Recombinant Arabidopsis thaliana Phosphoacetylglucosamine mutase (DRT101), partial

What is the molecular identity of Arabidopsis thaliana Phosphoacetylglucosamine mutase (DRT101)?

Arabidopsis thaliana Phosphoacetylglucosamine mutase (DRT101) is encoded by the At5g18070 gene and identified by UniProt accession P57750 (AGM1_ARATH). This enzyme, also known as At-PGM3, catalyzes the interconversion of N-acetylglucosamine-6-phosphate to N-acetylglucosamine-1-phosphate, a critical step in hexosamine metabolism . The protein functions primarily as a phosphomutase in carbohydrate metabolism pathways, with potential roles in cell wall biosynthesis and glycoprotein formation in plants. The DRT101 designation connects it to DNA-damage-repair/toleration pathways, suggesting possible functions in stress response mechanisms .

What expression systems are commonly used for producing recombinant DRT101?

Recombinant DRT101 can be produced in multiple expression systems, each offering specific advantages for different research applications:

For studies requiring specific tagging, biotinylated versions using AviTag-BirA technology are available (CSB-EP017869DOA-B), which enable directional coupling to streptavidin surfaces for interaction studies .

What are the optimal methods for assaying phosphoacetylglucosamine mutase activity in plant extracts?

The enzymatic activity of phosphoacetylglucosamine mutase can be assessed using a coupled spectrophotometric assay measuring the conversion of N-acetylglucosamine-6-phosphate to N-acetylglucosamine-1-phosphate. The general protocol involves:

• Plant tissue homogenization in buffer containing protease inhibitors

• Clarification by centrifugation (typically 15,000 × g for 15 minutes at 4°C)

• Assay reaction mixture preparation containing:

  • 50 mM HEPES buffer (pH 7.5)

  • 5 mM MgCl₂

  • 1 mM N-acetylglucosamine-6-phosphate

  • 0.2 mM glucose-1,6-diphosphate (activator)

  • Coupling enzymes for detection

• Initiation of reaction by adding plant extract

• Measurement of activity by monitoring product formation spectrophotometrically

For recombinant enzyme, purification strategies typically employ affinity chromatography using His-tag or biotinylated tag systems, followed by activity assays under controlled temperature and pH conditions to determine optimal enzymatic parameters .

How can CRISPR-Cas9 be employed for genetic modification of the DRT101 gene?

CRISPR-Cas9 approaches for studying DRT101 function involve:

• gRNA Design: Design guide RNAs targeting specific regions of the At5g18070 gene. Effective gRNA pairs should be designed considering the gene structure and potential off-target effects. Target selection should avoid regions with secondary structure that might impede Cas9 binding .

• Construct Assembly: The following components are essential:

  • Cas9 expression cassette with plant-optimized codons

  • sgRNA expression driven by a U6 promoter

  • Selectable marker (typically herbicide resistance)

  • Vector backbone suitable for Agrobacterium-mediated transformation

• Transformation Methodology:

  • Floral dip transformation for Arabidopsis (success rate ~1%)

  • Selection of transformants on appropriate selective media

  • PCR-based screening for identifying CRISPR-induced mutations

  • Confirmation of mutations through sequencing

• Phenotypic Analysis: Compare wild-type and mutant lines for changes in growth parameters, metabolite profiles, and specifically N-acetylglucosamine metabolism .

Successful implementation of CRISPR-Cas9 for DRT101 studies requires careful primer design and construct validation before plant transformation, with an expected success rate of approximately 86% for effective gRNA targeting .

How does DRT101 function integrate with broader carbohydrate metabolism in Arabidopsis?

Phosphoacetylglucosamine mutase (DRT101) operates within a complex metabolic network connecting hexosamine metabolism with glycolysis and cell wall biosynthesis. The enzyme catalyzes a reversible reaction in the UDP-N-acetylglucosamine biosynthetic pathway, which provides essential precursors for:

• Cell wall chitin-like glycans synthesis

• N-glycosylation of proteins

• GPI-anchor biosynthesis

• O-GlcNAc modifications of regulatory proteins

Similar to phosphoglycerate mutase (PGAM) in glycolysis, DRT101 requires a phosphorylated intermediate for activity and contributes to metabolic flexibility. Mutant studies of related mutases in glycolysis have demonstrated significant impacts on both sugar and amino acid profiles. Metabolomic analyses of mutase-deficient lines have revealed:

• Accumulation of substrate metabolites

• Decreased levels of pathway products

• Compensatory changes in parallel metabolic pathways

This suggests that DRT101 may play a similarly important role in balancing nitrogen and carbon metabolism through its function in amino sugar processing, potentially affecting protein glycosylation and cell wall integrity .

What insights can DRT101 research provide for understanding plant stress responses?

Research on DRT101 (DNA-damage-repair/toleration protein 101) offers important perspectives on stress adaptation mechanisms in plants:

• Stress Response Integration: The dual naming of this gene (DRT101/AGM1) suggests functional roles in both DNA repair and metabolic pathways, indicating potential integration of stress response and basic metabolism .

• Metabolic Reprogramming: Under stress conditions, alterations in DRT101 activity may contribute to cell wall remodeling and glycoprotein modifications that enhance stress tolerance. This connects basic carbohydrate metabolism to adaptive responses .

• Organelle Communication: Given the importance of glycosylation in protein targeting and organelle function, DRT101 may influence chloroplast-cytosol-nucleus communication during stress. This relates to findings on other mutases that tether mitochondria to chloroplasts in metabolic complexes .

• RNA Editing Connections: Some research suggests potential interconnections between DRT101 and RNA editing machinery in chloroplasts, though the exact mechanisms remain to be fully characterized .

Researchers investigating stress physiology should consider incorporating DRT101 analysis in their experimental designs, particularly when studying cell wall modifications, protein glycosylation changes, or nucleotide sugar metabolism alterations under stress conditions .

What experimental approaches can resolve contradictions in reported DRT101 localization and function?

Resolving discrepancies in DRT101 localization and function requires multi-faceted experimental approaches:

• Subcellular Localization Verification:

  • Combine fluorescent protein fusions (N- and C-terminal) with organelle markers

  • Validate with biochemical fractionation and immunoblotting

  • Use super-resolution microscopy to detect potential membrane associations

  • Perform protease protection assays to determine membrane topology

• Functional Complementation Analysis:

  • Test whether DRT101 can complement yeast or bacterial phosphoacetylglucosamine mutase mutants

  • Create domain swaps with other mutases to identify critical functional regions

  • Implement site-directed mutagenesis of catalytic residues

  • Assess enzyme activity across varying environmental conditions

• Interactome Mapping:

  • Perform co-immunoprecipitation coupled with mass spectrometry

  • Use split-reporter systems (BiFC, FRET) to confirm protein-protein interactions in vivo

  • Apply proximity labeling techniques (BioID) to identify transient interaction partners

  • Characterize multiprotein complexes through blue-native PAGE

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics data from DRT101 mutants

  • Apply flux analysis using isotopically labeled substrates

  • Develop computational models that incorporate enzyme kinetics and metabolite concentrations

These approaches should be implemented with careful consideration of developmental stage and environmental conditions, as DRT101 function may vary depending on these contexts .

How can researchers differentiate between direct and indirect effects in DRT101 mutant phenotypes?

