Recombinant Methylmalonyl-CoA carboxyltransferase 5S subunit, partial

What is Methylmalonyl-CoA carboxyltransferase 5S subunit and what is its function?

Methylmalonyl-CoA carboxyltransferase 5S subunit (UniProtKB Entry: Q70AC7) is a protein component of the transcarboxylase enzyme complex found in Propionibacterium freudenreichii subsp. shermanii. The 5S subunit specifically catalyzes the transfer of the carboxyl group from biotin of the 1.3S subunit to pyruvate to form oxaloacetate and 1.3S biotin . This enzyme possesses metal ion binding capabilities and functions as part of the methylmalonyl-CoA metabolic pathway, which is critical for propionate metabolism in various organisms . The complete protein consists of 505 amino acid residues with a molecular weight of approximately 55.65 kDa and a theoretical isoelectric point (pI) of 5.51 .

How does the Methylmalonyl-CoA pathway function in different organisms?

The Methylmalonyl-CoA pathway functions as a critical metabolic route for the conversion of propionyl-CoA to succinyl-CoA across diverse organisms. In bacteria like Propionibacterium, the pathway involves the 5S subunit of methylmalonyl-CoA carboxyltransferase, which facilitates specific carboxyl group transfers in propionate metabolism . In mycobacteria such as M. tuberculosis, this pathway is part of a larger long-chain acyl-CoA carboxylase (LCC) complex involving multiple subunits (AccA3, AccD4, AccD5, and AccE5) that collectively carboxylate various acyl-CoA substrates . In eukaryotes like C. elegans, the pathway includes homologs of mammalian enzymes: propionyl-CoA carboxylase subunits (pcca-1, pccb-1), methylmalonyl-CoA epimerase (mce-1), and methylmalonyl-CoA mutase (mmcm-1) . The conservation of this pathway across different organisms underscores its fundamental importance in intermediary metabolism, particularly in converting propionyl-CoA to succinyl-CoA for entry into the TCA cycle .

What structural characteristics define the Methylmalonyl-CoA carboxyltransferase 5S subunit?

The Methylmalonyl-CoA carboxyltransferase 5S subunit from Propionibacterium freudenreichii subsp. shermanii is characterized by a distinct protein structure optimized for its catalytic function. The full-length protein contains 505 amino acid residues with a molecular weight of 55649.06 Da and a theoretical isoelectric point of 5.51 . While the specific crystal structure details weren't provided in the search results, related carboxyltransferases like AccD4 and AccD5 from M. tuberculosis are known to form hexameric structures as determined by size exclusion chromatography . The 5S subunit contains metal ion binding domains essential for its catalytic function in transferring carboxyl groups . The protein sequence contains specific functional domains that facilitate interaction with the biotin-containing 1.3S subunit and enable the precise coordination required for carboxyl transfer reactions from the biotin moiety to pyruvate, forming oxaloacetate .

How do the kinetic parameters of Methylmalonyl-CoA carboxyltransferase compare in different enzyme complexes?

The kinetic parameters of Methylmalonyl-CoA carboxyltransferase vary significantly depending on the specific enzyme complex and substrate. In the LCC complex of M. tuberculosis, which contains AccD5 (a carboxyltransferase), substrate-specific kinetic parameters revealed significant differences. With propionyl-CoA as substrate, the complex exhibited a Km of 156 ± 22 μM and Vmax of 500 ± 39 nmol/min/mg, resulting in a catalytic efficiency (Kcat/Km) of 3205 M−1sec−1 . In contrast, with acetyl-CoA, the complex showed a higher Km of 300 ± 29 μM and lower Vmax of 250 ± 31 nmol/min/mg, yielding a catalytic efficiency of 833 M−1sec−1 . For long-chain substrates like C20-CoA, the complex demonstrated remarkably high affinity (Km = 32 ± 4 μM) but much lower Vmax (2.44 ± 0.5 nmol/min/mg), resulting in a catalytic efficiency of only 63 M−1sec−1 . These parameters differ from those observed in the dedicated ACCase 5 complex with the same AccD5 subunit, which showed Km values of 240 ± 35 μM for propionyl-CoA and 220 ± 55 μM for acetyl-CoA . This comparison demonstrates how the structural context of the same carboxyltransferase subunit can significantly influence substrate specificity and catalytic efficiency.

