Recombinant R2-like ligand binding oxidase (Mb0238)

What is R2-like ligand binding oxidase and how does it relate to Mb0238?

R2-like ligand binding oxidase (R2lox) is a ferritin-like protein that harbors a heterodinuclear manganese-iron active site. It was discovered due to its sequence resemblance with the R2 subunit of class Ic ribonucleotide reductase (RNR) . Mb0238 refers to a specific R2lox protein from Mycobacterium tuberculosis (also known as MtR2lox), which was one of the first R2lox proteins to have its crystal structure determined . R2lox proteins have been identified in various organisms, with some organisms encoding multiple isoforms .

The enzyme binds fatty acid ligands that coordinate the metal center and catalyzes the formation of a tyrosine-valine ether cross-link in the protein scaffold upon O₂ activation, though its precise physiological function remains unestablished .

What is the structural composition of the R2lox active site?

The mature cofactor of R2lox has been characterized spectroscopically as a Mn(III)/Fe(III) center bridged by a μ-hydroxo/bis-μ-carboxylato network . The assembly of this active site occurs upon dioxygen (O₂) activation and involves the formation of high-valent intermediates that catalyze a two-electron reaction to generate the tyrosine-valine ether cross-link near the metal center .

Unlike the related class Ic ribonucleotide reductase R2 subunit (R2c), which uses its Mn(IV)/Fe(III) center to catalyze single-electron transfer for producing free radicals essential in deoxyribonucleotide synthesis, R2lox appears to have a distinct catalytic function .

What ligands co-purify with recombinant R2lox proteins?

When recombinantly expressed in Escherichia coli, R2lox proteins co-purify with a mixture of fatty acids, primarily hydroxy fatty acids (HFAs) of C₁₆ and C₁₈ chain lengths . Mass spectrometry-based metabolomics has confirmed this mixture in both Geobacillus kaustophilus R2loxI (GkR2loxI) and Sulfolobus acidocaldarius R2loxI (SaR2loxI) . These fatty acids are not necessarily the natural substrates but may mimic the binding of the actual physiological substrates, becoming trapped in the binding pocket during heterologous expression .

Table 1: Major Hydroxy Fatty Acids Co-purifying with Recombinant R2lox

What are the optimal conditions for recombinant expression of R2lox proteins?

Based on successful structural studies of R2lox proteins, heterologous expression in E. coli has proven effective for producing functional protein . Though specific expression protocols for Mb0238 are not detailed in the provided search results, general recombinant protein expression strategies would apply.

For metalloproteins like R2lox, consider the following methodological approach:

• Select an appropriate E. coli expression strain (BL21(DE3) or derivatives) that minimizes proteolytic degradation

• Design constructs with affinity tags (His-tag is common) to facilitate purification

• Optimize expression temperature, usually lower temperatures (16-20°C) after induction to allow proper protein folding

• Consider supplementing the growth medium with metal ions (Mn²⁺ and Fe²⁺/Fe³⁺) to ensure proper metallocenter formation

• Use rich media (like TB or 2xYT) to maximize cell density and protein yield

What purification strategies are most effective for R2lox proteins?

While the search results don't explicitly detail purification protocols for R2lox, standard approaches for metalloproteins with similar characteristics would include:

• Initial capture using immobilized metal affinity chromatography (IMAC) if the construct contains a His-tag

• Further purification by ion exchange chromatography based on the theoretical pI of the protein

• Size exclusion chromatography as a polishing step to remove aggregates and ensure homogeneity

• Consideration of buffer conditions that maintain metallocenter integrity (avoiding strong chelators like EDTA)

For studying the native ligands, avoid harsh washing steps that might remove bound fatty acids if characterizing the co-purified ligands is of interest .

What techniques have been most informative for R2lox structural studies?

X-ray crystallography has been the primary technique for elucidating R2lox structures, with crystal structures solved for proteins from multiple organisms:

• Mycobacterium tuberculosis (MtR2lox) - PDB ID: 3EE4, 4AC8

• Geobacillus kaustophilus (GkR2loxI) - PDB ID: 4HR0

• Sulfolobus acidocaldarius (SaR2loxI) - PDB ID: 6QRZ

• Sulfolobus acidocaldarius (SaR2loxII) - 2.26 Å resolution structure

• Saccharopolyspora erythraea (SeR2lox) - 1.38 Å resolution structure (highest resolution to date)

Complementary techniques for R2lox characterization include:

• Mass spectrometry-based metabolomics for ligand identification

• Spectroscopic methods (e.g., EPR, Mössbauer) for metallocenter characterization

• Bioinformatic analyses for sequence comparisons and evolutionary relationships

What are the key structural features of R2lox revealed by crystallography?

Crystal structures have revealed several important features of R2lox proteins:

• A conserved ferritin-like four-helix bundle fold with extensive remodeling to accommodate ligand binding

• A heterodinuclear Mn-Fe center with a μ-hydroxo/bis-μ-carboxylato bridging network

• A binding pocket that accommodates long-chain fatty acids, primarily HFAs

• A tyrosine-valine ether cross-link near the metal center formed upon O₂ activation

• A flexible loop that may be involved in substrate recognition or gating

• In some homologs (e.g., SaR2loxII), an ordered C-terminus that shields a conserved positively charged patch on the protein surface, potentially involved in protein-protein or protein-membrane interactions

How does the redox state of the cofactor impact R2lox structure and function?

