Recombinant Proteus mirabilis Lipoyl synthase (lipA)

What is the function of Proteus mirabilis Lipoyl synthase (lipA)?

Proteus mirabilis Lipoyl synthase (LipA) catalyzes the insertion of two sulfur atoms at C6 and C8 positions of an n-octanoyllysyl residue attached to a lipoyl carrier protein (LCP). This reaction transforms the octanoyl moiety into the lipoyl cofactor, which is essential for several multienzyme complexes including pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and the glycine cleavage system. The reaction proceeds through radical-mediated chemistry, with LipA belonging to the radical S-adenosylmethionine (SAM) superfamily of enzymes .

The catalytic mechanism involves sequential hydrogen atom abstractions from C6 and C8 of the octanoyl substrate, initiated by 5′-deoxyadenosyl 5′-radical (5′-dA- ) generated from the reductive cleavage of SAM. After each abstraction, a sulfur atom from the auxiliary [4Fe-4S] cluster is inserted at the respective position, resulting in the characteristic dithiolane ring of the lipoyl cofactor .

What cofactors are required for recombinant P. mirabilis LipA activity?

For recombinant P. mirabilis LipA to exhibit catalytic activity, several essential cofactors are required:

• Two [4Fe-4S] clusters:

  • A radical SAM [4Fe-4S] cluster that binds and cleaves SAM

  • An auxiliary [4Fe-4S] cluster that serves as the sulfur donor for the reaction

• S-adenosylmethionine (SAM) - Two molecules are required per catalytic cycle, one for each sulfur insertion

• Electron donor system - Typically sodium dithionite or a physiological system like flavodoxin/flavodoxin reductase with NADPH to provide electrons for SAM cleavage

• Substrate - Either a natural lipoyl carrier protein with an n-octanoyl modification on a specific lysine residue or a synthetic peptide substrate containing an octanoyllysyl residue

The critical role of these cofactors is demonstrated by the requirement for anaerobic conditions during purification and assays to maintain the oxygen-sensitive [4Fe-4S] clusters in their active state .

How does the auxiliary cluster function during LipA catalysis?

The auxiliary [4Fe-4S] cluster in LipA plays a unique role as the direct sulfur donor during catalysis:

• Sulfur donation mechanism: The auxiliary cluster provides both sulfur atoms for insertion at C6 and C8 positions of the octanoyl substrate

• Cluster sacrifice: During turnover, the auxiliary cluster undergoes degradation as its sulfur atoms are extracted, limiting catalysis to one turnover in the absence of a regeneration system

• Intermediate formation: Evidence shows a covalent cross-link forms between LipA and its substrate during catalysis, likely involving the auxiliary cluster, when the second sulfur insertion is slowed

• Cluster state changes: Mössbauer spectroscopy reveals that after donating one sulfur atom, the auxiliary cluster converts to a species with spectroscopic properties similar to a reduced [3Fe-4S]⁰ cluster

This sacrificial nature of the auxiliary cluster creates a requirement for regeneration systems in vivo and in vitro for multiple turnovers to occur. The ability to form a covalent intermediate with substrates has been demonstrated experimentally through reactions where the second sulfur insertion is deliberately slowed using deuterium substitution at C8 or limiting SAM concentrations .

What methods can be used to express and purify active recombinant P. mirabilis LipA?

Obtaining active recombinant P. mirabilis LipA requires careful consideration of expression conditions and purification protocols:

Expression Strategy:

• Expression system selection:

  • E. coli BL21(DE3) or similar strains optimized for iron-sulfur proteins

  • Vector with inducible promoter (T7) and affinity tag (typically His6)

  • Co-expression with iron-sulfur cluster assembly machinery may improve yield

• Culture conditions:

  • Growth medium supplemented with iron source (50-100 μM ferric ammonium citrate)

  • Lower temperature induction (18-20°C) to improve proper folding

  • Microaerobic conditions to protect iron-sulfur clusters

  • Extended induction period (12-16 hours)

Purification Protocol:

• Anaerobic techniques:

  • All buffers must be degassed and contain reducing agents

  • Purification under anaerobic conditions (glove box) is optimal

  • Include glycerol (10%) and DTT (5 mM) in all buffers

• Protein quality assessment:

  • Iron and sulfide quantification (typically 8 Fe and 8 S per protein)

  • UV-visible spectroscopy (characteristic absorption at ~410 nm)

  • Activity assays using synthetic peptide substrates

Reconstitution of [4Fe-4S] Clusters:
If clusters are not fully incorporated during expression, in vitro reconstitution can be performed:

• Incubate with excess Fe²⁺ (FeCl₃) and S²⁻ (Na₂S) under reducing conditions

• Remove excess reagents by desalting or dialysis

• Verify cluster incorporation spectroscopically

This comprehensive approach ensures the isolation of catalytically competent enzyme with intact [4Fe-4S] clusters necessary for activity.

How can auxiliary cluster regeneration be optimized for multiple turnovers?

The auxiliary cluster of LipA is degraded during catalysis, limiting the enzyme to a single turnover unless regeneration systems are present. Based on the search results, several approaches can optimize cluster regeneration:

Iron-Sulfur Carrier Proteins:

• Identification of relevant carrier proteins:

  • In humans, NFU1 has been shown to form a tight complex with LIAS (human lipoyl synthase) and efficiently restore its auxiliary cluster during turnover

  • In E. coli, NfuA serves a similar function for bacterial LipA

  • For P. mirabilis, the homologous NFU protein should be identified and tested

• Experimental conditions for optimal regeneration:

  • Use carrier proteins in excess (10-20 fold) relative to LipA

  • The tight complex formation between NFU1 and LIAS suggests pre-incubation may enhance activity

  • Ensure proper redox conditions for cluster transfer

Quantitative Enhancement:
In experiments with human LIAS, the addition of NFU1 increased turnovers from 1 to more than 5 over 150 minutes . The reaction didn't show a clear burst phase followed by slower product formation, suggesting that auxiliary cluster regeneration is not rate-limiting when appropriate carrier proteins are present .

Additional Factors:

• Other proteins that may enhance regeneration:

  • ISCA1 and ISCA2 showed slight enhancement of LIAS turnover

  • BOLA3, despite being implicated in lipoyl cofactor biosynthesis in humans and yeast, showed no direct effect on Fe-S cluster transfer from NFU1 or GLRX5 to LIAS in vitro

• Tracking cluster regeneration:

  • Using ³⁴S-reconstituted NFU1 demonstrated that the sulfur atoms from the carrier protein are incorporated into the lipoyl product

These findings provide a framework for developing efficient regeneration systems for P. mirabilis LipA to achieve multiple catalytic cycles in vitro.

What experimental approaches can track the reaction intermediates in LipA catalysis?

Investigating the LipA reaction mechanism requires specialized techniques to detect transient intermediates:

Substrate Modification Strategies:

• Deuterium labeling:

  • Using [6,6-²H₂] or [8,8-²H₂] octanoyl substrates slows hydrogen abstraction

  • This approach can trap reaction intermediates, as demonstrated in studies where deuterium substitution at C8 slowed the second sulfur insertion and allowed detection of a covalent enzyme-substrate intermediate

• Limited SAM availability:

  • Reactions with limiting SAM concentrations can halt the reaction after the first sulfur insertion

  • This approach has successfully generated a covalent cross-linked species between LipA and substrate

Detection Methods:

• Chromatographic separation:

  • HPLC or LC-MS to separate and quantify reaction intermediates

  • Monitor both the 6-thiooctanoyl intermediate and final lipoyl product

  • Track 5'-deoxyadenosine formation as evidence of SAM cleavage

• Spectroscopic techniques:

  • Mössbauer spectroscopy reveals that the cross-linked intermediate contains a partially disassembled auxiliary cluster with properties similar to reduced [3Fe-4S]⁰ clusters

  • EPR spectroscopy can detect radical intermediates

• Protein-substrate adduct characterization:

  • The covalent cross-link between enzyme and substrate can be observed when proteins elute simultaneously by anion-exchange chromatography but separate under aerobic SDS-PAGE conditions

  • This behavior is consistent with linkage through the oxygen-sensitive auxiliary cluster

Intermediates Validation:
Using a small unlabeled (N⁶-octanoyl)-lysyl-containing peptide substrate, researchers demonstrated both chemical and kinetic competence of the cross-linked species, providing strong evidence that it represents a true reaction intermediate .

These approaches collectively provide a toolkit for detailed mechanistic investigations of P. mirabilis LipA catalysis.

How does lipoyl synthase activity change across bacterial growth phases?

Growth phase-dependent changes in lipoyl synthase activity reflect broader adaptations in bacterial physiology. Although specific data for P. mirabilis LipA is not provided in the search results, relevant findings from studies with Salmonella Typhimurium suggest important patterns:

Transcriptional Changes:

• RNAseq analysis revealed significant changes in gene expression upon entry into stationary phase, including genes involved in membrane permeability

• Cyclopropane fatty acid (CFA) synthase expression increased during stationary phase :

  • CFA synthase catalyzes the biosynthesis of cyclopropane fatty acids from olefinic fatty acids

  • This modification decreases membrane permeability, potentially affecting substrate access to LipA

Membrane Modifications:

• Stationary phase entry is associated with decreased membrane permeability :

  • Increased expression of CFA synthase leads to cyclopropane fatty acid formation

  • These modifications alter membrane fluidity and barrier properties

• The CFA enzymatic reaction shares mechanistic similarities with LipA:

  • Both enzymes use SAM as a cofactor

  • Both involve methyl transfer reactions

Implications for LipA Activity:

• Activity regulation:

  • Changes in membrane composition may affect access of substrates to LipA

  • Alteration in expression of LipA and related proteins across growth phases

• Metabolic shifts:

  • Changes in L-methionine and S-adenosyl-L-methionine synthesis pathways during growth phase transitions

  • These pathways produce SAM, an essential cofactor for LipA

Experimental Design for P. mirabilis LipA:
To investigate growth phase-dependent changes in P. mirabilis LipA activity, researchers should:

• Monitor lipA gene expression across growth phases using qRT-PCR

• Measure LipA protein levels by western blotting

• Assess enzyme activity using in vitro assays with samples harvested at different growth points

• Correlate activity with changes in membrane composition

This growth phase-dependent regulation may represent an important aspect of LipA function in bacterial physiology.

What is the role of iron-sulfur cluster carrier proteins in LipA function?

Iron-sulfur cluster carrier proteins play a crucial role in maintaining LipA catalytic activity by regenerating the auxiliary cluster that gets degraded during turnover:

Key Carrier Proteins:

• NFU1/NfuA:

  • Human NFU1 forms a tight complex with LIAS and efficiently restores its auxiliary cluster during turnover

  • NFU1 can provide multiple sulfur atoms for LIAS catalysis, supporting more than 5 turnovers with 10 μM LIAS when NFU1 is present at 200 μM (as monomer)

  • E. coli NfuA serves a similar function for bacterial LipA

  • The binding appears to be specific and physiologically relevant

• Other potential carriers:

  • ISCA1 and ISCA2 can enhance LIAS turnover, but only slightly

  • GLRX5 may also participate in cluster transfer

Interaction Mechanisms:

• Complex formation:

  • NFU1 and LIAS form a tight complex in vitro

  • This interaction has also been demonstrated in yeast two-hybrid studies

  • The complex formation suggests a direct protein-protein interaction mechanism for cluster transfer

• Sulfur transfer:

  • Studies using ³⁴S-reconstituted NFU1 demonstrated that sulfur atoms from the carrier protein are incorporated into the lipoyl product

  • The data suggests that potentially all four sulfides of the auxiliary cluster of LIAS can be used for lipoyl product formation

Physiological Significance:
The importance of these carrier proteins is highlighted by clinical observations:

• Mutations in human NFU1 cause fatal infantile encephalopathy and/or pulmonary hypertension, associated with defects in lipoylation

• BOLA3 deficiency causes a similar clinical presentation, though interestingly, in vitro studies suggest BOLA3 has no direct effect on Fe-S cluster transfer from NFU1 or GLRX5 to LIAS

These findings establish iron-sulfur cluster carrier proteins as essential components of the LipA catalytic cycle, acting as regeneration systems that enable multiple turnovers.

How does isotope labeling help elucidate the LipA reaction mechanism?

Isotope labeling has been instrumental in elucidating key aspects of the LipA reaction mechanism:

Sulfur Source Determination:

• ³⁴S labeling experiments:

  • Reconstituting NFU1 with ³⁴S allows tracking of sulfur transfer to LIAS and subsequently to the lipoyl product

  • These experiments confirmed that the sulfur atoms in the product originate from the auxiliary cluster

  • Data suggests that "potentially all four sulfides of the auxiliary cluster of LIAS can be used for lipoyl product formation"

• Quantitative analysis:

  • The observation of "almost 1.5 equiv of the ³²S-³²S-containing lipoyl product" when using NFU1 at natural abundance provides insights into the stoichiometry of cluster utilization

Reaction Intermediate Trapping:

• Deuterium labeling:

  • Using deuterium-substituted substrates at C8 significantly slows the second sulfur insertion

  • This approach enabled the observation of a covalent cross-link between LipA and the substrate

  • The intermediate's chemical and kinetic competence was demonstrated, confirming its role in the reaction pathway

• Limiting SAM conditions:

  • Reactions with limiting SAM concentrations also facilitated intermediate trapping

  • This technique complements the deuterium labeling approach

Structural Characterization:
Mössbauer spectroscopy of the cross-linked intermediate revealed that one of the [4Fe-4S] clusters (presumably the auxiliary cluster) is partially disassembled to a 3Fe-cluster with spectroscopic properties similar to reduced [3Fe-4S]⁰ clusters .

Table 1: Isotope Labeling Strategies for LipA Mechanism Studies

These isotope labeling studies have collectively established a detailed mechanistic understanding of the LipA reaction.

What is the evolutionary significance of LipA function across bacterial species?

The evolutionary conservation and significance of LipA reflect its central role in lipoyl cofactor biosynthesis:

Conservation Patterns:

• Pathway conservation:

  • The lipoyl cofactor biosynthetic pathway shows remarkable conservation across species from bacteria to humans

  • Two distinct pathways exist: an exogenous salvage pathway and an endogenous biosynthesis pathway

  • In E. coli and other bacteria, lipoate protein ligase A (LplA) handles the exogenous pathway, while LipA is central to the endogenous pathway

• Functional equivalence:

  • Human LIAS is functionally equivalent to bacterial LipA enzymes

  • The auxiliary [4Fe-4S] cluster mechanism is conserved across species

Mechanistic Conservation:

• Auxiliary cluster function:

  • The use of an auxiliary [4Fe-4S] cluster as a direct sulfur donor is conserved from bacteria to humans

  • This unusual "sacrificial" mechanism suggests strong evolutionary pressure to maintain this specific catalytic strategy

• Carrier protein interactions:

  • The requirement for iron-sulfur cluster carrier proteins (like NFU1/NfuA) for regeneration is conserved

  • The tight complex formation between NFU1 and LIAS is mirrored in bacterial systems

Metabolic Integration:
The endogenous lipoyl cofactor biosynthesis pathway in E. coli and likely P. mirabilis involves the bacterial-type acyl carrier protein (ACP) for constructing the C8 fatty acyl backbone . This integration with fatty acid biosynthesis represents an efficient use of cellular resources.

Clinical Relevance:
Defects in human lipoyl synthase or its associated proteins lead to severe metabolic disorders:

• Mutations in NFU1 cause fatal infantile encephalopathy and/or pulmonary hypertension

• BOLA3 deficiency produces similar clinical presentations

• These disorders highlight the essential nature of lipoyl cofactor biosynthesis across evolution

This evolutionary conservation underscores the fundamental importance of LipA and suggests that insights gained from studying P. mirabilis LipA will have broader implications for understanding lipoyl cofactor biosynthesis across species.