What experimental approaches can be used to determine the functional roles of individual subunits in carboxyltransferase complexes?

Several experimental approaches can be employed to determine the functional roles of individual subunits in carboxyltransferase complexes:

• In vitro reconstitution: By independently expressing and purifying individual subunits (e.g., using N-terminal His-tagged proteins and Ni2+ affinity chromatography) and systematically combining them in different combinations, you can determine which subunits are essential for activity. This approach was successfully used with the M. tuberculosis LCC complex to establish that all four subunits (AccA3, AccD4, AccD5, and AccE5) were required for activity .

• Substrate competition assays: To determine the catalytic role of specific subunits, researchers can conduct competition experiments using different substrates. For example, when investigating AccD5's role in the LCC complex, competition between long-chain acyl-CoAs and propionyl-CoA provided insights into subunit specificity .

• Inhibitor studies: Using subunit-specific inhibitors (like andrimid for AccD5) and measuring the impact on different activities can reveal the catalytic versus structural roles of specific subunits .

• RNA interference and gene deletion studies: In model organisms like C. elegans, RNA interference against specific pathway genes (e.g., mmcm-1, mmab-1, mmaa-1) combined with metabolite analysis can determine functional roles in vivo .

• Functional complementation: Testing whether genes from one organism can rescue phenotypes in another organism (e.g., C. elegans mmcm-1 delivery into human fibroblasts) helps validate conservation of function across species .

• Size exclusion chromatography: This technique helps determine the oligomeric state of purified subunits, providing structural insights that can inform functional roles .

What are the implications of Methylmalonyl-CoA carboxyltransferase pathway disruption across different model systems?

Disruption of the Methylmalonyl-CoA carboxyltransferase pathway has significant implications across different model systems:

In bacterial systems like Propionibacterium, disruption would directly impact pyruvate metabolism and oxaloacetate formation, potentially affecting central carbon metabolism and energy production .

In mycobacterial systems such as M. tuberculosis, disruption of the LCC complex (which includes carboxyltransferase subunits) would impair the generation of key substrates including methylmalonyl-CoA, required for methyl-branched lipid biosynthesis, and α-carboxy-C24–26-CoA, needed for mycolic acid biosynthesis . Since these lipids are essential components of the mycobacterial cell wall, pathway disruption would likely affect bacterial viability and virulence.

In C. elegans, disruptions in this pathway through deletion mutants of methylmalonyl-CoA mutase (mmcm-1), co(I)balamin adenosyltransferase (mmab-1), and methylmalonyl-CoA epimerase (mce-1) led to reduced 1-[14C]-propionate incorporation into macromolecules and increased methylmalonic acid production . These findings parallel the metabolic disturbances seen in human methylmalonic acidemia.

In humans, pathway disruptions cause methylmalonic acidemia (MMAemia, MIM 251000 & MIM 251100), characterized by methylmalonic acid accumulation in tissues and body fluids, with secondary metabolic perturbations including hyperglycinemia, hyperammonemia, and intermittent hypoglycemia . Clinical manifestations include developmental delay, renal disease, pancreatitis, and metabolic infarction of the basal ganglia, highlighting the critical nature of this pathway in human metabolism .

What are the optimal conditions for expressing and purifying recombinant Methylmalonyl-CoA carboxyltransferase 5S subunit?

The optimal conditions for expressing and purifying recombinant Methylmalonyl-CoA carboxyltransferase 5S subunit can be adapted from successful protocols used for similar carboxyltransferases:

Expression system: E. coli has been successfully used to express carboxyltransferase subunits from various organisms. For the related AccD4 and AccD5 subunits from M. tuberculosis, expression as N-terminal His-tagged proteins in E. coli yielded functional proteins .

Expression conditions: While not explicitly stated for the 5S subunit, typical conditions for similar proteins involve induction with IPTG (0.5-1 mM) at mid-log phase (OD600 ~0.6) followed by expression at lower temperatures (16-25°C) to enhance proper folding.

Purification strategy: Ni2+ affinity chromatography has been effective for purifying His-tagged carboxyltransferase subunits . A typical protocol would include:

• Cell lysis in appropriate buffer (often containing 20-50 mM Tris-HCl pH 8.0, 300-500 mM NaCl, 5-10% glycerol)

• Loading on Ni-NTA column

• Washing to remove non-specific binding proteins

• Elution with imidazole gradient (typically 50-250 mM)

• Size exclusion chromatography to remove aggregates and confirm oligomeric state

Oligomeric state assessment: Size exclusion chromatography should be performed to determine the oligomeric state. Related carboxyltransferases like AccD4 and AccD5 form hexamers , which may provide guidance for the expected elution profile of the 5S subunit.

Buffer optimization: For storage and enzymatic assays, appropriate buffers typically include 20-50 mM Tris-HCl or HEPES (pH 7.5-8.0), 100-150 mM NaCl, 10% glycerol, and 1-5 mM DTT or 2-mercaptoethanol to maintain reducing conditions.

What analytical methods can be used to assess the carboxyltransferase activity of the 5S subunit?

Several analytical methods can be employed to assess the carboxyltransferase activity of the 5S subunit:

Spectrophotometric assays: Coupling the carboxylation reaction to NAD(P)H oxidation through auxiliary enzymes allows for continuous monitoring of activity by measuring the decrease in absorbance at 340 nm. This approach provides real-time kinetic data.

Radioisotope-based methods: Incorporation of 14C-labeled substrates (such as [14C]-propionate) into products can quantify pathway flux, as demonstrated in C. elegans studies . The labeled products can be separated and quantified by liquid scintillation counting.

LC/MS analysis: For direct product identification and quantification, liquid chromatography coupled with mass spectrometry provides high sensitivity and specificity. This approach was successfully used to detect carboxy-C20-CoA products (m/z of 1104.4, 1126.4, and 1142.3) in the M. tuberculosis LCC complex reactions . MS/MS analysis of the charged ions can further confirm product identity.

Substrate competition assays: These can determine substrate preferences by measuring activity with different substrate combinations and concentrations .

Enzyme inhibition studies: Using specific inhibitors like andrimid (for AccD5) can help distinguish the contributions of different subunits in multi-component systems .

Michaelis-Menten kinetic analysis: By varying substrate concentrations and measuring initial reaction rates, key kinetic parameters (Km, Vmax, and catalytic efficiency) can be determined to characterize enzyme performance with different substrates .

How can researchers troubleshoot expression and activity issues with recombinant Methylmalonyl-CoA carboxyltransferase?

Researchers can employ several strategies to troubleshoot expression and activity issues with recombinant Methylmalonyl-CoA carboxyltransferase:

Expression troubleshooting:

• Codon optimization: Adapt codons to match the expression host preferences, particularly if expressing a bacterial protein like the 5S subunit from Propionibacterium in E. coli.

• Expression tags and fusion partners: Test different affinity tags (His, GST, MBP) and their positions (N- or C-terminal) to improve solubility and expression.

• Expression conditions: Modulate induction temperature (16-30°C), IPTG concentration (0.1-1 mM), and expression duration (4-24 hours) to optimize for soluble protein yield.

• Specialized expression strains: Use E. coli strains designed for improved expression of challenging proteins (e.g., BL21(DE3)pLysS, Rosetta for rare codons, or Arctic Express for cold-adapted chaperones).

Activity troubleshooting:

• Multisubunit reconstitution: If the 5S subunit requires interaction with other components (as observed in the M. tuberculosis LCC complex), optimize the molar ratios of components. For example, the optimal AccA3:AccD4:AccD5:AccE5 ratio was found to be 1:1:1:2 in the M. tuberculosis LCC complex .

