Recombinant Photobacterium profundum N-succinylglutamate 5-semialdehyde dehydrogenase (astD)

What is the biochemical function of N-succinylglutamate 5-semialdehyde dehydrogenase (astD)?

N-succinylglutamate 5-semialdehyde dehydrogenase (astD) catalyzes the oxidation of N-succinyl-L-glutamate 5-semialdehyde to N-succinyl-L-glutamate using NAD+ as a cofactor. The reaction proceeds as follows:

N-succinyl-L-glutamate 5-semialdehyde + NAD+ + H2O → N-succinyl-L-glutamate + NADH + 2 H+

This enzyme belongs to the family of oxidoreductases, specifically those acting on aldehyde or oxo groups with NAD+ or NADP+ as acceptor. The enzyme participates in arginine and proline metabolism pathways, playing a crucial role in nitrogen utilization .

The systematic name for this enzyme is N-succinyl-L-glutamate 5-semialdehyde:NAD+ oxidoreductase. Other common names include succinylglutamic semialdehyde dehydrogenase, N-succinylglutamate 5-semialdehyde dehydrogenase, SGSD, and AruD .

How does the structure of astD compare to related aminotransferases?

While specific structural data for Photobacterium profundum astD isn't directly provided in the available literature, comparison with related aminotransferases reveals important structural features. Aminotransferases like AstC from E. coli possess an αβ-fold protein structure with well-defined active sites that show high degrees of shape complementarity to their substrates .

These enzymes typically form dimeric structures, as observed in ArgD from Salmonella typhimurium (PDB codes 2PB2 and 2PB0). The active site regions are highly conserved across species with only minor variations in specific residues. For instance, the PLP (pyridoxal phosphate) binding site shows remarkable conservation, with variations limited to specific residues (e.g., Val225 sometimes appearing as isoleucine) .

Based on structural analyses of related enzymes, we would expect astD to maintain the core fold common to this enzyme family while potentially exhibiting specialized structural adaptations for functioning under high hydrostatic pressure conditions characteristic of deep-sea environments.

What expression systems are most effective for recombinant Photobacterium profundum astD?

For expressing recombinant enzymes from Photobacterium profundum, including astD, E. coli-based expression systems have proven effective. The optimization process should consider:

• Vector selection: Plasmid vectors with strong, inducible promoters (T7, tac) are typically used.

• Expression conditions: Lower temperatures (16-20°C) during induction often improve the solubility of recombinant proteins from psychrophilic/piezophilic organisms.

• Host strain selection: E. coli strains like BL21(DE3) or Rosetta(DE3) are commonly used for expression of proteins from marine bacteria.

The successful expression of other Photobacterium profundum genes in E. coli, as demonstrated with recD, suggests this approach would be viable for astD as well . When expressing genes from piezophilic (pressure-loving) organisms like P. profundum, special attention should be paid to potential differences in codon usage and protein folding mechanisms that might affect recombinant expression.

How does high hydrostatic pressure affect the structure and function of Photobacterium profundum enzymes like astD?

High hydrostatic pressure significantly impacts enzyme structure and function in deep-sea organisms like Photobacterium profundum. Research with P. profundum SS9 has demonstrated that specific adaptations are required for growth under high-pressure conditions (280-atm) .

For deep-sea enzymes including astD, pressure effects likely include:

• Structural adaptations: Increased flexibility in certain regions balanced with rigidity in others to maintain catalytic activity under pressure.

• Active site modifications: Specialized active site architecture that remains functional under pressure conditions that would typically inhibit enzymatic activity.

• Protein-protein interactions: Adaptations in quaternary structure that prevent pressure-induced dissociation.

Studies with P. profundum recD gene showed its essential role in high-pressure growth, suggesting specialized adaptations in DNA metabolism enzymes. When the P. profundum recD gene was introduced into E. coli recD mutants, it enabled growth under high-pressure conditions and prevented cell filamentation . This indicates that specific adaptations in P. profundum enzymes are transferable and functional in heterologous systems under pressure.

For astD specifically, we would expect similar pressure-adapted features that maintain enzymatic function in the deep-sea environment where P. profundum naturally thrives.

What methodological approaches should be used to determine kinetic parameters of astD under varying pressure conditions?

