Recombinant Danio rerio Metallophosphoesterase 1 (mppe1)

What is the basic function and structure of Danio rerio MPPE1?

Danio rerio MPPE1 (Metallophosphoesterase 1) belongs to the metallo-dependent phosphatase (MDP) superfamily. Based on structural and functional characterization of similar zebrafish metallophosphatases, MPPE1 likely contains a dinuclear metal center essential for catalytic activity . The enzyme is believed to catalyze the hydrolysis of phosphoester bonds in various substrates.

The catalytic domain likely features conserved motifs characteristic of the metallophosphoesterase family, including metal-coordinating residues (histidine, aspartate, and asparagine) arranged to position substrate and water molecules for nucleophilic attack. Studies of related zebrafish metallophosphatases suggest that MPPE1 may contain structural elements unique to the ADPRibase-Mn-like protein family that delimit the active site entrance .

How does the zebrafish MPPE1 gene compare to its human ortholog?

Zebrafish exhibit approximately 70% gene homology with humans, with 84% of genes associated with human diseases having zebrafish counterparts . For MPPE1 specifically, comparative genomic analysis would likely reveal conserved domains essential for metallophosphoesterase activity across vertebrates.

What expression patterns does MPPE1 show during zebrafish development?

While specific MPPE1 expression data is limited in the provided literature, zebrafish provide an excellent model for developmental expression studies due to their rapid, externally observable development and transparent embryos. Expression analysis techniques like whole-mount in situ hybridization can detect spatial and temporal expression patterns of MPPE1 throughout embryonic development.

Based on studies of related metallophosphoesterases, MPPE1 may show tissue-specific expression patterns, potentially including neural tissues given the role of zebrafish as models for neurological disorders . Researchers can leverage resources like the Zebrafish Genome Collection (ZGC) and the Zebrafish Gene Expression Database to compare expression patterns across developmental stages .

How do metal ion dependencies affect the catalytic activity of recombinant Danio rerio MPPE1?

The catalytic activity of metallophosphoesterases in zebrafish demonstrates complex metal ion dependencies that likely apply to MPPE1 as well. Related enzymes show a primary dependence on manganese (Mn²⁺), with a high-affinity binding site corresponding to the dinuclear metal center .

Analysis of similar zebrafish metallophosphatases reveals a complex response to increasing Mn²⁺ concentrations that differs depending on the substrate:

This complex behavior suggests multiple metal binding sites with differential effects on catalytic activity depending on the substrate . When expressing recombinant MPPE1, researchers should carefully optimize metal ion concentrations in activity assays and consider potential allosteric effects of secondary metal binding sites.

What are the molecular mechanisms of substrate recognition and catalysis by Danio rerio MPPE1?

Based on studies of related zebrafish metallophosphoesterases, MPPE1 likely employs a bidentate water molecule bridging the dinuclear metal center as the nucleophile for catalysis . Site-directed mutagenesis studies of similar enzymes highlight the importance of conserved residues like histidine (e.g., His-97 in ADPRibase-Mn) in substrate orientation and catalysis .

Substrate recognition likely involves structural elements unique to the metallophosphoesterase family that form the active site entrance. Molecular docking studies with potential substrates, combined with mutational analysis of residues lining the active site, would provide insights into the molecular determinants of substrate specificity for MPPE1.

A comprehensive understanding of these mechanisms would require:

• Crystal structure determination of MPPE1 with and without bound substrates

• Molecular dynamics simulations to analyze substrate binding modes

• Enzymatic assays with structure-based mutants to validate catalytic mechanisms

How do phylogenetic variations in MPPE1 structure-function relationships manifest across vertebrate species?

Comparative analysis of MPPE1 across vertebrate species would likely reveal evolutionarily conserved domains essential for core metallophosphoesterase activity, alongside species-specific variations that may reflect adaptation to different physiological contexts.

Studies of related metallophosphoesterases show significant functional differences between zebrafish and mammalian enzymes. For example, the catalytic efficiency toward different substrates varies markedly between rat and zebrafish ADPRibase-Mn, with the zebrafish enzyme showing stricter substrate specificity . Most notably, the zebrafish enzyme shows negligible activity toward cyclic ADP-ribose (cADPR), while the rat enzyme hydrolyzes this substrate efficiently .

These differences correlate with genomic adaptations – zebrafish lack homologs of the human CD38 or BST-1 cADPR synthetases/glycohydrolases , suggesting co-evolution of enzymatic networks involving metallophosphoesterases. For MPPE1 research, this highlights the importance of considering the broader metabolic and signaling context when extrapolating findings from zebrafish to mammalian systems.

What are the optimal expression systems and purification strategies for recombinant Danio rerio MPPE1?

For recombinant expression of Danio rerio MPPE1, researchers should consider:

Expression Systems:

• E. coli-based systems: Typically used for initial expression attempts due to simplicity and high yield. BL21(DE3) strains are recommended for metalloproteins, with optimization of induction conditions to enhance soluble expression.

• Eukaryotic systems: Consider insect cell or yeast expression if E. coli yields inactive protein, as these may provide better post-translational processing.

Purification Strategy:

• Immobilized metal affinity chromatography (IMAC) using His-tag fusion proteins

• Ion exchange chromatography for further purification

• Size exclusion chromatography for final polishing

Metal Reconstitution Considerations:
Since metallophosphoesterases may lose metal ions during purification, a critical step is metal reconstitution with controlled Mn²⁺ concentrations . Based on studies of similar zebrafish enzymes, proper metal incorporation is essential for activity assessment, with potential heteronuclear centers (Fe-Mn) requiring special attention .

