis Assay:

  • Reconstitute the complete MPT synthesis pathway in vitro using:

    • Precursor Z (substrate)

    • MOCS2A and MOCS2B (MPT synthase components)

    • MOCS3 with ATP and MgCl2

  • Detect MPT formation by conversion to Form A and fluorescence detection

  • Quantify using HPLC analysis with fluorescence detection

What spectroscopic and analytical techniques are most effective for characterizing MOCS3 structure and function?

Spectroscopic Techniques:

• UV-Visible Spectroscopy:

  • Monitor protein concentration and purity

  • Detect potential cofactors or prosthetic groups

  • Follow enzymatic reactions that involve chromogenic products

• Circular Dichroism (CD):

  • Assess secondary structure composition

  • Monitor protein folding and stability

  • Evaluate structural changes upon substrate binding

• Fluorescence Spectroscopy:

  • Study protein-protein interactions using FRET with fluorescently labeled MOCS3 and its interaction partners

  • As demonstrated with ECFP and EYFP fusions to study MOCS3 interactions with MOCS2A and URM1

Mass Spectrometry Techniques:

• ESI-MS/MS:

  • Identify post-translational modifications including persulfide formation

  • Confirm protein identity and integrity

  • Detect and characterize reaction intermediates

  • ESI-MS has successfully identified persulfide formation on the C412 residue in MOCS3-RLD

• Native MS:

  • Analyze protein-protein complexes

  • Study conformational states

Structural Techniques:

• X-ray Crystallography:

  • Determine high-resolution 3D structure

  • Identify active site residues and binding pockets

• NMR Spectroscopy:

  • Study protein dynamics

  • Map protein-protein interaction interfaces

  • Investigate structural changes during catalysis

Activity-Based Techniques:

• Site-Directed Mutagenesis:

  • Evaluate the role of specific residues in catalysis

  • Create variants to study mechanistic details

  • Create C412S mutants to confirm the role of this residue in persulfide formation and sulfur transfer

Which residues are critical for the sulfurtransferase activity of MOCS3?

The sulfurtransferase activity of MOCS3 is dependent on specific conserved residues:

Critical Catalytic Residue:

• Cysteine 412 (C412) in the six-amino acid active loop of the rhodanese-like domain is the principal catalytic residue that forms the persulfide intermediate essential for sulfur transfer

• Mutation of C412 completely abolishes the sulfurtransferase activity, demonstrating its critical role in catalysis

Structural Elements:

• The active site loop contains the conserved CXXGXR motif found in rhodanese-like proteins

• While MOCS3-RLD contains four cysteine residues, only C412 in the active loop is conserved across homologous proteins from different organisms

Non-catalytic Cysteines:

• C316 and C324 form a disulfide bridge that likely maintains structural integrity

• The remaining non-catalytic cysteines are not directly involved in the sulfur transfer reaction in vitro, as demonstrated by simultaneous mutagenesis studies

This table summarizes the roles of cysteine residues in MOCS3-RLD:

How does the mechanism of sulfur transfer by MOCS3 compare to other sulfurtransferases?

MOCS3 employs a mechanism similar to other sulfurtransferases but with distinct features:

Comparison with Other Sulfurtransferases:

• Mechanism Similarity to TtuA:

  • MOCS3 likely coordinates sulfide at a non-ligated metal center before transfer, similar to TtuA in 2-thiouridine synthesis

  • Both involve transfer of non-core sulfide to activated substrates

• Comparison with LarE Sulfur Insertases:

  • Some LarE homologs (like LarE from L. plantarum) function as sacrificial sulfur transferases using a cysteine residue that becomes dehydroalanine

  • Other LarE homologs (like LarE from T. maritima) use [4Fe-4S] clusters to coordinate and transfer sulfur

  • MOCS3 doesn't appear to use a [4Fe-4S] cluster but forms a persulfide intermediate on C412

• Similarity to Mitochondrial Rhodaneses:

  • MOCS3-RLD catalyzes thiosulfate:cyanide sulfurtransferase activity like classical rhodaneses

  • Unlike most mammalian rhodaneses that localize to mitochondria, MOCS3 is found in the cytosol

Sulfur Transfer Mechanism Table:

What is the specific role of MOCS3 in molybdenum cofactor biosynthesis in D. yakuba?

