Recombinant Iron-dependent extradiol dioxygenase (hsaC)

What is HsaC and what is its role in Mycobacterium tuberculosis?

HsaC is an iron-dependent extradiol dioxygenase enzyme from Mycobacterium tuberculosis H37Rv that plays a critical role in cholesterol metabolism. It functions as a ring-cleaving dioxygenase, catalyzing the cleavage of catecholic substrates during cholesterol degradation. The enzyme shares approximately 40% amino acid sequence identity with BphC (EC 1.13.11.39), a well-characterized type I extradiol dioxygenase that cleaves 2,3-dihydroxybiphenyl (DHB) . HsaC utilizes Fe(II) in a 2-His 1-carboxylate facial triad coordination environment to catalyze the cleavage of catechols and their analogues, which is a typical feature of extradiol dioxygenases . Studies with gene deletion mutants have established HsaC's importance in cholesterol degradation and its association with pathogenicity, making it a significant enzyme in mycobacterial metabolism and virulence .

What is the substrate specificity of HsaC?

HsaC demonstrates notable substrate specificity that distinguishes it from related extradiol dioxygenases. Based on steady-state kinetic studies, HsaC exhibits 90-times greater specificity for the steroid metabolite DHSA (3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione) over DHB (2,3-dihydroxybiphenyl), which is the preferred substrate of the related enzyme BphC . This substrate preference reflects HsaC's specialized role in steroid metabolism.

The specificity values (kcat/Km) clearly demonstrate this preference:

• DHSA: 15 ± 2 μM⁻¹s⁻¹

• DHDS: 1.4 ± 0.1 μM⁻¹s⁻¹

• DHB: 0.16 ± 0.02 μM⁻¹s⁻¹

The enzyme also shows decreased catalytic efficiency toward chlorinated substrates, with 2′,6′-diCl DHB and 4-Cl DHDS being cleaved very slowly (partition ratios <50) . This substrate preference pattern aligns with HsaC's biological function in cholesterol catabolism rather than in degradation of aromatic pollutants like polychlorinated biphenyls.

How should HsaC be purified and stabilized for experimental studies?

For effective purification and stabilization of HsaC from M. tuberculosis H37Rv, researchers should follow these methodological guidelines:

• Purify the enzyme anaerobically to >99% apparent homogeneity from a recombinant E. coli strain expressing the enzyme.

• Verify iron content (properly purified enzyme should contain approximately 0.92 equivalents of iron) .

• For steady-state kinetic assays, stabilize the enzyme by diluting it in 20 mM HEPES, 80 mM NaCl, pH 7.5 supplemented with:

  • 5% t-butanol

  • 2 mM dithiothreitol

  • 0.1 mg/ml bovine serum albumin

• Store the diluted enzyme on ice under an inert atmosphere to prevent oxidation.

• For kinetic studies, use buffer equilibrated with 5% oxygen in nitrogen to obtain better quality data and prevent oxidative inactivation of both the enzyme and substrate (particularly DHSA) .

This careful handling is essential because both HsaC and its substrates are susceptible to oxidative inactivation in air-saturated buffer, which can significantly affect experimental results.

What is the oxygen dependence of HsaC?

HsaC demonstrates interesting oxygen kinetics that are relevant to its physiological role in M. tuberculosis. The apparent KmO₂ of HsaC was determined to be 90 ± 20 μM, which is 13-fold less than that of the related enzyme BphC . This KmO₂ value is also nearly 3-times less than the concentration of O₂ in air-saturated buffer, indicating that HsaC requires relatively high oxygen concentrations for optimal activity .

Despite this lower oxygen affinity, the specificity of HsaC for O₂ (0.20 ± 0.01 μM⁻¹s⁻¹) is only 5-times less than that of BphC (1.0 ± 0.1 μM⁻¹s⁻¹) . This oxygen requirement is particularly relevant considering the hypoxic environment of tuberculous granulomas, where oxygen tensions can be significantly reduced compared to uninfected lungs . It's worth noting that HsaC's KmO₂ is almost two orders of magnitude greater than that of some extradiol dioxygenases isolated from hypoxic soil environments, suggesting that this enzyme and the cholesterol catabolic pathway of M. tuberculosis have not evolved to function optimally in extremely hypoxic environments .

How does the crystal structure of HsaC inform our understanding of its substrate binding?

The crystal structure of HsaC provides significant insights into its substrate binding mechanisms. HsaC has been crystallized in both substrate-free form and in complex with DHSA, revealing important structural features:

• The substrate-binding pocket of HsaC (550 ų) is larger than that of BphC (420 ų), accommodating the bulkier steroid substrate .

