Recombinant Sphyraena idiastes L-lactate dehydrogenase A chain (ldha)

What is Sphyraena idiastes L-lactate dehydrogenase A chain and what is its significance in evolutionary studies?

Sphyraena idiastes L-lactate dehydrogenase A chain (ldha) is the A subunit of lactate dehydrogenase isolated from Sphyraena idiastes, a temperate barracuda species found in the Pacific Ocean. This enzyme plays a central role in the Cori cycle, catalyzing the interconversion of pyruvate and lactate during anaerobic metabolism. The significance of this particular LDH lies in its evolutionary adaptations to environmental temperature. S. idiastes inhabits waters with an average temperature of 18°C, and its LDH has evolved specific kinetic properties optimal for this temperature range. Studying this enzyme provides insights into how relatively small genetic changes can produce significant functional adaptations to specific thermal environments .

The recombinant form allows researchers to study the precise structure-function relationships that underlie temperature adaptation in enzymes, making it an excellent model system for evolutionary biochemistry research. Comparative studies with LDH from other Sphyraena species that evolved in different thermal environments reveal how natural selection shapes enzyme kinetics in response to temperature challenges .

How does S. idiastes LDH differ from other Sphyraena species in terms of temperature adaptation?

S. idiastes LDH exhibits distinct temperature-dependent kinetic properties when compared to LDH from other Sphyraena species. These species inhabit different thermal environments along the Pacific coast of the Americas: S. argentea (18°C, temperate), S. lucasana (23°C, tropical), S. ensis (26°C, tropical), and S. idiastes (18°C, temperate) .

Electrophoretic studies have shown that the two temperate species have identical electrophoretic and kinetic properties, suggesting they may have identical M chains. In contrast, the tropical species show distinct electrophoretic mobility, indicating specific amino acid substitutions that alter charge while maintaining enzymatic function .

What are the standard methods for cloning and expressing recombinant S. idiastes ldha?

The standard methodology for cloning and expressing recombinant S. idiastes ldha involves several key steps:

• RNA Isolation and cDNA Synthesis: Total RNA is extracted from S. idiastes tissue, and reverse transcription is performed to generate cDNA .

• Gene Amplification: The ldha gene is amplified using PCR with primers designed based on conserved regions from related species. For iguanid species, researchers have successfully used primers designed by homology with ldha from Sceloporus undulatus and Dipsosaurus dorsalis .

• RACE Amplification: Rapid Amplification of cDNA Ends (RACE) may be employed to obtain the complete sequence, particularly for the 5' and 3' ends of the gene .

• Vector Construction: The full-length ldha cDNA is inserted into an expression vector (such as pTrcHis) using appropriate restriction sites. For example, primers can be designed to insert BamHI sites at the 5' end and EcoRI sites at the 3' end of the gene .

• Transformation: The recombinant plasmid is transformed into competent bacterial cells (such as E. coli TOP10) .

• Protein Expression: Transformed bacteria are cultured and protein expression is induced with IPTG (typically at 1 mmol L-1 concentration) .

• Protein Purification: The recombinant protein is purified using affinity chromatography, often facilitated by a poly-histidine tag incorporated into the expression construct. Purification typically involves:

  • Cell lysis with reagents like CellLytic B

  • Filtration of the lysate

  • Affinity chromatography using cobalt or nickel columns

  • Elution with imidazole

  • Desalting into an appropriate storage buffer

How is the kinetic activity of recombinant S. idiastes LDH typically measured?

Measuring the kinetic activity of recombinant S. idiastes LDH involves spectrophotometric assays that monitor the oxidation of NADH during the conversion of pyruvate to lactate. The standard methodology includes:

• Enzyme Preparation: Purified recombinant LDH is diluted to an appropriate concentration in a suitable buffer, typically potassium phosphate (pH 6.8-7.0) .

• Reaction Setup: A typical reaction mixture contains:

  • Buffer (often 50 mmol L-1 potassium phosphate)

  • Pyruvate (substrate, at various concentrations)

  • NADH (cofactor)

  • Purified enzyme

• Temperature Control: Assays are conducted at various temperatures to determine temperature-dependent kinetics, with particular attention to the physiologically relevant temperature for S. idiastes (18°C) .

• Data Collection: The decrease in absorbance at 340 nm (corresponding to NADH oxidation) is monitored over time using a spectrophotometer.