In Drosophila yakuba, as in other eukaryotes, MOCS3 plays a crucial role in the molybdenum cofactor (Moco) biosynthesis pathway:

MOCS3's Role in the Moco Biosynthesis Pathway:

• Activation of MOCS2A:

  • MOCS3 uses its N-terminal adenylation domain to catalyze ATP-dependent adenylation of the C-terminal carboxyl group of MOCS2A

  • This creates an acyl-adenylate intermediate at the C-terminus of MOCS2A

• Sulfur Transfer to MOCS2A:

  • The C-terminal rhodanese-like domain (MOCS3-RLD) transfers sulfur from a persulfide formed on C412 to the activated MOCS2A

  • This results in the formation of a thiocarboxylate group (-COSH) at the C-terminus of MOCS2A

• MPT Synthase Function:

  • The thiocarboxylated MOCS2A associates with MOCS2B to form active MPT synthase

  • This complex converts precursor Z to molybdopterin (MPT) by transferring two sulfur atoms to form the dithiolene group

Significance in D. yakuba Metabolism:

• Moco is essential for the activity of molybdoenzymes including sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase

• These enzymes are critical for various metabolic processes including purine catabolism and detoxification pathways

• D. yakuba MOCS3 (GE18783) functions in the cytosol, where the Moco biosynthesis pathway occurs

How does MOCS3 participate in tRNA thiolation and what is its significance?

MOCS3 is a key player in the tRNA thiolation pathway, which is critical for translation fidelity:

MOCS3's Role in tRNA Thiolation:

• Activation of URM1:

  • MOCS3 uses its N-terminal domain to adenylate the C-terminus of URM1, a ubiquitin-related modifier protein

  • Similar to its action on MOCS2A, this creates an acyl-adenylate intermediate

• Sulfur Transfer to URM1:

  • MOCS3 transfers sulfur via its rhodanese-like domain to form a thiocarboxylate group at the C-terminus of URM1

  • The activated URM1-COSH serves as the sulfur donor for tRNA modification

• tRNA Modification:

  • The thiocarboxylated URM1 participates in the thiolation of specific uridines in the wobble position of certain tRNAs

  • This results in the formation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at the wobble position (U34) of tRNAs for Lys, Glu, and Gln

Biological Significance:

• The thiolation of wobble uridines enhances codon-anticodon interactions

• This modification is crucial for translation efficiency and accuracy

• Defects in this pathway can lead to translation defects and proteotoxic stress

Interaction Requirements:

• The C-terminal double glycine motif of URM1 is essential for interaction with MOCS3

• Deletion of the C-terminal glycine of URM1 results in loss of interaction with MOCS3

• FRET studies have shown that these interactions occur primarily in the cytosol, though extension of the C-terminus of URM1 with an additional glycine alters the localization of MOCS3 from the cytosol to the nucleus

What structural features enable MOCS3 to interact with both MOCS2A and URM1?

The dual functionality of MOCS3 in interacting with both MOCS2A and URM1 is facilitated by several key structural features:

Structural Elements for Substrate Recognition:

• N-Terminal Adenylation Domain:

  • Contains the ATP-binding site for adenylation of both MOCS2A and URM1

  • Recognizes the C-terminal carboxyl groups of both substrate proteins

• Substrate Recognition Features:

  • Both MOCS2A and URM1 share a β-grasp fold structure

  • Both contain a highly conserved C-terminal double glycine motif that is critical for interaction with MOCS3

  • Deletion of the C-terminal glycine from either MOCS2A or URM1 results in loss of interaction with MOCS3

Interaction Specificity Determinants:

The following table summarizes the common features and specific requirements for MOCS3 interactions:

Experimental Evidence:

• FRET studies using enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) fusions have confirmed the interactions of MOCS3 with both MOCS2A and URM1 in living cells

• Fluorescence resonance energy transfer efficiency was determined by measuring the decrease in donor lifetime, providing quantitative evidence of these interactions

What are the current methodological challenges in studying D. yakuba MOCS3 function in vivo?

Researchers investigating D. yakuba MOCS3 function face several methodological challenges:

Technical Challenges:

• Protein Expression and Purification:

  • Ensuring proper folding of the dual-domain structure

  • Maintaining activity of both adenylyltransferase and sulfurtransferase domains

  • Preventing oxidation of the catalytic cysteine residue during purification

  • Addressing heterogeneity caused by post-translational modifications like N-terminal gluconoylation

• Activity Assays:

  • Developing sensitive assays to measure both adenylyltransferase and sulfurtransferase activities

  • Distinguishing between activities of the two domains

  • Ensuring physiological relevance of in vitro assays

• In Vivo Studies:

  • Limited genetic tools for D. yakuba compared to D. melanogaster

  • Challenges in creating tissue-specific or conditional knockouts

  • Difficulty in distinguishing phenotypes caused by disruption of Moco biosynthesis versus tRNA thiolation