• Two key structural differences contribute to this enlarged binding pocket:

  • The loop-helix-loop segment (residues 172-190) angles outwards and contains a 6-residue insertion compared to BphC, increasing the opening by up to 10 Å

  • The distal portion of the substrate-binding pocket is lined with fewer bulky residues (e.g., Leu174, Met207, and Val214 in HsaC versus Met175, Phe202, and His209 in BphC)

Most interestingly, crystallographic studies reveal that DHSA binds in different modes in the two molecules of the asymmetric unit:

• In molecule A: The catecholic ring coordinates the Fe in a bidentate manner

• In molecule B: The catecholic ring coordinates the Fe in a monodentate manner

The asymmetric bidentate binding in molecule A corresponds to what has been reported in other extradiol dioxygenase:substrate complexes. In this binding mode, the proximal hydroxyl (O4) binds the Fe trans to His145, and the distal hydroxyl (O3) binds trans to His215, displacing the two water molecules present in the resting state enzyme .

The monodentate binding observed in molecule B was unexpected but provides potential insights into the initial substrate-binding steps of extradiol dioxygenases. This configuration may represent an intermediate step in the substrate binding process, consistent with proposed mechanisms where initial binding of the catechol to the iron is monodentate .

Key crystallographic properties and refinement statistics are summarized in the following table:

What mechanisms contribute to the catalytic inactivation of HsaC?

HsaC is notably susceptible to oxidative inactivation during catalytic turnover, which is a critical consideration for researchers working with this enzyme. Several key mechanisms contribute to this inactivation:

The partition ratio data clearly illustrates this susceptibility to inactivation:

Unlike some other bacteria with meta-cleavage pathways that have recruited a ferredoxin to reduce adventitiously oxidized iron, BLAST searches indicate that the M. tuberculosis genome does not encode such a ferredoxin . This may explain the heightened susceptibility of HsaC to oxidative inactivation compared to other extradiol dioxygenases.

How does the catalytic mechanism of HsaC compare to other extradiol dioxygenases?

The catalytic mechanism of HsaC follows the general pathway established for extradiol dioxygenases but with specific characteristics that reflect its role in steroid degradation:

• Initial substrate binding: HsaC exhibits both bidentate and monodentate binding modes of the catecholic substrate to the Fe(II) center, with the monodentate mode likely representing an early intermediate in the binding process . This observation supports a proposed multi-step binding mechanism.

• Oxygen activation: Following substrate binding, O₂ binds to the Fe(II) center at a site defined by Val147, Phe192, and Ala203 (corresponding to Val148, Phe187, and Ala198 in BphC) . This leads to the formation of an Fe(II)-bound alkylperoxo intermediate.

• Catalytic steps: The mechanism proceeds through several proposed steps:

  • Heterolytic O-O bond cleavage

  • Criegee rearrangement involving 1,2-alkenyl migration

  • Formation of a lactone intermediate

  • Hydrolysis to form the ring-cleaved product

The HsaC mechanism appears to be fundamentally similar to that of other extradiol dioxygenases like BphC and homoprotocatechuate 2,3-dioxygenase (HPCD), but with structural adaptations to accommodate steroid substrates. The observation of both monodentate and bidentate binding modes in the crystal structure provides valuable experimental evidence supporting the multi-step binding of catecholic substrates proposed in the catalytic cycle .

Unique to HsaC is its significantly higher susceptibility to oxidative inactivation during turnover compared to other extradiol dioxygenases, which may reflect evolutionary adaptation to the specific physiological environment of M. tuberculosis .

What are the implications of HsaC research for tuberculosis pathogenesis and drug development?

Research on HsaC has significant implications for understanding tuberculosis pathogenesis and developing novel therapeutic approaches:

• Role in cholesterol metabolism: HsaC is a critical enzyme in the cholesterol degradation pathway of M. tuberculosis. Studies with hsaC-null gene deletion mutants have demonstrated the importance of this enzyme in cholesterol degradation and pathogenicity .

• Potential drug target: HsaC's essential role in cholesterol metabolism makes it a potential drug target. The detailed structural and mechanistic insights provided by crystallographic and kinetic studies offer a foundation for structure-based drug design .

• Inhibitor development: The study identified potential inhibitors of HsaC:

  • 2′,6′-diCl DHB, a PCB metabolite that potently inhibits and oxidatively inactivates BphC, also affects HsaC

  • 4-Cl DHDS, a chlorinated substrate analogue, shows potential as a competitive inhibitor

• Adaptation to host environment: HsaC's oxygen kinetics (KmO₂ = 90 ± 20 μM) suggest that while it is adapted to function in the relatively hypoxic environment of tuberculous granulomas, it has not evolved to function optimally in extremely hypoxic environments . This provides insights into the ecological niche occupied by M. tuberculosis during infection.

• Vulnerability to oxidative inactivation: HsaC's susceptibility to oxidative inactivation could be exploited in drug development. Compounds that promote the oxidation of the catalytically essential iron might serve as effective inhibitors .

The susceptibility of HsaC to inactivation by certain compounds, combined with its essential role in cholesterol metabolism, makes it a promising target for the development of novel anti-tuberculosis therapeutics.

What experimental techniques are optimal for measuring HsaC activity and inactivation?

For researchers studying HsaC, optimizing experimental techniques for measuring enzyme activity and inactivation is critical. Based on published research, the following methodological approaches are recommended:

• Enzyme preparation:

  • Purify HsaC anaerobically to >99% apparent homogeneity

  • Verify iron content (properly purified enzyme should contain approximately 0.92 equivalents of iron)

  • Stabilize the enzyme in 20 mM HEPES, 80 mM NaCl, pH 7.5 with additions of 5% t-butanol, 2 mM dithiothreitol, and 0.1 mg/ml bovine serum albumin

  • Store on ice under an inert atmosphere

• Activity assays:

  • Conduct kinetic studies using buffer equilibrated with 5% oxygen in nitrogen to prevent oxidative inactivation

  • For oxygen dependence studies, use DHDS as substrate due to ease of preparation (reactivity with O₂ will be similar for DHDS and DHSA as they have similarly substituted catecholic rings)

  • Measure steady-state kinetic parameters at 25°C in 20 mM HEPES, 80 mM NaCl, pH 7.0 (I = 0.1)

• Inactivation studies:

  • Determine partition ratios (moles of substrate consumed per mole of enzyme inactivated) to quantify susceptibility to inactivation

  • For comparative inactivation studies across substrates, standardize experimental conditions (oxygen concentration, pH, temperature)

  • When working with chlorinated compounds (like 2′,6′-diCl DHB or 4-Cl DHDS), account for their different modes of inactivation (steric vs. electronic)

The steady-state kinetic and inactivation parameters should be determined under standardized conditions to allow for meaningful comparisons across different substrates. The table below provides an example of the comprehensive data that should be collected:

How can structural studies of HsaC be optimized for capturing different catalytic states?

Capturing different catalytic states of HsaC is crucial for understanding its reaction mechanism. Based on successful approaches in the literature, researchers should consider these methodological strategies:

• Crystallization conditions:

  • For substrate-free HsaC: Use Cu-Kα radiation source with a wavelength of 1.542 Å

  • For HsaC:DHSA complex: Consider synchrotron sources (e.g., ALS 8.2.2) with a wavelength of 1.000 Å

  • Use space group P42₁2 with unit cell parameters approximately a = b = 123.7-124.3 Å, c = 106.3-106.7 Å

• Capturing different binding modes:

  • Soak crystals anaerobically with substrate to preserve the Fe(II) oxidation state

  • Examine multiple molecules in the asymmetric unit, as they may capture different catalytic states (as seen with the bidentate and monodentate binding modes in HsaC:DHSA complexes)

  • Different crystal packing forces may stabilize different intermediate states, so analyze multiple crystal forms if possible

• Validation techniques:

  • Use ligand-omit and Fo-Fc difference maps calculated using phases derived from the model in the absence of any ligands to validate the presence and orientation of bound substrates

  • When multiple binding modes are observed, consider alternative interpretations and validate through refinement (checking for positive residual density and temperature factors)

• Data collection and refinement:

  • Aim for high-resolution data (2.0-2.2 Å or better)

  • Use Rfree/Rfactor values to assess model quality (typically 22/18 for substrate-free and 26/19 for substrate-bound structures)

  • Analyze mean B values for protein, Fe, and substrate to evaluate the confidence in the model

The observation of different binding modes in different molecules of the asymmetric unit in HsaC:DHSA complexes demonstrates that crystallographic studies can capture mechanistically significant intermediates . This approach is reminiscent of successful studies with HPCD, where three different catalytic intermediates were trapped in different protein molecules of a single crystal .

How can researchers design effective inhibitors for HsaC based on structural and mechanistic insights?

Designing effective inhibitors for HsaC requires leveraging the structural and mechanistic insights gleaned from crystallographic and kinetic studies. Researchers should consider the following methodological approaches:

• Structure-based design strategies:

  • Target the Fe(II) coordination site: Develop compounds that can coordinate to the iron center in either a bidentate or monodentate manner

  • Exploit the larger binding pocket: Design compounds that fit the 550 ų substrate-binding pocket of HsaC, taking advantage of the outward-angled loop-helix-loop segment (residues 172-190)

  • Focus on the O₂-binding site: Design compounds that partially occlude the O₂-binding site defined by Val147, Phe192, and Ala203

• Mechanism-based inhibitor development:

  • Develop compounds that promote oxidative inactivation, given HsaC's susceptibility to this mode of inactivation