Determining kinetic parameters of astD under varying pressure conditions requires specialized equipment and methodological considerations:

Equipment requirements:

• High-pressure reaction vessels (capable of at least 280-atm pressure)

• Spectrophotometric capability to measure NADH formation in real-time under pressure

• Temperature control systems for isothermal measurements

Experimental approach:

• Monitor the reduction of NAD+ to NADH (absorbance at 340 nm) as a function of time

• Perform initial velocity measurements at varying substrate concentrations

• Conduct experiments at different pressure points (1, 100, 200, 280-atm)

Data analysis protocol:

• Use nonlinear regression to fit data to Michaelis-Menten equation at each pressure point

• Plot pressure versus kinetic parameters (kcat, KM, kcat/KM)

• Analyze pressure-dependent changes in activation volume

When interpreting results, researchers should consider that pressure effects on enzyme kinetics are typically analyzed through transition state theory and activation volume calculations, which can provide insights into the compressibility of the enzyme-substrate complex.

How can site-directed mutagenesis be used to investigate pressure adaptation mechanisms in astD?

Site-directed mutagenesis represents a powerful approach to investigate the molecular basis of pressure adaptation in astD. Based on comparative studies with other pressure-adapted enzymes, researchers should:

• Identify candidate residues: Focus on charged residues, particularly those in loop regions or at subunit interfaces, as these often contribute to pressure adaptation.

• Design mutagenesis strategy: Create individual point mutations (single amino acid substitutions) and analyze their effects on pressure tolerance.

• Analyze pressure-temperature stability profiles: Compare wild-type and mutant enzymes across a range of pressures and temperatures to identify stability shifts.

• Experimental validation protocol:

  • Express wild-type and mutant proteins

  • Purify using affinity chromatography

  • Measure enzymatic activity at various pressures

  • Determine structural changes using spectroscopic methods under pressure

Lessons from the characterization of P. profundum recD could inform this approach, as it was shown that specific genetic adaptations enabled function under high pressure. The study of recD revealed that even single point mutations (like the one found in EC1002 strain creating a stop codon) can dramatically affect pressure tolerance .

What are the best methods for assessing substrate specificity of recombinant astD?

Assessing substrate specificity of recombinant astD requires a systematic approach combining computational and experimental methods:

Experimental protocol:

• Substrate panel preparation: Prepare a diverse panel of potential substrates, including:

  • N-succinyl-L-glutamate 5-semialdehyde (native substrate)

  • Structurally similar aldehydes (varying acyl chain length)

  • Non-acylated glutamate semialdehyde

• Activity assay optimization:

  • Monitor NAD+ reduction at 340 nm

  • Maintain consistent enzyme concentration across assays

  • Control temperature and buffer conditions rigorously

• Kinetic parameter determination:

  • Determine kcat and KM for each substrate

  • Calculate catalytic efficiency (kcat/KM) to rank substrate preference

Analysis approach:
Create a comparative table of substrate specificities, as demonstrated with related enzymes:

Drawing parallels from studies on AstC from E. coli, we would expect astD to show preference for acylated substrates over non-acylated ones. As observed with AstC, which prefers N-succinylornithine over ornithine, the specificity might be governed by steric and desolvation effects rather than specific interactions between substrate and enzyme .

How should researchers design experiments to investigate the effects of pressure on astD oligomeric state?

Investigating pressure effects on astD oligomeric state requires specialized approaches combining structural and functional analyses:

Experimental design strategy:

• Protein preparation:

  • Express and purify astD to high homogeneity

  • Verify initial oligomeric state using size-exclusion chromatography

• High-pressure analytical techniques:

  • High-pressure size-exclusion chromatography

  • Analytical ultracentrifugation under pressure

  • Small-angle X-ray scattering under variable pressure

• Functional correlation:

  • Measure enzymatic activity in parallel with structural studies

  • Correlate changes in oligomeric state with alterations in catalytic efficiency

• Cross-linking studies:

  • Perform chemical cross-linking at various pressures

  • Analyze products by SDS-PAGE to detect pressure-dependent oligomerization changes

Studies of pressure effects on proteins from Photobacterium profundum show that pressure can significantly impact quaternary structure. For E. coli and other mesophiles, elevated pressure inhibits protein quaternary structure and function, often leading to altered cellular morphology . Deep-sea adapted enzymes like those from P. profundum would be expected to maintain their native oligomeric state at high pressures, and experimental designs should focus on identifying the molecular features that enable this stability.

What strategies can address expression and solubility challenges with recombinant astD?

