Recombinant Human Acetolactate synthase-like protein (ILVBL)

What is ILVBL and what are its key functions?

ILVBL (IlvB Acetolactate Synthase Like) is a protein-coding gene located on chromosome 19p13.1 that encodes a 632-amino-acid protein sharing similarity with thiamine pyrophosphate-binding proteins identified in bacteria, yeast, and plants . Functionally, it serves as an endoplasmic reticulum 2-OH acyl-CoA lyase involved in the cleavage (C1 removal) reaction in fatty acid alpha-oxidation through a thiamine pyrophosphate (TPP)-dependent mechanism . The protein is particularly involved in the phytosphingosine degradation pathway, demonstrating its importance in lipid metabolism . ILVBL shows the highest homology with bacterial enzymes such as the B isozyme of the large catalytic subunit of Escherichia coli acetohydroxy-acid synthase (AHAS) and the oxalyl-CoA decarboxylase of Oxalobacter formigenes .

How does ILVBL relate to other proteins in its family?

ILVBL belongs to a family of thiamine pyrophosphate (TPP)-binding proteins with significant sequence conservation across species. It shares structural and functional similarities with acetolactate synthases from multiple organisms including Bacillus subtilis (PDB ID: 4RJJ), Arabidopsis thaliana (PDB ID: 1Z8N), Klebsiella pneumoniae (PDB ID: 1OZG), and Saccharomyces cerevisiae (PDB ID: 1T9B) . The protein contains highly conserved active site residues, including K38 and Q485, which participate in catalytic mechanisms similar to those observed in K. pneumoniae, while Q122, Q422, M481, and Y490 are important for ThDP localization, as confirmed in B. subtilis . An important paralog of ILVBL is HACL1, which performs similar metabolic functions . Understanding these evolutionary relationships provides insight into the conserved functional mechanisms of ILVBL.

What are the optimal methods for expressing recombinant human ILVBL?

For successful expression of recombinant human ILVBL, researchers should consider a systematic approach based on established protocols for similar TPP-dependent enzymes. Expression in E. coli systems has proven effective for bacterial homologs of acetolactate synthase, as demonstrated in studies with B. licheniformis ALS . When expressing human ILVBL, consider using a pET expression system with BL21(DE3) E. coli strains, which provide tight expression control through IPTG induction.

The expression protocol should include:

• Codon optimization for the host organism

• Addition of an affinity tag (His6) for purification

• Culture at 28-30°C rather than 37°C after induction to improve protein folding

• Supplementation with thiamine pyrophosphate (TPP) and Mg²⁺ in the growth medium

Expression yield can be monitored through SDS-PAGE and Western blotting. For human ILVBL specifically, supplementing with 0.5 mM ThDP and 2 mM MgCl₂ in the buffer system is critical for maintaining enzymatic activity during and after purification, as these cofactors are essential for the protein's catalytic function .

How can researchers assess and improve the acid tolerance of recombinant ILVBL?

To assess acid tolerance of recombinant ILVBL, researchers should implement a systematic approach similar to that used for bacterial acetolactate synthases. The methodology involves:

• Incubating the purified enzyme at different pH values (typically pH 4.0-8.0) for a standardized period (e.g., 2 hours)

• Measuring residual enzyme activity under optimal conditions

• Calculating the half-life of enzyme activity at acidic pH values

For improving acid tolerance, structure-based mutagenesis has proven effective. The conventional method involves converting alkaline residues to acidic residues . Based on homology modeling and sequence alignment analysis, researchers should identify conserved alkaline residues on the protein surface. For example, in B. licheniformis ALS, mutations like N210D and H399D significantly improved acid tolerance by:

• Increasing the negative charge on the protein surface

• Forming new hydrogen bond networks that stabilized the protein structure

• Modifying the electrostatic potential around the active site

When applying this approach to human ILVBL, researchers should:

• Generate a reliable homology model using SWISS-MODEL

• Identify surface-exposed alkaline residues through conservation analysis

• Design single and double mutants

• Test mutants for acid tolerance and catalytic efficiency

A successful mutation strategy should extend the half-life at acidic pH while maintaining comparable catalytic parameters (Km and Vmax) to the wild-type enzyme.