• Calculation of Kinetic Parameters:

  • Initial velocities are determined at different substrate concentrations

  • Michaelis-Menten or Lineweaver-Burk plots are constructed

  • Km (Michaelis constant) and Vmax (maximum velocity) are calculated

  • Catalytic rate constants (kcat) are determined using the equation: kcat = Vmax/[E], where [E] is enzyme concentration

  • Catalytic efficiency is assessed using the kcat/Km ratio

By performing these measurements across a range of temperatures, researchers can construct temperature profiles for the enzyme's activity and determine the thermal optimum and temperature dependence of various kinetic parameters.

What molecular mechanisms underlie the temperature adaptation of S. idiastes LDH compared to tropical Sphyraena species?

The temperature adaptation of S. idiastes LDH involves several molecular mechanisms that affect both catalytic efficiency and thermal stability. Research on LDH across various ectothermic vertebrates has revealed several key principles that likely apply to S. idiastes:

These adaptations in S. idiastes LDH demonstrate the remarkable precision with which natural selection can fine-tune enzyme function in response to environmental temperature, even with very small genetic changes.

How can site-directed mutagenesis be used to investigate the structure-function relationship in S. idiastes LDH?

Site-directed mutagenesis offers a powerful approach to investigate the specific amino acid residues responsible for the temperature-adaptive properties of S. idiastes LDH. A comprehensive research strategy would include:

• Identification of Target Residues: By comparing the sequences of LDH from S. idiastes (temperate, 18°C) with tropical Sphyraena species (S. lucasana, 23°C; S. ensis, 26°C), researchers can identify candidate residues that may contribute to temperature adaptation. Prime targets would be residues that differ between temperate and tropical species, particularly those in or near the active site, at subunit interfaces, or in regions affecting conformational flexibility.

• Construction of Mutants: Once target residues are identified, site-directed mutagenesis can be performed on the cloned S. idiastes ldha gene. This typically involves:

  • Designing primers that incorporate the desired mutation

  • PCR-based mutagenesis using these primers

  • Transformation of the mutated construct into expression hosts

  • Verification of mutations by DNA sequencing

  • Expression and purification of mutant proteins

• Functional Characterization: The purified mutant proteins should be subjected to comprehensive kinetic analysis, including:

  • Determination of Km and kcat values at different temperatures

  • Construction of temperature profiles for catalytic efficiency (kcat/Km)

  • Assessment of thermal stability through activity retention after heat exposure

  • Measurement of activation energies for the catalyzed reaction

• Structural Analysis: Where possible, structural characterization of wild-type and mutant proteins can provide insights into the mechanistic basis of observed kinetic differences. Techniques might include:

  • X-ray crystallography

  • Circular dichroism spectroscopy to assess secondary structure changes

  • Fluorescence spectroscopy to detect alterations in tertiary structure

  • Hydrogen-deuterium exchange mass spectrometry to identify flexible regions

By creating mutations that convert residues in S. idiastes LDH to their counterparts in tropical Sphyraena species (and vice versa), researchers can establish causal relationships between specific amino acid substitutions and adaptive changes in enzyme function.

How does temperature affect the conformational dynamics of S. idiastes LDH during catalysis?

The conformational dynamics of LDH during catalysis are crucial for understanding its temperature adaptation. Studies on LDH from various species suggest that temperature affects several aspects of protein dynamics that are likely relevant to S. idiastes LDH:

Understanding these dynamics requires specialized techniques such as:

• Temperature-dependent enzyme kinetics

• Hydrogen-deuterium exchange mass spectrometry

• Molecular dynamics simulations

• NMR spectroscopy for investigating protein motions

• Time-resolved X-ray crystallography or cryo-electron microscopy

By comparing the conformational dynamics of S. idiastes LDH with those of tropical Sphyraena species, researchers can gain insights into how evolutionary changes in protein flexibility contribute to temperature adaptation.

What physiological consequences might result from the specific kinetic properties of S. idiastes LDH?

The specific kinetic properties of S. idiastes LDH likely have significant physiological consequences for the organism, affecting its metabolic function and ecological adaptation. Based on studies of LDH in other fish species, several physiological effects can be inferred:

• Swimming Performance: LDH plays a crucial role in anaerobic metabolism during intense swimming. The specific kinetic properties of S. idiastes LDH may influence swimming endurance and recovery from exhaustive exercise. Studies in Fundulus heteroclitus have shown that different LDH genotypes are associated with differences in swimming performance at specific temperatures .

