Recombinant Panicum miliaceum Alanine aminotransferase 2

What is Panicum miliaceum Alanine aminotransferase 2 and what reaction does it catalyze?

AlaAT2 from Panicum miliaceum (proso millet) is an enzyme that catalyzes the reversible transamination reaction between alanine and 2-oxoglutarate to form pyruvate and glutamate following the Ping Pong Bi-Bi mechanism . The enzyme plays key roles in carbon and nitrogen metabolism in plants, particularly during stress responses. The reaction can be summarized as:

L-alanine + 2-oxoglutarate ⇌ pyruvate + L-glutamate

To study this reaction, researchers typically employ spectrophotometric assays coupled with lactate dehydrogenase to monitor NADH oxidation, or direct product quantification through chromatographic methods.

What is the molecular structure and sequence characteristics of P. miliaceum AlaAT2?

P. miliaceum AlaAT2 (UniProt: P34106) is a 482 amino acid protein that likely functions as a homodimer based on structural models available in the SWISS-MODEL Repository . The complete amino acid sequence has been determined and is available in protein databases . The enzyme contains a PLP (pyridoxal phosphate) binding site characteristic of aminotransferases.

Based on structural analysis, two SWISS-MODEL templates (3tcm.1.A and 2egy.1.A) have been used to model P. miliaceum AlaAT2 structure, with QMEAN scores of 0.90 and 0.58 respectively, and the higher-quality model suggesting a homo-2-mer quaternary structure .

How does AlaAT2 relate to other aminotransferase enzymes in plants?

Plant AlaATs can be classified into different subgroups with distinct functions. For example, in Populus, AlaAT3 and AlaAT4 (subgroup A) encode true alanine aminotransferases, while AlaAT1 and AlaAT2 (subgroup B) encode glutamate:glyoxylate aminotransferases (GGAT) .

P. miliaceum AlaAT2 shares an identical sequence with AlaAT from Panicum hallii (UniProt: A0A2S3IMC6) . When studying AlaAT across species, it's important to consider evolutionary relationships and potential functional divergence. Previous research has shown that cytosolic and mitochondrial isoforms of AspAT from Panicum miliaceum can induce the expression of endogenous Pepcase when expressed in transgenic tobacco plants .

What expression systems are most effective for producing functional recombinant P. miliaceum AlaAT2?

Based on commercial production methods, recombinant P. miliaceum AlaAT2 has been successfully expressed in a baculovirus expression system . This eukaryotic system offers advantages for plant proteins by providing appropriate post-translational modifications and folding machinery.

For laboratory-scale expression, several methods could be considered:

• Bacterial expression (E. coli) with codon optimization

• Yeast expression systems (S. cerevisiae or P. pastoris)

• Plant-based transient expression

Each system presents tradeoffs between yield, functionality, and ease of purification. When comparing expression systems, researchers should systematically evaluate:

• Protein yield

• Enzymatic activity

• Proper folding and oligomerization

• Presence of appropriate cofactors (PLP)

What purification strategies yield the highest activity for recombinant AlaAT2?

While specific purification protocols for P. miliaceum AlaAT2 are not detailed in the search results, purification of AlaAT from other organisms like Pyrococcus furiosus has been achieved through multistep chromatography .

A recommended purification workflow would include:

• Initial capture by affinity chromatography (if using tagged constructs)

• Intermediate purification by ion exchange chromatography

• Polishing by size exclusion chromatography to confirm dimeric state

Critical considerations during purification include:

• Maintaining PLP cofactor in buffers

• Optimizing pH and ionic strength based on protein properties

• Including reducing agents to prevent oxidation of critical cysteine residues

• Monitoring activity throughout purification steps

Commercial preparations typically achieve >85% purity as assessed by SDS-PAGE .

How can substrate specificity of P. miliaceum AlaAT2 be comprehensively characterized?

A thorough substrate specificity analysis would include:

• Testing various amino acid donors:

  • Alanine (primary substrate)

  • Other amino acids (glutamate, aspartate, etc.)

• Testing various keto acid acceptors:

  • 2-oxoglutarate

  • Pyruvate

  • Other keto acids

• Determining kinetic parameters:

  Parameter	Alanine + 2-oxoglutarate	Glutamate + Pyruvate
  Km	To be determined	To be determined
  kcat	To be determined	To be determined
  kcat/Km	To be determined	To be determined

Similar studies with AlaAT from other organisms have shown that the kcat/Km values for alanine and pyruvate formation can be similar (41 and 33 s⁻¹ mM⁻¹ respectively for P. furiosus AlaAT), suggesting the enzyme may not be biased toward either direction .

What role does AlaAT2 play in plant responses to hypoxic stress?

AlaAT plays a crucial role in plant responses to hypoxia:

• During hypoxia, plants accumulate alanine as a nitrogen-efficient storage compound

• Upon return to normoxic conditions, AlaAT is critical for breaking down accumulated alanine

• Studies in Arabidopsis have shown that AlaAT1 knockout mutants (alaat1-1) accumulate more alanine during early hypoxia and show delayed alanine breakdown during recovery

To investigate this role in P. miliaceum, researchers should:

• Monitor alanine levels under hypoxic conditions and during recovery

• Track AlaAT2 expression and activity throughout stress and recovery phases

• Perform comparative analyses with knockout/knockdown mutants if available

• Consider metabolic flux analysis using isotopically labeled substrates

How does AlaAT2 contribute to nitrogen use efficiency in plants?

• It facilitates nitrogen redistribution through reversible amino group transfer

• Expression studies in Populus have shown that some AlaAT genes are induced by exogenous nitrogen and exhibit diurnal fluctuation patterns

• PnAlaAT3 gene expression could be regulated by glutamine or its related metabolites in roots

To investigate nitrogen use efficiency aspects:

What approaches can effectively assess the tissue-specific expression of AlaAT2?