What are the critical catalytic residues and cofactor binding sites in ILVBL?

Based on structural studies of homologous acetolactate synthases, ILVBL contains several critical catalytic residues and cofactor binding sites essential for its enzymatic function. The protein requires thiamine pyrophosphate (ThDP) and Mg²⁺ as cofactors for catalytic activity .

Key residues identified through homology modeling and structural comparison include:

The active site structure is highly conserved across species, with the ThDP binding pocket forming a crucial part of the catalytic center . Studies on bacterial homologs have shown that mutations near the active site, such as H399D in B. licheniformis ALS, can alter ThDP binding and change Mg²⁺ coordination from 6 to 4, potentially affecting catalytic efficiency but also improving protein stability under acidic conditions .

Researchers investigating human ILVBL should focus on these conserved motifs when designing site-directed mutagenesis experiments to probe structure-function relationships or to engineer enzymes with modified properties.

How can structural modeling inform the design of ILVBL mutations for improved functionality?

Structural modeling provides critical insights for rational design of ILVBL mutations to enhance functionality. Using comparative modeling based on homologous proteins with known crystal structures can yield reliable structural predictions. For ILVBL, researchers should:

• Generate a homology model using multiple templates (e.g., structures from B. subtilis, A. thaliana, K. pneumoniae, and S. cerevisiae)

• Validate the model quality using metrics such as GMQE, QMEAN, and MolProbity scores

• Analyze surface electrostatic potential to identify regions susceptible to pH changes

• Examine hydrogen bonding networks for opportunities to enhance stability

When selecting mutation sites, focus on three key areas:

• Surface-exposed alkaline residues that could be converted to acidic residues to improve acid tolerance

• Residues near but not directly in the active site that could enhance substrate specificity

• Residues involved in subunit interactions that might affect oligomeric stability

The success of this approach is demonstrated in bacterial ALS, where mutations like N210D and H399D significantly enhanced acid tolerance by increasing negative surface charge and forming new stabilizing hydrogen bonds . The N210D mutation altered surface electrostatic potential, while H399D formed a new hydrogen bond with G448, improving local conformational stability without disrupting catalytic function .

For visualization and analysis, researchers should use molecular modeling software such as PyMOL or UCSF Chimera to compare electrostatic surface potentials before and after virtual mutations, as shown in studies where surface charge distribution significantly affected protein stability in acidic environments .

What is the evidence linking ILVBL gene polymorphisms to aspirin-exacerbated respiratory disease?

Research has established a significant association between ILVBL gene polymorphisms and aspirin-exacerbated respiratory disease (AERD). A comprehensive study involving 141 AERD patients and 995 aspirin-tolerant asthmatic (ATA) subjects demonstrated that specific single nucleotide polymorphisms (SNPs) in the ILVBL gene on chromosome 19p13.1 correlate with both AERD risk and the percent decline in forced expiratory volume in one second (FEV1) after oral aspirin challenge .

The investigation methodology involved:

• Subject recruitment from nine university hospitals in Korea

• Diagnosis confirmation according to the Global Initiative for Asthma (GINA) guidelines

• AERD and ATA determination using oral aspirin challenge (OAC) tests

• Selection and genotyping of nine SNPs with minor allele frequencies above 0.05 in the ILVBL gene region

The study focused on polymorphisms in the region from 2 kb upstream to 0.5 kb downstream of ILVBL, specifically selecting SNPs from the Asian population database of the International HapMap Project . This methodological approach ensures a comprehensive examination of genetic variations across the ILVBL locus, providing robust evidence for its association with AERD. Although the direct mechanistic link between ILVBL (which is likely involved in branched-chain amino acid or pyruvate metabolism) and aspirin metabolism remains unclear, these genetic associations suggest a potential role for ILVBL in the pathophysiology of AERD .

How might ILVBL be involved in the pathophysiology of other diseases?

ILVBL has been associated with multiple diseases beyond aspirin-exacerbated respiratory disease, including conduct disorder and potentially various metabolic conditions . Given its role in fatty acid alpha-oxidation and the phytosphingosine degradation pathway, ILVBL dysfunction may impact lipid metabolism and subsequently contribute to disease development through several potential mechanisms:

• Altered lipid metabolism leading to accumulation of toxic intermediates

• Disruption of membrane structure and function due to abnormal lipid composition

• Impaired signaling pathways that rely on lipid-derived second messengers

• Dysregulation of inflammatory responses through altered eicosanoid metabolism

The connection between ILVBL and aspirin sensitivity suggests potential involvement in inflammatory pathways. Since ILVBL shows homology with acetolactate synthases involved in branched-chain amino acid metabolism, its dysfunction might also affect amino acid homeostasis, potentially impacting neurotransmitter synthesis in the case of conduct disorder .

Research approaches to investigate these connections should include:

• Transcriptomic analysis of ILVBL expression in affected tissues

• Metabolomic profiling to identify altered metabolic pathways

• Functional studies using siRNA knockdown or CRISPR/Cas9 gene editing

• Animal models with tissue-specific ILVBL mutations to study phenotypic effects

Understanding these broader disease associations requires interdisciplinary approaches combining biochemistry, genetics, and clinical research to establish causative relationships rather than mere correlations.

How can site-directed mutagenesis be optimized to enhance ILVBL enzymatic properties?

Optimizing site-directed mutagenesis for ILVBL requires a sophisticated approach combining structural analysis, computational prediction, and iterative experimentation. Based on successful strategies applied to bacterial acetolactate synthases, researchers should implement a multi-phase optimization protocol:

Phase 1: In silico prediction and design

• Generate a high-quality homology model of human ILVBL using multiple templates

• Perform molecular dynamics simulations at varying pH conditions to identify structurally unstable regions

• Calculate pKa shifts of ionizable residues under different conditions

• Use algorithms like PROPKA or H++ to identify residues with abnormal titration behavior

• Design mutation candidates focusing on three categories:

  • Surface charge modifications (H→D, K→E, R→D)

  • Hydrogen bond network enhancements

  • Stability-enhancing secondary structure modifications

Phase 2: Experimental screening and validation

• Create a library of single mutants using overlap extension PCR

• Express mutants in parallel using a high-throughput expression system

• Develop a rapid screening assay for acid tolerance and catalytic efficiency

• Test promising candidates under varying conditions of:

  • pH (4.0-8.0)

  • Temperature (25-50°C)

  • Substrate concentration

  • Cofactor availability

Phase 3: Combinatorial optimization

• Combine beneficial mutations that affect different aspects of protein structure

• Test for additive or synergistic effects on stability and activity

• Analyze structural changes using circular dichroism and fluorescence spectroscopy

• Determine enzyme kinetics parameters for promising double or triple mutants

This approach has proven successful with bacterial acetolactate synthases where double mutants like N210D–H399D demonstrated significantly improved acid tolerance, extending half-life at pH 4.0 from 0.8 hours (wild-type) to 2.2 hours . The mutation strategy should aim to increase negative surface charge while maintaining or enhancing local structural stability near the active site, as demonstrated by the formation of new hydrogen bonds that improved conformational stability in previous studies .

What are the challenges in correlating ILVBL genetic variants with clinical phenotypes?

Correlating ILVBL genetic variants with clinical phenotypes presents several methodological challenges that researchers must address through rigorous experimental design and statistical analysis:

Challenge 1: Phenotypic heterogeneity and definition
Different studies may use varying criteria to define clinical phenotypes such as aspirin-exacerbated respiratory disease (AERD). The gold standard should include:

• Oral aspirin challenge tests with standardized protocols

• Objective measurements (e.g., FEV1 decline)

• Clear inclusion/exclusion criteria for patient cohorts

• Comprehensive clinical characterization

Challenge 2: Population stratification and ethnicity effects
ILVBL variants may show population-specific effects, as demonstrated by the focus on Asian populations in some studies . Researchers should:

• Include adequate sample sizes from multiple ethnic backgrounds

• Perform ancestry informative marker analysis

• Use genomic control or principal component analysis to correct for population stratification

• Validate findings across different populations

Challenge 3: Gene-gene and gene-environment interactions
ILVBL functions within complex metabolic pathways and may interact with other genes or environmental factors:

• Analyze gene-gene interactions using statistical approaches like multifactor dimensionality reduction

• Assess gene-environment interactions through detailed environmental exposure history

• Consider epigenetic modifications through methylation analysis

• Use systems biology approaches to model pathway interactions

Challenge 4: Functional validation of genetic associations
Moving beyond statistical associations requires functional validation:

• Develop cell models expressing different ILVBL variants

• Use patient-derived cells when possible

• Implement CRISPR/Cas9 to create isogenic cell lines differing only in the ILVBL variant

• Perform metabolomic profiling to identify affected pathways

What novel technologies could advance ILVBL research beyond current methodological limitations?

Several cutting-edge technologies offer promising avenues to overcome current limitations in ILVBL research:

Cryo-electron microscopy (cryo-EM)
While traditional X-ray crystallography has been challenging for ILVBL, cryo-EM could determine its structure without crystallization. Researchers should:

• Purify ILVBL to high homogeneity (>95%)

• Optimize buffer conditions to prevent protein aggregation

• Use state-of-the-art direct electron detectors for high-resolution imaging

• Apply 3D classification and refinement for structural determination

AlphaFold2 and other AI-based structure prediction
Recent advances in AI-based protein structure prediction offer unprecedented accuracy:

• Generate AlphaFold2 models of human ILVBL

• Compare predictions with homology models based on bacterial templates

• Validate structural predictions through limited proteolysis and hydrogen-deuterium exchange

• Use predicted structures to guide rational enzyme engineering

Single-cell multi-omics approaches
To understand tissue-specific roles of ILVBL:

• Apply single-cell RNA-seq to identify cell types expressing ILVBL

• Use spatial transcriptomics to map expression patterns in tissues

• Combine with proteomics to correlate transcript and protein levels

• Integrate with metabolomics to identify cell-specific metabolic changes

CRISPR-based functional genomics
For systematic functional analysis:

• Implement CRISPR interference (CRISPRi) for graduated gene knockdown

• Use CRISPR activation (CRISPRa) to upregulate ILVBL expression

• Create precise point mutations mimicking disease-associated variants

• Develop high-throughput CRISPR screens for identifying synthetic lethal interactions

These approaches would substantially advance our understanding of ILVBL beyond what has been achieved with traditional methods in bacterial homologs, where techniques such as enzyme activity assays and basic mutagenesis have predominated .

How might understanding ILVBL function contribute to therapeutic approaches for related diseases?

Understanding ILVBL function could enable novel therapeutic approaches for diseases like aspirin-exacerbated respiratory disease (AERD) and potentially other conditions associated with ILVBL dysfunction. Strategic research directions should focus on:

Precision medicine approaches based on ILVBL genotyping

• Develop rapid, cost-effective genotyping assays for clinically relevant ILVBL variants

• Conduct prospective clinical trials to determine if genotype-guided therapy improves outcomes

• Establish predictive algorithms incorporating ILVBL genotype and other factors to estimate disease risk

• Design preventive interventions for high-risk individuals based on genetic profiles

Small molecule modulators of ILVBL activity

• Perform virtual screening against the ILVBL active site to identify potential inhibitors or activators

• Synthesize and test candidate compounds in enzyme activity assays

• Evaluate effects in cellular and animal models of disease

• Optimize lead compounds for pharmacokinetic properties and specificity

Metabolic bypass strategies

• Identify metabolic pathways that can compensate for altered ILVBL function

• Test dietary interventions that modify substrate availability or product formation

• Investigate supplementation with downstream metabolites to bypass ILVBL-dependent steps

• Develop targeted metabolic therapies for specific ILVBL-related metabolic defects

Gene therapy and advanced biologics

• Design gene therapy approaches for severe ILVBL deficiencies

• Develop antisense oligonucleotides for modulating ILVBL expression

• Create engineered enzymes with enhanced properties for enzyme replacement therapy

• Investigate mRNA-based approaches for temporary ILVBL supplementation

The foundational knowledge from bacterial homologs, where mutations like N210D and H399D significantly altered enzyme properties and improved productivity in fermentation settings , provides a template for rational enzyme engineering that could be applied therapeutically. Additionally, understanding the genetic associations between ILVBL variants and disease phenotypes enables stratification of patients for clinical trials and personalized treatment approaches.