• Oxygen Delivery: LDH activity can indirectly affect oxygen delivery to tissues. In Fundulus, LDH genotypes influence erythrocyte ATP levels, which in turn affect hemoglobin's oxygen affinity through the Bohr effect. Similar mechanisms might operate in S. idiastes, optimizing oxygen delivery at its environmental temperature .

• Development Timing: Different LDH genotypes in Fundulus are associated with differences in hatching time (LDH-B4 fish: 11.9 days; LDH-B2/B6: 12.4 days; LDH-B6: 12.8 days) . The specific properties of S. idiastes LDH might similarly influence developmental timing, potentially adapting it to the seasonal patterns in its temperate habitat.

• Metabolic Flux Balance: The Km and kcat values of LDH influence the balance between glycolytic and gluconeogenic fluxes. The temperature-adapted properties of S. idiastes LDH likely help maintain appropriate metabolic balance at its environmental temperature of 18°C .

These physiological consequences demonstrate how molecular adaptations in a single enzyme can propagate to affect whole-organism performance and fitness in specific thermal environments. Understanding these connections requires integrating biochemical analyses with physiological studies at the organismal level.

What experimental conditions are critical for accurately measuring the temperature-dependent kinetics of S. idiastes LDH?

Accurate measurement of temperature-dependent kinetics for S. idiastes LDH requires careful control of several experimental parameters:

• Temperature Control: Given that S. idiastes LDH is adapted to function at approximately 18°C, precise temperature control is essential . Researchers should:

  • Use water-jacketed reaction vessels or temperature-controlled spectrophotometers

  • Allow sufficient time for temperature equilibration before initiating reactions

  • Measure the actual temperature in the reaction mixture

  • Include temperature points that span the physiological range of S. idiastes (approximately 13-23°C) and extend beyond it for comparative purposes

• Buffer Considerations:

  • Select buffers with minimal temperature dependence of pKa (e.g., phosphate buffers)

  • Adjust pH at each experimental temperature to maintain consistent protonation states

  • Standardize ionic strength across different temperatures

  • Account for temperature effects on substrate and cofactor solubility

• Enzyme Stability: Ensure that any observed temperature effects reflect intrinsic kinetic properties rather than progressive denaturation:

  • Verify enzyme stability at each experimental temperature over the time course of the assay

  • Include appropriate controls for time-dependent activity loss

  • For measurements at higher temperatures, minimize pre-incubation time

• Substrate Concentrations: For Michaelis-Menten kinetics, use an appropriate range of substrate concentrations:

  • Include concentrations spanning from approximately 0.2× to 5× the Km value

  • Ensure NADH concentrations are non-limiting (typically 0.1-0.2 mM)

  • Account for potential substrate inhibition at high pyruvate concentrations

• Data Analysis:

  • Apply appropriate enzyme kinetic models (Michaelis-Menten, Lineweaver-Burk, Eadie-Hofstee)

  • Use Arrhenius plots to determine activation energies

  • Calculate catalytic efficiencies (kcat/Km) at each temperature

  • Compare parameters with those of other Sphyraena species under identical conditions

By carefully controlling these conditions, researchers can obtain reliable data on the temperature dependence of S. idiastes LDH kinetics, facilitating meaningful comparisons with LDH from other species and providing insights into the molecular basis of temperature adaptation.

What approaches can be used to study the thermal stability of S. idiastes LDH?

Studying the thermal stability of S. idiastes LDH requires a multi-faceted approach that examines different aspects of protein denaturation and functional loss. Based on research methodologies applied to other LDHs, the following approaches are recommended:

• Activity-Based Methods:

  • Thermal inactivation kinetics: Incubate the enzyme at various temperatures for defined periods, then measure residual activity at a standard temperature (e.g., 25°C)

  • Construct thermal inactivation profiles showing activity retention versus temperature

  • Determine T50 (temperature at which 50% activity is retained) and compare with LDH from tropical Sphyraena species

  • Calculate inactivation rate constants and corresponding activation energies for the unfolding process

• Spectroscopic Methods:

  • Circular dichroism (CD) spectroscopy to monitor temperature-induced changes in secondary structure

  • Fluorescence spectroscopy to detect alterations in tertiary structure, particularly around tryptophan residues

  • UV-visible spectroscopy to monitor turbidity associated with protein aggregation

  • Dynamic light scattering to assess temperature-dependent changes in protein size distribution

• Calorimetric Methods:

  • Differential scanning calorimetry (DSC) to determine the melting temperature (Tm) and enthalpy of unfolding

  • Isothermal titration calorimetry (ITC) to assess thermodynamic parameters of substrate binding at different temperatures

• Molecular Dynamics Simulations:

  • Simulate protein behavior at different temperatures

  • Identify regions of the protein that unfold first at elevated temperatures

  • Compare dynamics of S. idiastes LDH with those of tropical Sphyraena species

When comparing thermal stability between S. idiastes LDH and LDH from tropical Sphyraena species, it is important to note that temperate-adapted enzymes generally show lower thermal stability than tropical-adapted ones . This reflects an evolutionary trade-off between catalytic efficiency at low temperatures and thermal stability.