• Metal ion requirements: Since the 5S subunit is involved in metal ion binding , ensure appropriate metal ions (Mg2+, Mn2+, or Zn2+) are included in assay buffers.

• Substrate solubility issues: For long-chain acyl-CoA substrates, solubility can be problematic. The M. tuberculosis study noted that C24-CoA showed limited availability to the enzyme due to low solubility . Consider using detergents or carrier proteins to improve substrate availability.

• Coupled enzyme systems: For indirect assays, verify that coupling enzymes are not rate-limiting by using excess amounts.

• Assay sensitivity: For low-activity preparations, extend reaction times and use more sensitive detection methods like LC/MS instead of spectrophotometric assays.

How can understanding Methylmalonyl-CoA carboxyltransferase inform therapeutic strategies for methylmalonic acidemia?

Understanding Methylmalonyl-CoA carboxyltransferase and related enzymes in the propionyl-CoA metabolic pathway can significantly inform therapeutic strategies for methylmalonic acidemia through several approaches:

Gene therapy approaches: The successful partial restoration of propionate flux in human fibroblasts from methylmalonic acidemia patients through lentiviral delivery of C. elegans mmcm-1 demonstrates the potential of gene therapy approaches . Similar strategies targeting the carboxyltransferase components could be explored.

Enzyme replacement therapy: Detailed characterization of the structure and function of pathway enzymes could enable the development of stabilized recombinant enzyme formulations for replacement therapy.

Metabolic bypass strategies: Understanding the complete pathway reveals potential metabolic bypass routes. For instance, the older hypothesis that patients with mutations in methylmalonyl-CoA epimerase (MCEE) might be protected via a free MMA shunt that bypasses the reaction could inform similar bypass strategies .

Small molecule chaperones: Structural understanding of the carboxyltransferase could guide the design of small molecules that stabilize mutant but potentially functional enzymes in patients, improving their residual activity.

Dietary management optimization: Detailed enzyme kinetics, such as those determined for different acyl-CoA substrates in the M. tuberculosis system , could inform more precise dietary restrictions and supplements for patients with specific pathway deficiencies.

Biomarker development: Understanding the metabolic consequences of pathway disruption can identify novel biomarkers for disease progression and treatment efficacy, beyond the established methylmalonic acid measurements.

What is the current understanding of the evolutionary conservation of Methylmalonyl-CoA metabolism across species?

The methylmalonyl-CoA metabolic pathway exhibits remarkable evolutionary conservation across species, reflecting its fundamental importance in carbon metabolism:

Bacterial systems: In Propionibacterium freudenreichii, the pathway involves the specialized 5S subunit of methylmalonyl-CoA carboxyltransferase, which catalyzes specific carboxyl group transfers in propionate metabolism . This represents one evolutionary solution for handling propionate metabolism.

Mycobacterial adaptation: In M. tuberculosis, the pathway has evolved into a multifunctional long-chain acyl-CoA carboxylase (LCC) complex capable of carboxylating various acyl-CoA substrates ranging from acetyl-CoA and propionyl-CoA to long-chain acyl-CoAs . This represents an adaptation that allows a single complex to generate multiple substrates required for complex lipid biosynthesis.

Eukaryotic conservation: C. elegans studies revealed a "full complement of mammalian homologues for the conversion of propionyl-CoA to succinyl-CoA," including propionyl-CoA carboxylase subunits (pcca-1, pccb-1), methylmalonyl-CoA epimerase (mce-1), and methylmalonyl-CoA mutase (mmcm-1) . This demonstrates strong conservation between nematodes and mammals.

Functional conservation: The ability of C. elegans mmcm-1 to partially restore propionate flux in human methylmalonic acidemia patient fibroblasts further confirms functional conservation across distant evolutionary lineages .

Epimerase conservation: The striking conservation of the methylmalonyl-CoA epimerase gene throughout the phyla suggests it plays a critical role in intermediary metabolism, despite earlier hypotheses that it might be dispensable in some contexts .

This evolutionary conservation underscores the pathway's ancient origins and essential metabolic function, while species-specific adaptations reflect different metabolic demands across diverse organisms.