Recombinant Cat Malate dehydrogenase, cytoplasmic (MDH1)

What is the predicted molecular weight and structure of feline cytoplasmic MDH1?

Based on comparative analysis with other mammalian MDH1 proteins, feline cytoplasmic MDH1 is predicted to have a molecular weight of approximately 36-38 kDa in its native form. Like other mammalian MDH1 enzymes, cat MDH1 likely functions as a homodimer with each monomer containing the characteristic conserved Rossman fold for NAD+ binding .

The expected amino acid sequence length is around 334 amino acids, similar to human MDH1. While specific feline sequence information is limited, phylogenetic analysis suggests high conservation among mammalian MDH1 proteins, with expected sequence homology of >85% with other mammalian cytosolic MDH1 proteins .

What expression systems are most effective for producing recombinant cat MDH1?

E. coli expression systems have proven most efficient for recombinant MDH1 production across multiple species. For optimal expression of cat MDH1, the following approaches are recommended:

• Vector selection: pET-based expression vectors containing T7 promoters show high expression levels

• Fusion tags: N-terminal His-tag or GST-tag significantly improves solubility and facilitates purification

• Host strains: BL21(DE3) or Rosetta(DE3) strains are preferred, particularly when codon optimization is employed

• Expression conditions: Induction with 0.5-1.0 mM IPTG at OD600 0.6-0.8, followed by expression at 16-18°C for 16-18 hours maximizes soluble protein yield

For mammalian MDH1 proteins, including predicted cat MDH1, this approach typically yields 15-20 mg of purified protein per liter of bacterial culture with >90% purity after affinity chromatography .

What purification strategy provides the highest purity and yield for recombinant cat MDH1?

A multi-step purification approach is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged proteins or glutathione-agarose for GST-tagged proteins

• Intermediate purification: Ion-exchange chromatography using DEAE-Sepharose at pH 9.5

• Polishing step: Size exclusion chromatography using Superdex 200 in a buffer containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl

This approach typically yields protein with >95% purity as determined by SDS-PAGE. For optimal stability during purification, adding 5-10% glycerol and 1-2 mM DTT to all buffers is recommended .

What are the expected kinetic parameters for recombinant cat MDH1?

While specific kinetic parameters for cat MDH1 have not been extensively characterized, extrapolation from closely related mammalian MDH1 enzymes suggests the following approximate values:

Note that like other mammalian MDH1 enzymes, cat MDH1 is expected to show approximately 1.7-fold higher catalytic efficiency (k_cat) for oxaloacetate reduction compared to malate oxidation, with nearly 350-fold preference in terms of catalytic specificity (k_cat/K_m) .

How does cat MDH1 activity vary with pH and temperature?

Based on studies of mammalian MDH1 enzymes, cat MDH1 is expected to demonstrate the following pH and temperature dependencies:

pH optima:

• Malate → Oxaloacetate direction: pH 8.5-9.5

• Oxaloacetate → Malate direction: pH 7.0-7.5

Temperature characteristics:

• Temperature optimum: 35-40°C

• Thermostability: Maintains >50% activity after 30 minutes at 40°C

• Complete inactivation occurs at temperatures >55°C for extended periods

The enzyme shows highest stability in the pH range of 7.0-8.0, with significant activity loss below pH 6.0 or above pH 10.0 .

How can I accurately measure the activity of recombinant cat MDH1?

Several validated methods are available for measuring MDH1 activity:

• Spectrophotometric NADH oxidation/NAD⁺ reduction assay:

  • Monitor absorbance change at 340 nm (ε = 6,220 M^-1cm^-1)

  • Reaction mixture: 50 mM potassium phosphate buffer (pH 7.5), 0.2 mM NADH, 0.2-2.0 mM oxaloacetate for forward reaction

  • Reaction mixture: 50 mM CAPS buffer (pH 9.5), 2.0 mM NAD⁺, 5-50 mM malate for reverse reaction

• Coupled colorimetric assay:

  • Based on the reduction of tetrazolium salt (MTT) in an NADH-coupled reaction

  • Absorbance measured at 565 nm

  • Linear detection range: 0.5-65 U/L for 20-minute reaction at 37°C

  • More stable color development and less interference from sample components

For accurate activity measurements, it is essential to:

• Maintain temperature at 25°C or 37°C (specify in reporting)

• Include appropriate enzyme-free controls

• Calculate initial rates using only the linear portion of progress curves

• Express activity in μmol/min/mg protein (specific activity)

What are the key active site residues in cat MDH1 and how do they contribute to catalysis?

