OXSM Human

What is OXSM and what cellular roles does it play?

OXSM (3-Oxoacyl-ACP Synthase Mitochondrial), also known as Type II Mitochondrial Beta-Ketoacyl Synthase, is a critical enzyme in the mitochondrial fatty acid synthesis (mtFAS II) pathway. This enzyme functions as a beta-ketoacyl synthetase that catalyzes chain elongation reactions essential for mitochondrial fatty acid production .

The protein consists of 455 amino acids (positions 28-459) with a molecular mass of 48.1kDa and contains conserved catalytic domains that are responsible for its enzymatic activity . OXSM is a key component in the production of octanoic acid, which serves as the precursor for lipoic acid (LA) synthesis . LA is a potent antioxidant that improves mitochondrial function and provides beneficial effects for preventing several metabolic and degenerative diseases .

How does OXSM's function differ from cytoplasmic fatty acid synthase (FASN)?

While both OXSM and cytoplasmic FASN catalyze fatty acid synthesis reactions, they differ in several important aspects:

OXSM plays a specialized role in producing fatty acid intermediates crucial for mitochondrial function rather than bulk fatty acid synthesis for energy storage .

What are the structural features of OXSM that enable its catalytic activity?

OXSM contains several conserved structural domains essential for its beta-ketoacyl synthase activity:

• The active site includes a catalytic triad typical of thiolase-fold enzymes

• Substrate binding pockets that accommodate both the acyl-ACP and malonyl-ACP substrates

• Interface regions that enable interaction with mitochondrial acyl carrier protein (mACP)

• Conserved motifs for binding to the phosphopantetheine arm of ACP

These structural features enable OXSM to catalyze the condensation reaction between acyl substrates bound to mACP and malonyl-mACP, extending the growing fatty acid chain by two carbon atoms in each catalytic cycle .

What are the most reliable expression systems for recombinant OXSM?

Based on published methodologies, Escherichia coli remains the preferred expression system for recombinant human OXSM production . The following protocol has been verified for high-yield expression:

• Construct design: The OXSM coding sequence (residues 28-459) with an N-terminal His6-tag in a pET-based vector

• Expression strain: BL21(DE3) or equivalent for high-yield protein production

• Culture conditions: LB media, induction with 0.5-1.0 mM IPTG at OD600 of 0.6-0.8

• Induction parameters: 18-25°C for 16-20 hours to enhance proper folding

• Cell harvesting: Centrifugation at 5000×g for 15 minutes at 4°C

For optimal yield and activity, researchers should include protease inhibitors during cell lysis and maintain reducing conditions throughout purification to preserve enzymatic activity .

What purification strategies yield the highest quality OXSM protein?

A multi-step chromatography approach has been established for obtaining highly pure and active OXSM:

• Initial capture using Ni-NTA affinity chromatography targeting the His-tag

  • Binding buffer: 20 mM Tris-HCl (pH 8.0), 0.15 M NaCl, 10% glycerol, 20 mM imidazole

  • Elution buffer: Same with 250-500 mM imidazole gradient

• Secondary purification using ion exchange chromatography

  • Resource Q or equivalent anion exchange column

  • Buffer: 20 mM Tris-HCl (pH 8.0) with gradient of 0-500 mM NaCl

• Final polishing with size exclusion chromatography

  • Superdex 200 or equivalent

  • Running buffer: 20 mM Tris-HCl (pH 8.0), 0.15 M NaCl, 1 mM DTT, 10% glycerol

Final preparation should be stored at -20°C with addition of carrier protein (0.1% HSA or BSA) to prevent aggregation and preserve activity during long-term storage .

What methodologies effectively measure OXSM enzymatic activity?

Several complementary approaches can be employed to assess OXSM activity:

When designing these assays, researchers should include appropriate controls and validate the linearity of response to ensure accurate activity measurements .

How does OXSM interact with mitochondrial acyl carrier protein (mACP)?