Quality Control:

• SDS-PAGE and Western blotting for purity assessment

• Mass spectrometry for identity confirmation

• Metal content analysis by atomic absorption spectroscopy

• Circular dichroism for secondary structure verification

What assay methods are most effective for characterizing MPPE1 enzymatic activity?

Based on studies of related metallophosphoesterases, effective assay methods include:

Colorimetric Phosphate Detection:

• Malachite green assay for inorganic phosphate released during catalysis

• Para-nitrophenyl phosphate (pNPP) assay for general phosphatase screening

Substrate-Specific Methods:

• HPLC-based assays for direct detection of substrate consumption and product formation

• Coupled enzyme assays linking MPPE1 activity to easily measurable secondary reactions

Kinetic Analysis Considerations:

• Optimize metal ion concentrations based on the complex metal dependence observed in related enzymes

• Control temperature and pH carefully, testing multiple buffers for optimal activity

• Include control reactions without enzyme and with heat-inactivated enzyme

• Consider potential allosteric effects when analyzing kinetic data

Representative Experimental Conditions Based on Related Metallophosphoesterases:

• Buffer: 50 mM HEPES-KOH (pH 7.5)

• Temperature: 37°C

• Metal cofactors: 1-10 μM Mn²⁺ (optimal range)

• Substrate concentration range: 0.1-10 × Km

How can gene editing approaches be applied to study MPPE1 function in zebrafish models?

Zebrafish provide excellent opportunities for gene editing approaches to study MPPE1 function:

CRISPR/Cas9 Knockout Strategy:

• Design sgRNAs targeting early exons of the mppe1 gene

• Inject Cas9 protein and sgRNA into one-cell stage embryos

• Screen F0 embryos for mosaic mutations and raise to adulthood

• Outcross potential founders and screen F1 offspring for germline transmission

• Establish stable knockout lines through selective breeding

The efficiency of this approach has been demonstrated for other zebrafish genes involved in neurological disorders, such as cacna1aa and gabra1 .

Phenotypic Characterization:

• Morphological analysis: Assess developmental defects, especially in tissues where MPPE1 is expressed

• Behavioral testing: Utilize established protocols for assessing zebrafish behavior, particularly if neurological phenotypes are expected

• Molecular and biochemical analysis: Examine changes in relevant pathways through transcriptomics and proteomics

Rescue Experiments:
To confirm phenotype specificity, perform rescue experiments by:

• Generating mRNA encoding wild-type MPPE1

• Co-injecting with CRISPR components or injecting into knockout embryos

• Assessing phenotypic rescue as confirmation of specificity

How does MPPE1 function potentially relate to human neurological disorders?

Zebrafish have emerged as valuable models for human neurological disorders due to significant conservation in brain structure and function . While specific roles of MPPE1 in neurological disorders remain to be fully characterized, several lines of evidence suggest potential relevance:

• Conserved gene families: 84% of genes associated with human diseases have zebrafish counterparts , suggesting conservation of MPPE1 function across species.

• Neurological model system: Zebrafish are established models for various neurological conditions, including epilepsy syndromes (Dravet syndrome, pyridoxine-dependent epilepsy) , Parkinson's disease , and anxiety/depression .

• Metallophosphatase activity: Related metallophosphatases in mammals are involved in signaling pathways relevant to neurological function, such as those involving ADP-ribose and calcium regulation .

Future research directions might include:

• Conditional knockout of mppe1 in specific neural cell types

• Examination of electrophysiological properties in mppe1-deficient zebrafish neurons

• Assessment of potential interactions between MPPE1 and other proteins implicated in neurological disorders

• Screening for small molecule modulators of MPPE1 activity with potential therapeutic applications

What are the most promising approaches for resolving contradictions in the current MPPE1 literature?

Although specific contradictions in MPPE1 literature are not explicitly mentioned in the search results, research on related metallophosphoesterases reveals areas where conflicting data might emerge:

Metal Dependency Discrepancies:
Related zebrafish metallophosphatases show complex relationships with metal ions, with potential heteronuclear centers (Fe-Mn) and multiple binding sites affecting activity differently depending on substrate . For MPPE1, researchers should:

• Standardize metal content analysis methods

• Systematically vary metal ion types and concentrations across experimental conditions

• Consider multiple binding sites with potentially distinct effects

Substrate Specificity Variations:
Different research groups might report varying substrate preferences for MPPE1. To resolve such discrepancies:

• Conduct side-by-side comparisons using standardized assay conditions

• Determine full kinetic parameters (kcat, Km) rather than simply reporting activity

• Consider the impact of expression system and purification method on enzyme properties

Structural Interpretations:
Conflicting structural models might emerge from different approaches:

• Compare crystallographic data with solution-based structural studies

• Validate structural predictions through targeted mutagenesis

• Consider the impact of different metal occupancy on structure

How can high-throughput approaches advance our understanding of MPPE1 biology?

High-throughput approaches offer powerful tools for exploring MPPE1 biology comprehensively:

Small Molecule Screening:

• Develop fluorescence-based activity assays amenable to plate reader formats

• Screen compound libraries for inhibitors or activators of MPPE1

• Use chemical biology approaches to identify novel MPPE1 substrates or interaction partners

Large-Scale Genetic Screens:
Zebrafish are particularly amenable to genetic screens, allowing:

• Forward genetic screens to identify modifiers of mppe1 knockout phenotypes

• CRISPR-based screens targeting potential interaction partners

• Enhancer/suppressor screens in sensitized backgrounds

Omics Integration:

• Transcriptomics: RNA-seq analysis of mppe1-deficient zebrafish to identify dysregulated pathways

• Proteomics: Proximity labeling approaches to map the MPPE1 interactome

• Metabolomics: Identification of altered metabolites in mppe1 mutants to infer substrate preferences

Data Integration Framework:
A comprehensive understanding will require integration across multiple data types: