Recombinant Oceanobacillus iheyensis Cardiolipin synthase (cls)

What is Oceanobacillus iheyensis and why is its Cardiolipin synthase significant?

Oceanobacillus iheyensis is an alkaliphilic and extremely halotolerant Bacillus-related species that was isolated from deep-sea sediment collected at a depth of 1050 meters on the Iheya Ridge. This Gram-positive bacterium is strictly aerobic, rod-shaped, motile by peritrichous flagella, and spore-forming . It has remarkable adaptability to extreme environments, capable of growing at salinities of 0-21% (w/v) NaCl at pH 7.5 and 0-18% at pH 9.5, with an optimum NaCl concentration of 3% for growth at both pH values .

The cardiolipin synthase (cls) from O. iheyensis is particularly significant because it functions in these extreme conditions. Cardiolipin is a critical phospholipid in bacterial membranes that contributes to membrane stability and functionality, especially under stress conditions. Understanding how this enzyme functions in extreme environments can provide insights into membrane adaptation mechanisms and potential biotechnological applications.

What are the structural characteristics of recombinant O. iheyensis Cardiolipin synthase?

The recombinant full-length Oceanobacillus iheyensis Cardiolipin synthase protein consists of 479 amino acids (1-479aa) . The protein has a UniProt ID of Q8EM16 and is also known by synonyms cls, OB3045, Cardiolipin synthase, and CL synthase .

The complete amino acid sequence is:
MGITSLLLGLTFVLNIALAISIIFLERKDPTSSWAWVMVLLFIPILGFFLYLIFGKPISN RKIFSWDKKSRLGVKTTVQSQLRLLEENQFEFNQPDLIEHKDLVYLHLKNDEAIYTQNNG VDIFTDGQTKFDALLEDIEKAKKHIHIQYYIMRSDGLGNRLADMLIKKVNEGVEVRVLYD DMGSRSLKNSYIKRLKRAGVMVEAFFPSRFIVNFKINYRNHRKLAIIDGYIGYLGGFNVG DEYLGINKKFGYWRDTHLRVIGDAVQSMQTRFILDWNQASRDTILYNEDYYQTVSAGNVG MQIVTSGPDSEYEQIKNGYIKMIMEANDYICIQTPYFIPDESLRDALKIAVLSGVHVKIM IPNKPDHPFVYWATLSYCGDLIQAGAEIFIYQNGFLHAKTIIVDGRIASVGTANIDVRSF RLNFEVNGFLYDSEVVNRLQNEFDADLEKSTQMTRKLYDQRSIGIRFKESISRLISPVL

While the detailed three-dimensional structure has not been fully characterized in the provided search results, the protein is likely to contain transmembrane domains consistent with its role in membrane lipid synthesis.

How is recombinant O. iheyensis Cardiolipin synthase typically expressed?

Recombinant Oceanobacillus iheyensis Cardiolipin synthase is typically expressed in Escherichia coli expression systems. The commercially available recombinant protein is produced with an N-terminal His-tag fusion to facilitate purification . The full-length protein (residues 1-479) is expressed, which ensures that all functional domains are present and the protein maintains its native activity.

The expression system utilizes standard molecular biology techniques:

• The cls gene is cloned into an appropriate expression vector with a His-tag sequence.

• The construct is transformed into a suitable E. coli strain.

• Expression is induced under optimized conditions.

• Cells are harvested and lysed to release the recombinant protein.

• The protein is purified using affinity chromatography targeting the His-tag.

What are the recommended storage and handling conditions for the recombinant protein?

The purified recombinant Oceanobacillus iheyensis Cardiolipin synthase protein is typically provided as a lyophilized powder . For optimal stability and activity, the following storage and handling recommendations should be observed:

• Store the lyophilized protein at -20°C/-80°C upon receipt.

• Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles.

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL.

• Add 5-50% glycerol (final concentration) and aliquot for long-term storage at -20°C/-80°C.

• For working aliquots, store at 4°C for up to one week .

The reconstituted protein is typically stored in a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0 , which helps maintain protein stability.

How does the function of Cardiolipin synthase relate to O. iheyensis' extremophile nature?

Oceanobacillus iheyensis thrives in highly alkaline and saline environments, which poses significant bioenergetic challenges. The membrane composition, particularly the presence of cardiolipin, is crucial for maintaining membrane integrity and functionality under these extreme conditions.

Cardiolipin synthase produces cardiolipin, a dimeric phospholipid that plays essential roles in:

• Membrane stabilization: Cardiolipin creates regions of high curvature in the membrane, which can influence protein organization and function.