  • Consider two distinct inhibition strategies based on known mechanisms:
    a. Steric inhibition: Similar to 2′,6′-diCl DHB, which partially blocks the O₂-binding site
    b. Electronic modification: Similar to 4-Cl DHDS, utilizing electron-withdrawing groups on the catecholic ring

• Substrate analog development:

  • Use DHDS as a starting point for inhibitor design, as it incorporates key features of the natural substrate DHSA (including the methyl group on the catecholic ring and a saturated 2-carbon bridge between the two ring systems)

  • Modify the catecholic moiety to increase binding affinity while preventing catalytic turnover

  • Ensure the bicycloalkanone moiety is positioned similarly to DHSA in both binding modes, as this appears to be a determinant in initial enzyme-substrate complex formation

• Screening and validation methodologies:

  • Test candidate inhibitors for both competitive inhibition (Kic values) and inactivation efficiency (partition ratios)

  • Verify inhibition mechanisms through crystallographic studies of enzyme-inhibitor complexes

  • Assess specificity by comparing effects on HsaC versus related enzymes like BphC

The significantly different Km values of HsaC for chlorinated compounds compared to BphC (approximately 1,000-fold greater) suggest that achieving selectivity for HsaC over other extradiol dioxygenases is feasible . This selective inhibition would be valuable for targeting M. tuberculosis while minimizing effects on commensal bacteria.

What is the relationship between HsaC function and oxygen availability in tuberculous granulomas?

The relationship between HsaC function and oxygen availability in tuberculous granulomas represents an important area for future research. Current data indicates that HsaC has a KmO₂ of 90 ± 20 μM, which is below the oxygen concentration in air-saturated buffer but significantly higher than oxygen levels in hypoxic tuberculous granulomas .

Future investigations should address:

• HsaC activity under physiologically relevant oxygen concentrations:

  • Measure enzymatic activity at oxygen concentrations matching those in tuberculous granulomas (approximately 3% of uninfected lungs)

  • Determine if HsaC activity becomes rate-limiting for cholesterol metabolism under hypoxic conditions

• Adaptation mechanisms:

  • Investigate whether M. tuberculosis employs alternative mechanisms to compensate for reduced HsaC activity under hypoxic conditions

  • Explore potential post-translational modifications that might alter HsaC's oxygen affinity in vivo

  • Examine whether gene expression levels of HsaC are upregulated under hypoxic conditions to compensate for reduced specific activity

• Reductase partners:

  • Search for potential electron-transfer proteins that might reduce adventitiously oxidized HsaC iron, similar to the ferredoxin recruited by the xylene catabolic pathway of Pseudomonas putida mt-2

  • Determine if M. tuberculosis uses alternative mechanisms to protect HsaC from oxidative inactivation during infection

• Implications for persistence:

  • Investigate whether limitations in HsaC activity under hypoxic conditions contribute to the shift toward dormancy and persistence observed in M. tuberculosis within granulomas

  • Examine the correlation between oxygen availability, HsaC activity, and cholesterol metabolism rates during different phases of infection

This research area is particularly relevant given the importance of cholesterol metabolism for M. tuberculosis survival in the host and the hypoxic nature of the granuloma environment where the bacterium persists.

How can the different substrate binding modes of HsaC inform our understanding of extradiol dioxygenase catalysis?

The observation of both bidentate and monodentate binding modes in HsaC:DHSA crystal structures provides a unique opportunity to investigate the catalytic mechanism of extradiol dioxygenases. Future research should explore:

• Temporal sequence of binding events:

  • Develop time-resolved crystallographic approaches to determine if the monodentate binding mode is indeed an early intermediate that precedes the bidentate mode

  • Use rapid kinetic approaches (stopped-flow spectroscopy) to characterize the kinetics of substrate binding steps

  • Determine whether all substrates follow the same binding progression or if alternative pathways exist

• Factors influencing binding mode selection:

  • Investigate how substrate structure affects the preference for monodentate versus bidentate binding

  • Explore how protein dynamics and active site architecture influence binding mode selection

  • Examine whether binding mode correlates with catalytic efficiency or susceptibility to inactivation

• Computational studies:

  • Use molecular dynamics simulations to model the transition between monodentate and bidentate binding modes

  • Calculate energy profiles for different binding modes to determine the most favorable pathway

  • Predict how mutations in the active site might affect binding mode preferences

• Comparative analysis across extradiol dioxygenases:

  • Determine if the binding modes observed in HsaC are universal features across extradiol dioxygenases

  • Compare with other enzymes like HPCD where multiple catalytic intermediates have been observed in crystal structures

  • Develop a unified model of substrate binding for extradiol dioxygenases

This research direction could significantly advance our understanding of the fundamental mechanisms of extradiol dioxygenase catalysis, with potential implications for enzyme engineering and inhibitor design.