Researchers working with recombinant astD may encounter expression and solubility challenges due to its origin from a piezophilic (pressure-adapted) organism. The following strategies can help address these issues:

Solubility enhancement strategies:

• Expression optimization:

  • Lower induction temperature (16-20°C)

  • Reduce inducer concentration

  • Extend expression time (24-48 hours)

• Fusion tag selection:

  • Test multiple solubility tags (MBP, SUMO, Trx)

  • Optimize tag position (N-terminal vs C-terminal)

  • Include appropriate linker sequences

• Buffer optimization:

  • Screen various buffer systems (HEPES, Tris, phosphate)

  • Test stabilizing additives (glycerol, arginine, trehalose)

  • Optimize ionic strength and pH

• Co-expression approaches:

  • Co-express with chaperones (GroEL/ES, DnaK/J)

  • Consider co-expression with interacting partners from the same metabolic pathway

How can researchers identify and troubleshoot catalytic discrepancies in astD kinetic studies?

When conducting kinetic studies with astD, researchers may encounter discrepancies in catalytic parameters. A systematic troubleshooting approach includes:

Identification and resolution protocol:

• Enzyme quality assessment:

  • Verify protein purity by SDS-PAGE (>95% homogeneity)

  • Confirm absence of interfering activities

  • Check enzyme stability over the course of assay

• Assay condition verification:

  • Ensure pH stability throughout reaction

  • Control temperature precisely

  • Verify linear range of detection method

• Substrate quality control:

  • Test substrate purity

  • Check for substrate degradation during storage

  • Verify substrate solubility in assay conditions

• Data analysis approach:

  • Compare initial velocity determination methods

  • Apply appropriate regression models

  • Consider allosteric effects if Michaelis-Menten kinetics aren't observed

Referencing studies of related enzymes, such as ArgD and AstC, can provide valuable insights. For these enzymes, researchers observed kcat values of 0.22–0.82 s−1 and 0.61–1.6 s−1 for ornithine and acetylornithine respectively, with KM values ranging significantly (4,500-640 μM for ornithine and 150-37 μM for acetylornithine) . These benchmarks can help identify whether discrepancies in astD studies are within expected ranges for this enzyme family.

How can computational approaches aid in understanding astD adaptation to high pressure?

Computational approaches offer powerful tools for investigating the molecular basis of astD adaptation to high pressure environments:

Recommended computational protocols:

• Molecular dynamics simulations:

  • Perform simulations at varying pressures (1-1000 atm)

  • Analyze protein conformational changes under pressure

  • Calculate volume fluctuations and compressibility

• Homology modeling and structural analysis:

  • Generate structural models based on related enzymes

  • Identify potential pressure-sensitive regions

  • Compare with mesophilic homologs to identify adaptive features

• Quantum mechanics/molecular mechanics (QM/MM) studies:

  • Investigate pressure effects on transition state energetics

  • Calculate activation volumes

  • Model pressure effects on catalytic mechanisms

Computational enzyme design approaches, as described in studies of other enzymes, can be adapted to understand astD pressure adaptation. These methods have successfully guided the redesign of enzymes to improve catalytic efficiency and substrate specificity .

For astD specifically, computational approaches could help identify how this enzyme maintains catalytic function at 280-atm pressure, where most mesophilic enzymes would be inhibited. Molecular dynamics simulations could reveal pressure-induced conformational changes and how the enzyme's structure mitigates these effects.

What insights can comparative studies between astD and homologous enzymes from non-piezophilic organisms provide?

Comparative studies between astD from the piezophilic Photobacterium profundum and homologous enzymes from non-piezophilic organisms can reveal critical adaptations for function under high pressure:

Comparative analysis framework:

• Sequence-based comparisons:

  • Identify conserved catalytic residues

  • Locate piezophile-specific substitutions

  • Calculate amino acid composition differences

• Structural comparisons:

  • Analyze cavity volumes and distributions

  • Compare flexibility of key regions

  • Examine salt bridge and hydrogen bond networks

• Functional comparisons:

  • Measure enzyme kinetics under varying pressures

  • Determine pressure stability profiles

  • Assess temperature-pressure adaptation trade-offs

The available data on AstC and ArgD from E. coli and S. typhimurium provide useful comparison points. These enzymes share 58% identity but show functional interchangeability . Similarly, comparing astD with its mesophilic counterparts could reveal whether functional conservation exists despite adaptation to different pressure regimes.

Such comparative studies with recD from P. profundum revealed that introducing this gene into E. coli recD mutants enabled growth under high pressure and prevented cell filamentation . This suggests that individual proteins from piezophiles can confer pressure-tolerance properties when expressed in mesophilic systems, highlighting the importance of protein-specific adaptations.