Based on highly conserved active site architecture across mammalian MDH1 proteins, the cat MDH1 active site likely contains these critical residues:

• Substrate binding pocket:

  • Arg92, Arg98, and Arg162: Form ionic interactions with carboxylate groups of malate/oxaloacetate

  • His187: Acts as a catalytic residue involved in proton transfer

  • Ser242: Contributes to substrate stabilization through hydrogen bonding

• NAD⁺/NADH binding site:

  • Gly11, Ala13, Gly14, Gln15, Ile16: Form the classical Rossmann fold that binds the adenine portion

  • Asp42: Forms hydrogen bonds with ribose hydroxyls

  • Val87, Gly88, Ala89, Met90: Interact with the nicotinamide portion

  • Ile108, Val129, Asn131: Stabilize binding through hydrophobic and hydrogen bonding interactions

• Catalytic residues:

  • His187 and Asp158: Form a catalytic dyad essential for proton transfer

  • Arg162: Polarizes the carbonyl group of the substrate

  • Asn131: Positions the nicotinamide ring for optimal hydride transfer

These residues create a precise microenvironment that lowers the activation energy for hydride transfer between substrate and cofactor, with the reaction proceeding through an ordered bi-bi mechanism .

How do post-translational modifications affect cat MDH1 activity?

While cat-specific data is limited, studies on mammalian MDH1 enzymes indicate these critical post-translational modifications:

• Acetylation:

  • Lysine 118 (K118) is a key acetylation site that regulates enzyme activity

  • Acetylation reduces catalytic efficiency by disrupting substrate binding

  • Deacetylation by HDAC inhibitors like ACY1215 can rescue enzyme activity

• Phosphorylation:

  • Multiple serine/threonine residues can be phosphorylated

  • Phosphorylation generally increases enzyme activity by enhancing substrate binding affinity

  • Regulated by cellular kinases including PKA and CaMKII

• Other modifications:

  • Oxidation of cysteine residues can inactivate the enzyme under oxidative stress

  • SUMOylation has been reported to affect protein stability and subcellular localization

Researchers studying cat MDH1 should consider these potential modification sites and their impact on enzyme function, particularly in studies involving metabolic regulation or disease models .

How does the tertiary and quaternary structure of cat MDH1 contribute to its function?

Mammalian MDH1 proteins, including predicted cat MDH1, adopt a consistent structural organization:

• Tertiary structure features:

  • Each monomer (approximately 35-37 kDa) contains two domains

  • N-terminal domain forms a Rossmann fold that binds NAD⁺/NADH

  • C-terminal domain contains the substrate binding site

  • A flexible loop (residues 90-100) undergoes conformational change upon substrate binding

• Quaternary structure characteristics:

  • Functions as a homodimer with extensive intersubunit contacts

  • Dimer interface involves approximately 18 residues including Tyr18, Met55, Glu56, Gln58, Asp59, Cys60, and others

  • Salt bridges and hydrogen bonds stabilize the dimer interface

  • The dimer structure creates a microenvironment that enhances catalytic efficiency

• Functional implications:

  • Loop closure after substrate binding is essential for catalysis

  • Dimer formation provides thermal stability and resistance to denaturation

  • Conformational changes during catalysis follow an induced-fit mechanism

  • The quaternary structure allows for potential allosteric regulation

Crystal structures of human MDH1 (PDB: 4WLV) show that the enzyme can bind substrate analogs such as malonate even in the absence of cofactor, suggesting a flexible binding mechanism that may be conserved in cat MDH1 .

How can I design site-directed mutagenesis experiments to study cat MDH1 structure-function relationships?