• Osmotic adaptation: The lipid composition of the membrane, including cardiolipin content, helps regulate the cell's response to osmotic stress in high salinity environments.

• pH homeostasis: Membrane composition affects the proton permeability and contributes to the cell's ability to maintain a cytoplasmic pH below the external pH in alkaline environments .

The genome of O. iheyensis encodes numerous proteins associated with the regulation of intracellular osmotic pressure and pH homeostasis . Cardiolipin synthase is likely part of this adaptation machinery, contributing to the bacterium's ability to maintain membrane function under extreme conditions.

What experimental approaches can be used to characterize the enzyme kinetics?

To characterize the enzyme kinetics of Oceanobacillus iheyensis Cardiolipin synthase, researchers can employ several methodologies:

• Spectrophotometric assays:

  • Monitor the consumption of substrates or production of byproducts spectrophotometrically

  • Use coupled enzyme assays to link cardiolipin production to a detectable signal

• Radiometric assays:

  • Use radiolabeled substrates to track the formation of cardiolipin

  • Separate reaction products by thin-layer chromatography and quantify radioactivity

• HPLC analysis:

  • Separate and quantify reaction products using HPLC

  • Can be coupled with mass spectrometry for detailed product characterization

• pH and salt concentration studies:

  • Characterize enzyme activity across a range of pH values (7.0-10.5) and salt concentrations (0-21% NaCl)

  • Determine optimal conditions that reflect the enzyme's natural environment

• Temperature-dependent studies:

  • Assess activity across different temperatures to determine optimal conditions

  • Investigate thermal stability relevant to the deep-sea origin of the organism

How can structural biology approaches enhance our understanding of this enzyme?

Structural biology approaches can provide critical insights into the function and mechanism of Oceanobacillus iheyensis Cardiolipin synthase:

• X-ray crystallography:

  • Determine the three-dimensional structure at atomic resolution

  • Identify active site residues and substrate binding pockets

  • Reveal structural adaptations for extremophile conditions

• Cryo-electron microscopy:

  • Visualize the protein in different conformational states

  • Study membrane integration of the enzyme

• NMR spectroscopy:

  • Investigate protein dynamics in solution

  • Characterize substrate binding events

• Molecular dynamics simulations:

  • Model protein behavior in different pH and salt conditions

  • Predict structural changes during catalysis

• Site-directed mutagenesis:

  • Confirm the role of predicted active site residues

  • Investigate the importance of specific amino acids in extremophile adaptation

  • Create variants with modified catalytic properties

How does O. iheyensis Cardiolipin synthase compare to homologs from other bacteria?

Comparative analysis between Oceanobacillus iheyensis Cardiolipin synthase and homologs from other bacteria can reveal important evolutionary adaptations and functional differences:

The genome of O. iheyensis contains many genes potentially associated with adaptation to highly alkaline and saline environments . Comparative analysis with three Bacillus species and two other Gram-positive species has been performed to identify candidate genes involved in alkaliphily .

These comparisons suggest that O. iheyensis Cardiolipin synthase likely contains specific amino acid substitutions or structural features that optimize its function in extreme environments, potentially including:

• Increased proportion of acidic amino acids on the protein surface

• Specialized salt bridges or ion-binding sites

• Modifications in the active site to maintain catalytic efficiency at high pH

• Structural adaptations to maintain proper folding and stability at high salt concentrations

What is the relationship between Cardiolipin synthase and ATP synthase in alkaliphilic bacteria?

In alkaliphilic bacteria like Oceanobacillus iheyensis, there appears to be an important relationship between membrane lipid composition (influenced by Cardiolipin synthase) and ATP synthase function:

• Bioenergetic challenges:

  • Alkaliphilic bacteria face a significant bioenergetic challenge because at high external pH, the protonmotive force (PMF) is too low to account for the observed ATP synthesis .

  • The PMF is lowered because these bacteria maintain a cytoplasmic pH well below the external pH, creating an energetically adverse pH gradient .

• Membrane composition and energy coupling:

  • Cardiolipin plays a crucial role in organizing and stabilizing respiratory chain complexes and ATP synthase in the membrane.

  • Evidence suggests that alkaliphiles may use membrane-associated microcircuits between H+ pumping complexes and ATP synthases .

  • These microcircuits likely depend upon proximity of pumps and synthases, specific membrane properties, and adaptations of the participating enzyme complexes .

• Adaptations of ATP synthase:

  • ATP synthesis in alkaliphiles depends upon alkaliphile-specific adaptations of the ATP synthase .

  • The membrane lipid environment, influenced by Cardiolipin synthase activity, may play a role in these adaptations.

This relationship highlights the important interplay between membrane composition and bioenergetic processes in extremophilic bacteria.

What assays can be used to measure Cardiolipin synthase activity?

Several assays can be employed to measure the activity of recombinant Oceanobacillus iheyensis Cardiolipin synthase:

• Direct measurement of cardiolipin formation:

  • Thin-layer chromatography (TLC) with phospholipid staining

  • Liquid chromatography-mass spectrometry (LC-MS)

  • Use of fluorescently labeled or radiolabeled substrates

• Coupled enzyme assays:

  • Monitoring release of CMP or other reaction byproducts

  • Linking to secondary reactions that produce measurable signals

• Binding assays to evaluate substrate interactions:

  • Isothermal titration calorimetry (ITC)

  • Surface plasmon resonance (SPR)

  • Fluorescence-based binding assays

• pH-dependent activity profiling:

  • Measurement of activity across a pH range of 7.0-10.5 to determine pH optima

  • Comparison with non-alkaliphilic cardiolipin synthases

• Salt-dependent activity profiling:

  • Measurement of activity across salt concentrations from 0-21% NaCl

  • Determination of optimal salt conditions for enzyme function

How can recombinant Cardiolipin synthase be incorporated into model membrane systems?

To study the function of Oceanobacillus iheyensis Cardiolipin synthase in a more native-like environment, researchers can incorporate the recombinant enzyme into various model membrane systems:

• Liposome reconstitution:

  • Prepare liposomes with lipid compositions resembling bacterial membranes

  • Incorporate purified recombinant cls protein using detergent-mediated reconstitution

  • Measure enzyme activity within the liposomal system

• Nanodiscs:

  • Incorporate cls into nanodiscs for a more defined membrane environment

  • Allow for controlled lipid composition and better accessibility for structural studies

• Giant unilamellar vesicles (GUVs):

  • Larger membrane systems that can be visualized by microscopy

  • Allow for studies of lipid domain formation and protein clustering

• Supported lipid bilayers:

  • Form lipid bilayers on solid supports for surface-sensitive techniques

  • Useful for atomic force microscopy and other surface characterization methods

• Proteoliposome arrays:

  • High-throughput systems for screening different lipid compositions

  • Useful for determining optimal membrane environments for enzyme activity

What are the unexplored aspects of O. iheyensis Cardiolipin synthase that warrant investigation?

Several aspects of Oceanobacillus iheyensis Cardiolipin synthase remain unexplored and represent promising avenues for future research:

• Structural determinants of extremophile adaptation:

  • Determination of high-resolution structures in different pH and salt conditions

  • Identification of specific residues involved in extremophile adaptation

  • Comparison with mesophilic homologs to identify key differences

• Substrate specificity:

  • Characterization of the enzyme's preference for different phospholipid substrates

  • Investigation of how substrate specificity may contribute to membrane adaptation

• Regulation mechanisms:

  • Study of how the enzyme's activity is regulated in response to environmental changes

  • Investigation of potential post-translational modifications

• Interaction partners:

  • Identification of proteins that interact with Cardiolipin synthase

  • Characterization of potential membrane complexes involving the enzyme

• Biotechnological applications:

  • Exploration of the enzyme's potential for synthesis of novel lipids

  • Development of extremophile-derived enzymes for industrial applications

How might understanding this enzyme contribute to synthetic biology applications?

Understanding Oceanobacillus iheyensis Cardiolipin synthase could contribute to various synthetic biology applications:

• Designer membrane engineering:

  • Creation of synthetic membranes with enhanced stability under extreme conditions

  • Development of liposomal drug delivery systems with improved pH and salt tolerance

• Extremophile chassis development:

  • Engineering of bacterial strains with enhanced tolerance to alkaline and high-salt conditions

  • Optimization of membrane composition for bioproduction in extreme environments

• Enzyme engineering:

  • Development of modified cardiolipin synthases with novel substrate specificities

  • Creation of enzymes capable of synthesizing novel phospholipids with unique properties

• Biomimetic materials:

  • Design of biomimetic membranes inspired by extremophile adaptations

  • Development of nanostructured materials with enhanced stability

• Bioenergetic applications:

  • Insights into ATP synthesis under adverse conditions

  • Development of optimized energy-generating systems based on extremophile principles