Experimental Approaches to Address Challenges:

• CRISPR/Cas9 System Optimization:

  • Adapt CRISPR/Cas9 protocols developed for D. melanogaster to D. yakuba

  • Design guide RNAs specific to D. yakuba MOCS3 sequence

  • Create domain-specific mutations to separate functions

• Cellular Systems:

  • Develop D. yakuba cell culture systems for in vivo studies

  • Use fluorescently tagged proteins to study localization and interactions

  • Employ substrate-specific assays to differentiate between pathways

• Cross-Species Complementation:

  • Test functionality by expressing D. yakuba MOCS3 in other model organisms with MOCS3 mutations

  • Compare activity with human MOCS3 in rescue experiments

How do mutations in key residues affect the dual functionality of MOCS3?

The effects of mutations on MOCS3 dual functionality provide insights into structure-function relationships:

Critical Residues and Mutation Effects:

• Adenylation Domain Mutations:

  • Mutations in the PP-loop motif (similar to the SGGXDS motif found in related enzymes ) disrupt ATP binding and adenylation activity

  • Affects both MOCS2A and URM1 activation pathways

  • Results in loss of both Moco biosynthesis and tRNA thiolation functions

• Rhodanese Domain Mutations:

  • C412S mutation: Completely abolishes sulfurtransferase activity by preventing persulfide formation

  • C316A/C324A double mutation: Disrupts the disulfide bridge, potentially affecting protein stability but not directly impacting catalysis

  • Active site loop mutations: Alter the microenvironment of C412, affecting persulfide formation and transfer efficiency

• Interface Residues Mutations:

  • Mutations at the domain interface may impact communication between adenylation and rhodanese domains

  • Can cause domain-specific or global conformational changes affecting one or both functions

Mutation Impact Table:

What is the evolutionary conservation pattern of MOCS3 across Drosophila species and beyond?

Evolutionary analysis of MOCS3 reveals important patterns of conservation:

Conservation Across Drosophila Species:

MOCS3 shows significant conservation across Drosophila species, reflecting its essential function:

• D. yakuba MOCS3 (GE18783) shows high sequence similarity to homologs in related species including D. melanogaster, D. grimshawi (GH10959), D. persimilis (GL26133), and D. willistoni (GK18675)

• The gene is consistently annotated as Uba4 across Drosophila species

• The dual-domain architecture is preserved throughout the genus

Broader Evolutionary Context:

• Conservation Across Taxa:

  • MOCS3 (UBA4) homologs are found across diverse eukaryotes from yeast to humans

  • The protein is present in vertebrates including mouse (Mocs3), pig (MOCS3), and zebrafish (mocs3 uba4 zgc:55696)

  • Even plant species contain MOCS3 homologs, as seen in rice (Oryza sativa) where it's annotated as MOCS3 CNX5 UBA4

• Domain-Specific Conservation:

  • The N-terminal adenylation domain shows homology to bacterial MoeB/ThiF proteins

  • The rhodanese-like domain shows greater sequence divergence except for the active site loop containing the catalytic cysteine

  • The catalytic cysteine (C412 in human MOCS3) is nearly universally conserved

• Functional Conservation vs. Adaptation:

  • The dual functions in Moco biosynthesis and tRNA thiolation appear conserved across eukaryotes

  • Species-specific adaptations may exist in substrate recognition regions

  • In bacteria, these functions are typically performed by separate proteins (e.g., MoeB in Bacillus subtilis)

This evolutionary conservation underscores the fundamental importance of MOCS3's dual roles in cellular metabolism across diverse species, from insects to mammals and plants.

What are the most promising areas for future research on D. yakuba MOCS3?

Several research directions hold particular promise for advancing our understanding of D. yakuba MOCS3:

• Structural Biology: Obtaining crystal structures of D. yakuba MOCS3, both full-length and individual domains, in complex with substrates MOCS2A and URM1.

• In vivo Regulation: Investigating how MOCS3 activity is regulated between its dual pathways and whether there are mechanisms for prioritizing one pathway over the other under specific cellular conditions.

• Comparative Functional Studies: Systematically comparing the biochemical properties of MOCS3 across Drosophila species to identify adaptive changes and their functional significance.

• Development of Specific Inhibitors: Creating chemical tools to selectively inhibit either the adenylyltransferase or sulfurtransferase activity to dissect the relative contributions of each pathway.

• Physiological Consequences: Exploring the effects of MOCS3 dysfunction on Drosophila development, lifespan, and stress responses, particularly focusing on phenotypes that might distinguish between Moco deficiency and defects in tRNA modification.