Based on studies of AlaAT genes in other plants, several complementary approaches are recommended:

• Promoter-reporter fusion analysis:

  • Generate transgenic plants with AlaAT2 promoter:GUS constructs

  • Perform histochemical staining under various conditions

  • Studies in Arabidopsis showed that both AlaAT genes are expressed predominantly in vascular tissues

• Transcript analysis:

  • qRT-PCR from different tissues

  • RNA-seq for genome-wide expression context

  • Northern blot analysis (has been used to identify 1.2-kb transcripts for aat gene in P. furiosus)

• Protein localization:

  • Immunohistochemistry with specific antibodies

  • Fluorescent protein fusions to track subcellular localization

These approaches should be performed under different environmental conditions and developmental stages for comprehensive understanding.

How is AlaAT2 expression regulated in response to environmental stressors?

Several regulatory patterns have been observed for AlaAT genes in plants:

• Nitrogen availability:

  • AlaAT genes in Populus (PnAlaAT1, PnAlaAT2, PnAlaAT3, PnAlaAT4) are induced by exogenous nitrogen

  • PnAlaAT3 expression in roots can be regulated by glutamine or its related metabolites

• Hypoxic stress:

  • Arabidopsis AlaAT1 and AlaAT2 are induced during hypoxia at the transcriptional level

  • Increased enzyme activity has been detected in hypoxically treated roots

• Diurnal regulation:

  • Some AlaAT genes (PnAlaAT1, PnAlaAT2) exhibit diurnal fluctuation in leaves

To investigate regulatory mechanisms in P. miliaceum AlaAT2:

• Perform promoter analysis to identify key regulatory elements

• Test stress-responsive expression under controlled conditions

• Consider chromatin immunoprecipitation to identify transcription factors

• Examine potential post-translational modifications

How can CRISPR-based genome editing be applied to study AlaAT2 function in Panicum miliaceum?

Recent advances in genome editing offer powerful approaches to study AlaAT2 function:

• Gene knockout strategies:

  • Complete gene disruption to create loss-of-function mutants

  • Analysis of metabolic and physiological consequences

• Base editing approaches:

  • Introduction of specific amino acid changes to alter catalytic properties

  • Modification of regulatory sites without disrupting protein expression

• Enhancer insertion:

  • Modifying expression patterns through regulatory element engineering

  • Stress-specific expression enhancement

• Rapid stabilization through speed breeding:

  • Accelerating the development of homozygous edited lines

These approaches would help establish causal relationships between AlaAT2 function and specific plant phenotypes, particularly related to stress tolerance.

What methodologies can effectively measure the impact of AlaAT2 modifications on metabolic flux?

• Isotope labeling experiments:

  • 13C labeling to track carbon flux

  • 15N labeling to monitor nitrogen distribution

  • Combined approaches to understand C/N interactions

• Metabolomics approaches:

  • Targeted analysis of amino acid pools

  • Untargeted approaches to identify unexpected metabolic shifts

  • Time-course analyses during stress and recovery phases

• Multi-omics integration:

  • Correlating transcript, protein, and metabolite changes

  • Network analysis to identify key regulatory nodes

  • Mathematical modeling to predict metabolic outcomes

These approaches would help place AlaAT2 function in the broader context of plant metabolism under both normal and stress conditions.

What are the optimal conditions for maintaining recombinant AlaAT2 activity during storage?

According to product information for recombinant P. miliaceum AlaAT2:

• Temperature recommendations:

  • Store at -20°C for standard storage

  • Use -20°C to -80°C for extended storage

  • Liquid preparations have a shelf life of approximately 6 months at these temperatures

  • Lyophilized forms maintain stability for up to 12 months

• Buffer and additive considerations:

  • Adding 5-50% glycerol (recommended final concentration: 50%) helps prevent freezing damage

  • Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL

  • Aliquot to avoid repeated freeze-thaw cycles

• Activity preservation:

  • Consider including PLP cofactor in storage buffers

  • Test activity periodically during long-term storage

  • Optimize protein concentration for maximum stability

How can understanding AlaAT2 function contribute to developing climate-resilient crops?

Recent research highlights AlaAT's importance in crop resilience to climate change :

• Stress response roles:

  • Hypoxia tolerance during flooding events

  • Drought resistance mechanisms

  • Heat and salinity stress adaptation

• Nitrogen efficiency improvements:

  • Enhanced nitrogen uptake and utilization

  • Better nitrogen remobilization during senescence

  • Reduced fertilizer requirements

• Genetic engineering opportunities:

  • Targeted modifications using genome editing approaches

  • Expression optimization in specific tissues or under specific conditions

  • Integration with other stress tolerance mechanisms

These applications are particularly relevant for crops like millet that are already adapted to marginal environments but may face increasing climate challenges.

What unknown aspects of AlaAT2 function warrant further investigation?

Several knowledge gaps remain in our understanding of AlaAT2 function:

• Regulatory networks:

  • Transcription factors controlling expression

  • Post-translational modifications affecting activity

  • Protein-protein interactions and metabolic channeling

• Evolutionary adaptations:

  • Functional differences between C3 and C4 plant AlaATs

  • Selection pressures driving AlaAT diversification

  • Specialized roles in different plant lineages

• Physiological integration:

  • Coordination with other stress response pathways

  • Cross-talk between carbon and nitrogen signaling networks

  • Temporal dynamics during stress and recovery phases

Addressing these questions will require interdisciplinary approaches combining molecular biology, biochemistry, genetics, and systems biology.