When designing mutagenesis experiments for cat MDH1 structure-function studies, consider these approaches:

• Key residues for targeted mutagenesis:

  • Catalytic residues: His187, Asp158 (convert to Ala or Asn to disrupt catalysis)

  • Substrate binding: Arg92, Arg98, Arg162 (convert to Lys to maintain charge but alter geometry)

  • Cofactor binding: Asp42, Ile108 (modify to alter NAD⁺/NADH specificity)

  • Dimer interface: Tyr18, Glu56 (mutate to disrupt dimerization)

• Experimental approach:

  • Use QuickChange site-directed mutagenesis or Gibson Assembly methods

  • Validate mutations by DNA sequencing

  • Express mutant proteins using the same conditions as wild-type

  • Purify using identical protocols to ensure comparable results

  • Characterize using comprehensive kinetic analysis with both substrates and both directions

• Expected outcomes and analysis:

  • Active site mutations: Expect 10-1000 fold reductions in k_cat or increases in K_m

  • Dimer interface mutations: May affect stability more than activity

  • Surface mutations: Could alter regulatory properties without changing catalytic parameters

  • Analyze results through Michaelis-Menten kinetics and thermal stability assays

When reporting results, calculate ΔΔG values for substrate binding and compare catalytic efficiency (k_cat/K_m) rather than individual parameters to fully understand the mutation effects .

How does cat MDH1 compare structurally and functionally to MDH1 from other species?

Comparative analysis of mammalian MDH1 enzymes reveals:

Notable observations from comparative studies:

• Mammalian MDH1 enzymes show extremely high sequence conservation in catalytic regions

• C. elegans MDH-1 demonstrates higher thermostability due to additional intersubunit salt bridges

• Species differences are primarily in surface-exposed regions rather than the active site

• Kinetic parameters are remarkably similar across mammalian species, suggesting functional conservation

For researchers working with cat MDH1, these similarities suggest that mammalian model systems provide good approximations, but cat-specific studies would be valuable to identify any unique properties .

What are the optimal storage and stability conditions for recombinant cat MDH1?

To maintain maximum stability and activity of recombinant cat MDH1:

• Short-term storage (1-2 weeks):

  • Store at 4°C in 20 mM Tris-HCl pH 7.5-8.0, 150 mM NaCl

  • Include protective additives: 10% glycerol, 1 mM DTT or 5 mM β-mercaptoethanol

  • Add 0.02% sodium azide to prevent microbial growth

• Long-term storage (months to years):

  • Store at -80°C in small aliquots (50-100 μL) to avoid freeze-thaw cycles

  • Include cryoprotectants: 20% glycerol or 5% trehalose

  • Maintain protein concentration between 1-5 mg/mL

  • Flash-freeze in liquid nitrogen before transferring to -80°C

• Stability enhancers:

  • Addition of 0.1-0.5 mM NAD⁺ significantly improves thermal stability

  • 1 mM EDTA helps prevent metal-catalyzed oxidation

  • pH 7.5-8.0 provides optimal stability balance

Under these conditions, recombinant mammalian MDH1 proteins typically retain >90% activity for 6 months at -80°C and >80% activity after 5 freeze-thaw cycles .

How can I study the role of cat MDH1 in cellular metabolism using recombinant protein?

Several advanced approaches are available:

These approaches can reveal how cat MDH1 contributes to cytosolic NAD⁺ regeneration, glycolytic flux, and the malate-aspartate shuttle efficiency in feline cells, with potential insights into species-specific metabolic adaptations .

Why might recombinant cat MDH1 show lower activity than expected, and how can this be resolved?