OXSM interacts specifically with human mitochondrial acyl carrier protein (mACP, also known as NDUFAB1) to catalyze fatty acid elongation reactions . This interaction involves:

• Recognition of the phosphopantetheine prosthetic group of mACP, which carries the growing acyl chain

• Specific protein-protein interactions between conserved surface regions of OXSM and mACP

• Proper orientation of substrates to enable the condensation reaction

Recent research demonstrates that mACP exhibits substrate sequestration and chain-flipping activity, which are crucial for protecting reactive intermediates and properly presenting them to partner enzymes like OXSM . This study provides an efficient approach toward understanding these fundamental protein-protein interactions, ultimately leading to insights into mitochondrial diseases such as fatty acid oxidation deficiencies .

What is the mechanistic relationship between OXSM and lipoic acid biosynthesis?

OXSM plays a critical role in lipoic acid biosynthesis through the following mechanism:

• OXSM catalyzes condensation reactions in the mtFAS II pathway, leading to the production of octanoyl-ACP

• The octanoyl group is transferred to specific protein lysine residues in mitochondrial enzyme complexes

• Subsequently, sulfur atoms are inserted into the octanoyl moiety by lipoic acid synthase

• The resulting lipoic acid functions as a cofactor for key mitochondrial enzymes including pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes

Disruption of OXSM function results in decreased lipoic acid content, which impairs these enzyme complexes, leading to mitochondrial dysfunction, increased reactive oxygen species production, and reduced cell viability .

What gene manipulation strategies are most effective for studying OXSM function?

Several genetic approaches have been validated for manipulating OXSM expression:

Verification of manipulation should include qPCR analysis using validated primers , western blotting with specific antibodies, and functional assays measuring lipoic acid production and mitochondrial function .

How can researchers distinguish between OXSM-specific effects and general mitochondrial dysfunction?

Distinguishing OXSM-specific effects from general mitochondrial dysfunction requires careful experimental design:

• Parallel manipulation of multiple mtFAS II pathway components

  • Compare OXSM knockdown with manipulation of other pathway enzymes

  • Identify common versus unique phenotypes

• Rescue experiments

  • Supplement with lipoic acid to bypass OXSM function

  • Reintroduce wild-type versus mutant OXSM to identify structure-function relationships

• Targeted metabolite analysis

  • Measure specific intermediates of the mtFAS II pathway

  • Quantify lipoylation of specific mitochondrial proteins

• Time-course studies

  • Determine the temporal sequence of events following OXSM disruption

  • Identify primary versus secondary consequences

• Compartment-specific markers

  • Monitor specific mitochondrial functions (membrane potential, respiration, ROS production)

  • Assess effects on other cellular compartments to determine specificity

This multi-faceted approach allows researchers to differentiate direct consequences of OXSM dysfunction from downstream or compensatory responses .

What model systems best reflect human OXSM function in health and disease?

Several experimental systems have been validated for studying human OXSM function:

• Cell culture models:

  • Human cell lines with high mitochondrial content (HepG2, primary hepatocytes)

  • Cells with active metabolism requiring functional mitochondria (myoblasts, neurons)

  • Patient-derived cells harboring OXSM mutations or variants

• In vitro reconstitution:

  • Purified recombinant OXSM and mACP for mechanistic studies

  • Isolated mitochondria for organelle-level functional analysis

• Animal models:

  • Mice with conditional OXSM knockout for tissue-specific studies

  • Zebrafish for developmental analysis of OXSM function

• Yeast models:

  • S. cerevisiae with human OXSM complementation

  • Simplified genetic system with conserved mtFAS II pathway

Each model system offers unique advantages, and the choice should be guided by the specific research question and desired experimental readouts .

How do fatty acid synthase inhibitors affect OXSM activity?

The fatty acid synthase inhibitor C75 (4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid) has been shown to affect OXSM and mitochondrial function:

• C75 shares conserved catalytic domains with FASN that are also present in OXSM, enabling potential binding and inhibition

• C75 treatment decreases cellular lipoic acid content, impairs mitochondrial function, increases reactive oxygen species, and reduces cell viability

• These effects parallel those observed with OXSM knockdown, suggesting OXSM inhibition as a potential mechanism

• Importantly, lipoic acid supplementation efficiently inhibits C75-induced mitochondrial dysfunction and oxidative stress

These findings indicate that FASN inhibitors designed as potential anticancer agents may have unintended effects on mitochondrial function through OXSM inhibition, which could contribute to both their therapeutic effects and adverse reactions .