How can the physiological relevance of in vitro findings on S. idiastes LDH be validated?

Bridging the gap between in vitro biochemical data and physiological function requires approaches that connect molecular properties to organismal performance. For S. idiastes LDH, several strategies can validate the physiological relevance of laboratory findings:

• Comparative Studies Across Thermal Gradients:

  • Compare LDH kinetic properties from S. idiastes populations across different thermal environments

  • Correlate enzyme properties with habitat temperature

  • Perform reciprocal transplant or common garden experiments to distinguish genetic adaptation from acclimation

• Tissue-Specific Analysis:

  • Measure LDH activity in different tissues of S. idiastes

  • Determine isozyme distribution patterns across tissues

  • Correlate tissue-specific LDH properties with metabolic demands

• Physiological Performance Tests:

  • Measure swimming performance at different temperatures

  • Assess recovery from exhaustive exercise

  • Determine critical thermal maxima and minima

  • Correlate these whole-organism parameters with LDH properties

• Metabolic Context:

  • Measure in vivo concentrations of pyruvate and lactate

  • Determine whether substrate concentrations fall within the range where S. idiastes LDH kinetic properties would be most relevant

  • Assess the balance between aerobic and anaerobic metabolism under different conditions

• Genetic Correlation Studies:

  • If polymorphisms exist in S. idiastes ldha, correlate genotypes with physiological traits

  • Studies in Fundulus heteroclitus have shown that LDH genotypes correlate with hatching time (LDH-B4 fish: 11.9 days; LDH-B6 fish: 12.8 days)

  • Similar approaches could reveal connections between S. idiastes LDH properties and life history traits

These approaches can provide strong evidence for the adaptive significance of the specific kinetic properties observed in S. idiastes LDH, connecting molecular evolution to ecological adaptation and demonstrating how selection acts on enzyme function to enhance organismal fitness in specific thermal environments.

What techniques can reveal the structural basis of temperature adaptation in S. idiastes LDH?

Understanding the structural basis of temperature adaptation in S. idiastes LDH requires techniques that can provide detailed information about protein structure and dynamics. Several complementary approaches are recommended:

• X-ray Crystallography:

  • Determine the three-dimensional structure of S. idiastes LDH at high resolution

  • Compare with structures from tropical Sphyraena species

  • Identify subtle differences in active site architecture, loop regions, and subunit interfaces

  • Co-crystallize with substrates or substrate analogs to examine binding interactions

• Molecular Dynamics Simulations:

  • Model the dynamics of S. idiastes LDH at different temperatures

  • Compare flexibility patterns with those of tropical Sphyraena LDHs

  • Identify regions with differential flexibility that may contribute to temperature adaptation

  • Simulate the effects of specific amino acid substitutions on protein dynamics

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Measure the rate of hydrogen-deuterium exchange in different regions of the protein

  • Identify areas with differential flexibility between S. idiastes LDH and tropical Sphyraena LDHs

  • Assess how temperature affects exchange rates in different protein regions

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • For specific domains or peptides, NMR can provide detailed information about dynamics

  • Compare chemical shift perturbations at different temperatures

  • Measure relaxation parameters related to protein motion

• Ancestral Sequence Reconstruction:

  • Infer ancestral sequences at key nodes in the Sphyraena phylogeny

  • Express and characterize reconstructed ancestral enzymes

  • Identify the historical sequence of mutations that led to temperature adaptation

• Comparative Analysis with Other Temperature-Adapted LDHs:

  • Compare S. idiastes LDH with LDHs from other cold-adapted organisms

  • Identify common structural features that contribute to cold adaptation

  • Distinguish convergent from divergent evolutionary solutions

These techniques, used in combination, can reveal how specific structural features of S. idiastes LDH contribute to its optimal function at 18°C and provide insights into the molecular mechanisms of enzymatic temperature adaptation more broadly.