Several factors can contribute to reduced activity in recombinant MDH1 preparations:

• Protein misfolding during expression:

  • Reduce expression temperature to 16-18°C

  • Co-express with molecular chaperones (GroEL/GroES)

  • Include 1% glucose in expression media to reduce basal expression level

  • Use auto-induction media for gentler protein expression

• Cofactor loss during purification:

  • Supplement purification buffers with 0.1 mM NAD⁺

  • Dialyze against buffers containing 0.05 mM NAD⁺

  • Include a reconstitution step with excess NAD⁺ followed by removal of unbound cofactor

• Oxidative damage:

  • Include 1-5 mM DTT or TCEP in all buffers

  • Perform all purification steps under nitrogen atmosphere when possible

  • Add 0.1-0.5 mM EDTA to chelate metal ions that catalyze oxidation

• Specific activity determination issues:

  • Ensure oxaloacetate solutions are freshly prepared (unstable in solution)

  • Verify pH of assay buffers (small changes significantly affect activity)

  • Calibrate spectrophotometer with NADH standards

  • Test multiple enzyme concentrations to ensure linearity of response

If activity issues persist, analyze the protein by native PAGE to confirm proper oligomeric state, as monomeric MDH1 shows significantly reduced activity compared to the dimeric form .

What are the critical factors for successful crystallization of recombinant cat MDH1?

For successful crystallization of mammalian MDH1 proteins:

• Sample preparation:

  • Achieve protein concentration of 10-15 mg/mL in 10 mM Tris-HCl pH 7.5, 50 mM NaCl

  • Remove His-tag using TEV protease to eliminate tag interference

  • Confirm monodispersity by dynamic light scattering (>95% monodisperse)

  • Apply final polishing step using ion exchange chromatography

• Crystallization conditions:

  • Initial screening: Commercial sparse matrix screens at 4°C and 18°C

  • Promising conditions: 0.1-0.2 M malonate buffer pH 6.0-8.0, 15-25% PEG 3350 or 3-6 M sodium formate

  • Addition of 0.5-2.0 mM NAD⁺ or NADH often improves crystal quality

  • Microseeding from initial crystals significantly enhances results

• Co-crystallization with ligands:

  • For substrate complexes: Use 5-10 mM malate or 1-2 mM oxaloacetate

  • For inhibitor complexes: Include compounds at concentrations 5-10× their Ki values

  • For cofactor complexes: 2-5 mM NAD⁺ or NADH

  • Allow 1-2 hours pre-incubation before setting up crystallization

• Data collection considerations:

  • Crystals typically diffract to 1.6-2.5 Å resolution

  • Use 20-25% glycerol or ethylene glycol as cryoprotectant

  • Collect complete datasets (>95% completeness) with redundancy >3

  • Process data with attention to potential twinning issues

Based on experience with human and other mammalian MDH1 proteins, initial crystallization hits can typically be optimized to produce diffraction-quality crystals within 1-2 months .

How can I accurately determine substrate specificity and inhibitor profiles for recombinant cat MDH1?

For comprehensive characterization of substrate specificity and inhibition:

• Substrate specificity analysis:

  • Test structurally related compounds: α-ketoglutarate, lactate, isocitrate

  • Determine full Michaelis-Menten parameters for each alternative substrate

  • Calculate specificity constants (k_cat/K_m) to quantify preference

  • Compare relative activities at physiological substrate concentrations

  Expected relative activity with different substrates:

  Substrate	Relative Activity (%)
  Malate/Oxaloacetate	100
  α-Hydroxymalonate	10-15
  Lactate/Pyruvate	<1
  Isocitrate	<0.1
  α-Ketoglutarate	<0.1
  Other α-hydroxy acids	<0.1

• Inhibitor profiling:

  • Screen initial inhibition at fixed concentration (100 μM)

  • For active compounds, determine IC₅₀ values

  • For potent inhibitors, determine inhibition mechanism (competitive, noncompetitive, uncompetitive)

  • Calculate Ki values using appropriate equations based on inhibition mechanism

  Common inhibitor classes and expected potency:

  Inhibitor Class	Typical Ki Range (μM)	Inhibition Mechanism
  Dicarboxylic acid analogs	10-100	Competitive vs. substrate
  Nucleotide analogs	5-50	Competitive vs. NAD(H)
  Metal ions (Zn<sup>2+</sup>, Cu<sup>2+</sup>)	50-500	Noncompetitive
  Thiol-reactive compounds	1-20	Irreversible

• Advanced analysis techniques:

  • Isothermal titration calorimetry to determine binding thermodynamics

  • Surface plasmon resonance for binding kinetics

  • Differential scanning fluorimetry to assess ligand-induced thermal stabilization

  • X-ray crystallography to confirm binding modes of substrates and inhibitors

These comprehensive approaches provide a complete profile of cat MDH1 specificity and inhibition characteristics, essential for understanding its metabolic role and potential as a drug target .