What methods can detect OXSM inhibition versus cytoplasmic FASN inhibition?

Differentiating between OXSM and FASN inhibition requires specialized experimental approaches:

These complementary approaches provide a comprehensive assessment of whether an inhibitor primarily affects OXSM, FASN, or both enzymes .

How can OXSM function be targeted for research and potential therapeutic applications?

Several strategies can be employed to modulate OXSM function for research or therapeutic purposes:

• Structure-based inhibitor design:

  • Target unique structural features of OXSM that differ from FASN

  • Develop compounds that specifically disrupt OXSM-mACP interaction

  • Design allosteric inhibitors that selectively affect OXSM function

• Mitochondrial targeting:

  • Create mitochondrially-targeted delivery systems for OXSM modulators

  • Leverage differences in mitochondrial versus cytoplasmic environment

• Genetic modulation approaches:

  • Develop antisense oligonucleotides or siRNAs specific to OXSM

  • Employ CRISPR-based technologies for precise editing of OXSM

• Metabolic bypass strategies:

  • Lipoic acid supplementation to compensate for reduced OXSM function

  • Alternative pathway activation to support mitochondrial function

For any therapeutic application, careful evaluation of effects on mitochondrial function is essential, as OXSM inhibition may have widespread consequences for cellular energy metabolism and redox balance .

What are the current knowledge gaps in OXSM research?

Despite significant progress, several important aspects of OXSM biology remain to be elucidated:

• High-resolution structural information about human OXSM and its complexes with mACP

• Complete characterization of the regulatory mechanisms controlling OXSM expression and activity

• Tissue-specific roles of OXSM in different metabolic contexts

• Comprehensive understanding of OXSM variants and their impact on human health

• Development of selective pharmacological tools to modulate OXSM function

• Integration of OXSM activity with other mitochondrial pathways and cellular metabolic networks

Addressing these knowledge gaps will require interdisciplinary approaches combining structural biology, biochemistry, genetics, and systems biology .

How might OXSM dysfunction contribute to human disease?

Emerging evidence suggests OXSM dysfunction may play roles in several human diseases:

• Mitochondrial disorders:

  • Defects in lipoic acid synthesis affecting energy metabolism

  • Compromised function of lipoylated enzyme complexes

• Neurodegenerative diseases:

  • Increased oxidative stress and impaired mitochondrial quality control

  • Energy deficits in high-energy-demanding neuronal cells

• Metabolic disorders:

  • Disruption of mitochondrial fatty acid metabolism

  • Altered cellular redox balance affecting insulin signaling

• Cancer:

  • Metabolic reprogramming of mitochondrial function

  • Potential role in tumor cell survival under stress conditions

Understanding these connections may provide new insights into disease mechanisms and potential therapeutic approaches targeting mitochondrial fatty acid synthesis .

What technological advances would accelerate OXSM research?

Several technological developments would significantly advance OXSM research:

• Structural biology tools:

  • Cryo-EM structures of OXSM in complex with mACP and other pathway components

  • Time-resolved structural techniques to capture catalytic intermediates

• Metabolic flux analysis:

  • Improved methods for tracking mitochondrial fatty acid synthesis in intact cells

  • Single-cell approaches to capture heterogeneity in OXSM function

• Organellar proteomics:

  • More sensitive detection of low-abundance mitochondrial proteins

  • Methods to capture transient protein-protein interactions in intact mitochondria

• Genetic models:

  • Conditional and tissue-specific OXSM knockout mouse models

  • Patient-derived cellular models with precise genetic editing

• Chemical biology tools:

  • Development of selective OXSM probes and activity-based protein profiling tools

  • Mitochondrially-targeted small molecule modulators of OXSM function

These technological advances would enable more comprehensive investigation of OXSM biology and its role in human health and disease .