Differentiating between direct and indirect effects in DRT101 mutant phenotypes requires systematic experimental design:

• Genetic Complementation Strategies:

  • Reintroduce wild-type DRT101 under native promoter

  • Create catalytically inactive versions (point mutations) to separate structural from enzymatic roles

  • Develop tissue-specific and inducible expression systems to temporally control DRT101 function

  • Compare phenotypes with heterologous complementation using E. coli phosphoacetylglucosamine mutase

• Metabolic Bypass Approaches:

  • Supply predicted metabolic products exogenously to rescue specific aspects of mutant phenotypes

  • Create double mutants affecting parallel pathways to identify compensatory mechanisms

  • Use inhibitors of specific pathway steps to mimic or enhance mutant effects

• Time-Course Analyses:

  • Monitor transcriptomic, proteomic, and metabolomic changes at early time points after inducible DRT101 disruption

  • Compare with later timepoints to distinguish primary from secondary effects

  • Implement kinetic modeling of metabolite changes

• Tissue-Specific Phenotyping:

  • Use tissue-specific promoters to drive CRISPR-Cas9 editing of DRT101

  • Compare cell-type specific effects through single-cell transcriptomics

  • Apply laser-capture microdissection coupled with metabolite analysis

These approaches help establish causality rather than correlation in observed phenotypes, particularly important given the potential interconnectedness of hexosamine metabolism with multiple cellular processes .

What are the potential connections between DRT101 and chloroplast function in Arabidopsis?

The potential functional relationships between DRT101 and chloroplast biology represent an emerging area of investigation:

• Metabolic Interaction Nodes:

  • Similar to other glycolytic enzymes that form complexes tethering organelles together, DRT101 may participate in cytosol-chloroplast metabolic channeling of hexose phosphate isomers .

  • The enzyme potentially contributes to UDP-GlcNAc production, which may influence protein glycosylation patterns of chloroplast-targeted proteins .

• RNA Editing Connections:

  • Some research suggests potential roles of DRT101 in RNA processing pathways that affect chloroplast gene expression

  • The appearance in contexts with other chloroplast RNA editing factors (like OTP proteins) suggests possible functional relationships .

• Stress Response Integration:

  • Under stress conditions, changes in carbohydrate metabolism involving DRT101 may affect chloroplast function

  • The DNA-damage-repair/toleration designation suggests potential roles in maintaining chloroplast genome integrity .

• Experimental Approaches to Explore These Connections:

  • Co-localization studies with chloroplast markers

  • Analysis of chloroplast morphology and function in DRT101 mutants

  • Examination of chloroplast protein glycosylation patterns

  • Investigation of potential protein-protein interactions with chloroplast envelope proteins

The interconnection between nucleotide sugar metabolism and chloroplast function remains underexplored, with DRT101 potentially serving as an important link between these processes .

How might biotechnology applications benefit from improved understanding of DRT101 function?

Advanced understanding of DRT101 function could enable several biotechnological applications:

• Engineering Plant Glycosylation Profiles:

  • Modulation of DRT101 expression could alter UDP-GlcNAc pools, affecting protein glycosylation patterns

  • This could be leveraged to modify cell wall properties, potentially improving biofuel production efficiency

  • Targeted changes in glycosylation could enhance plant stress tolerance through optimized protein stability and function

• Metabolic Engineering Applications:

  • Manipulation of hexosamine metabolism through DRT101 could redirect carbon flux toward valuable secondary metabolites

  • Enhanced production of N-acetylglucosamine-derived compounds with pharmaceutical applications

  • DRT101 could serve as a regulatory node for controlling carbon allocation between growth and defense pathways

• Molecular Farming Opportunities:

  • Optimization of glycosylation for recombinant protein production in plant systems

  • Enhanced production of properly glycosylated biopharmaceuticals through chloroplast transformation coupled with DRT101 modulation

  • Production of novel glycoconjugates with improved therapeutic properties

• Experimental Approaches to Develop These Applications:

  • Structure-function analysis to engineer DRT101 variants with altered kinetic properties

  • Multigene engineering combining DRT101 modifications with downstream pathway enzymes

  • Development of inducible expression systems for temporal control of hexosamine metabolism

These applications represent the translation of fundamental research on DRT101 into practical biotechnology solutions, underscoring the importance of basic research on plant metabolic enzymes .