How is recombinant MDH1 being used to study its role in disease models?

MDH1 is increasingly recognized as a critical metabolic enzyme with implications in several disease states:

• Cancer metabolism:

  • MDH1 supports glycolysis in rapidly proliferating cancer cells

  • It generates cytosolic NAD⁺ needed for sustained glycolytic flux

  • Recombinant MDH1 is used in reconstituted metabolic networks to study metabolic rewiring

  • Correlation between MDH1 amplification and poor clinical prognosis has been established

• Liver disease models:

  • Decreased MDH1 expression and activity is observed in acute liver failure

  • Acetylation of MDH1 affects energy metabolism in liver

  • Studies with wild-type and acetylation-deficient mutants (K118R) demonstrate regulation of hepatic metabolism

  • Histone deacetylase inhibitors affect MDH1 activity and may have therapeutic potential

• Neurodegenerative diseases:

  • Elevated MDH1 activity observed in Alzheimer's disease models

  • Recombinant MDH1 used to study effects of amyloid-β on enzyme function

  • Potential role in maintaining neuronal redox balance under investigation

These studies often employ recombinant MDH1 for in vitro models, enzyme replacement strategies, or as controls for measuring endogenous activity, highlighting the importance of pure, well-characterized recombinant protein preparations .

What approaches are being developed to use recombinant MDH1 as a therapeutic target?

Several innovative approaches target MDH1 for therapeutic applications:

• Small molecule inhibitor development:

  • Structure-based design of selective MDH1 inhibitors

  • Focus on differences between cytosolic MDH1 and mitochondrial MDH2 in the α7-α8 loop

  • Virtual screening against MDH1 crystal structures identifies novel chemical scaffolds

  • Testing of inhibitors in cancer cell lines shows promising anti-proliferative effects

• Protein-based therapeutics:

  • Engineered MDH1 variants with enhanced catalytic efficiency

  • Chimeric proteins combining MDH1 with targeting domains

  • PEGylated or nanoparticle-encapsulated MDH1 for improved pharmacokinetics

  • Enzyme replacement strategies for metabolic disorders

• Regulation of post-translational modifications:

  • Modulators of MDH1 acetylation status

  • HDAC inhibitors rescue MDH1 activity in disease models

  • Identification of critical acetylation sites (K118) as therapeutic targets

  • Development of small molecules that specifically prevent or mimic acetylation

These approaches utilize recombinant MDH1 for initial screening, mechanistic studies, and proof-of-concept experiments before advancing to cellular and animal models .

How do evolutionary differences in MDH1 across species impact its function and potential applications?

Evolutionary analysis of MDH1 provides insights into its functional conservation and adaptation:

• Sequence and structural conservation:

  • Core catalytic residues show near-perfect conservation across all eukaryotes

  • Substrate binding pocket architecture is highly preserved

  • Mammalian MDH1 enzymes share >85% sequence identity

  • Differences mainly occur in surface-exposed loops

• Species-specific adaptations:

  • C. elegans MDH-1 shows enhanced thermostability through additional salt bridges

  • Thermophilic organisms contain MDH1 variants with specialized stabilizing features

  • Plant MDH1 enzymes show adapted kinetic parameters reflecting different metabolic requirements

  • These adaptations may be exploited for biotechnological applications

• Evolutionary radiation and horizontal gene transfer:

  • MDH evolved across all domains of life with conserved core function

  • Gene duplication and horizontal gene transfer contributed to modern MDH diversity

  • MDH1 and lactate dehydrogenase (LDH) share evolutionary origins

  • Understanding this evolutionary history helps predict functional properties of unstudied MDH1 orthologs

This evolutionary perspective provides a framework for understanding species-specific variations in cat MDH1 and informs both fundamental research and biotechnological applications of recombinant MDH1 proteins .