Research has shown that the redox state of the metallocofactor significantly impacts R2lox structure. Specifically, changes in the redox state lead to structural alterations that likely create routes for O₂ and substrate access to the active site . This suggests a mechanism whereby the redox state might regulate substrate binding and catalytic activity.

Researchers investigating this aspect should consider methods to control and monitor the redox state, such as:

• Using specific reducing or oxidizing agents to manipulate the metal center

• Employing anaerobic techniques to prevent uncontrolled oxidation

• Utilizing spectroscopic methods (EPR, Mössbauer) to characterize the metal center's oxidation state

• Conducting crystallography under different redox conditions to capture structural changes

What approaches can distinguish between substrate and non-substrate ligands co-purified with R2lox?

Distinguishing true substrates from adventitiously bound ligands is a significant challenge in R2lox research. The current evidence suggests that the C₁₆ and C₁₈ HFAs that co-purify with recombinant R2lox may not be physiological substrates but rather mimics that bind due to their abundance in the expression host .

Methodological approaches to address this question include:

• Redox cycling experiments with purified enzyme to observe potential product formation (although previous attempts did not show product formation with copurified fatty acids)

• Comparative metabolomics between wild-type enzyme and catalytically inactive variants

• Testing activity with a diverse panel of potential substrates, particularly those with hydroxyl groups since HFAs are predominantly co-purified

• Structural studies comparing binding modes of different ligands

• Isotope labeling techniques to track potential transformations of bound ligands

What is the significance of the tyrosine-valine ether cross-link in R2lox proteins?

The tyrosine-valine ether cross-link formed in R2lox upon O₂ activation is a distinctive feature that differentiates it from related proteins . While the precise functional significance remains under investigation, several hypotheses can be considered:

• The cross-link may stabilize the metal center, similar to cross-links in other di-metal ferritin-like proteins where a valine-phenylalanine cross-link has been proposed to stabilize the metal center

• It could be involved in modulating substrate binding or product release

• The cross-link formation may be coupled to the actual catalytic function of the enzyme

• It may serve as a means of irreversible activation or control of enzyme activity

Researchers should consider comparative studies between cross-link-containing and cross-link-deficient variants to elucidate its role.

What evidence supports potential protein-protein or protein-membrane interactions of R2lox?

Several structural features suggest that R2lox proteins may engage in interactions with other proteins or membranes:

• In SaR2loxII, an ordered C-terminus shields a conserved positively charged patch on the protein surface that could potentially mediate such interactions

• A similar feature was observed in one particular structure of MtR2lox (PDB ID: 4AC8), though with the C-terminal segment forming a long α-helix

• The dynamic behavior between ordered and disordered states of the R2lox C-terminal tail likely plays a role in the physiological function of the enzyme

• A parallel can be drawn with the phylogenetically related RNR R2 subunit, where the C-terminus is crucial for interaction with its partner R1

These observations suggest that R2lox might interact with partner proteins, possibly for the delivery of poorly water-soluble long-chain aliphatic compounds, similar to how aldehyde deformylating oxygenase (ADO) interacts with an acyl-acyl carrier protein reductase .

How do sequence variations between R2lox homologs correlate with substrate specificity?

Analysis of R2lox structures reveals diversity in binding pocket shapes across homologs, along with low residue conservation in the distal part of the binding pocket . This structural and sequence variability suggests potential differences in substrate specificity between R2lox homologs.

For researchers investigating this aspect, the following methodological approaches are recommended:

• Comparative structural analysis of binding pockets across multiple homologs

• Docking studies with various potential substrates to predict binding preferences

• Site-directed mutagenesis of non-conserved residues in the binding pocket to alter specificity

• Activity assays with homologs from different organisms using a panel of potential substrates

• Phylogenetic analysis correlated with structural features to identify evolutionary patterns in substrate recognition

What are promising strategies to elucidate the physiological function of R2lox?

Given that the physiological function of R2lox remains unknown despite significant structural characterization, several research strategies could help resolve this question:

• Genetic approaches:

  • Gene knockout or silencing in native organisms

  • Phenotypic analysis of mutants lacking functional R2lox

  • Transcriptomic profiling to identify conditions that upregulate R2lox expression

• Biochemical approaches:

  • Broader substrate screening, particularly focusing on hydroxylated fatty acids and related compounds

  • Investigation of potential signaling roles rather than purely catalytic functions

  • Exploration of potential roles in lipid metabolism, given the fatty acid binding properties

• Interactome studies:

  • Identification of protein interaction partners through pull-down assays or proximity labeling

  • Investigation of potential membrane associations

  • Examination of the role of the dynamic C-terminal region in protein-protein interactions

How can researchers optimize metal incorporation for functional studies of R2lox?

The heterodinuclear Mn-Fe center is crucial for R2lox function, making proper metal incorporation essential for functional studies. While specific protocols aren't detailed in the search results, the following methodological considerations are important:

• Metal supplementation during expression:

  • Supplementing growth media with appropriate ratios of Mn²⁺ and Fe²⁺/Fe³⁺

  • Timing the addition of metals relative to protein induction

• In vitro metal reconstitution:

  • Protocols for removing adventitiously bound metals

  • Controlled addition of Mn²⁺ and Fe²⁺ under anaerobic conditions

  • Activation with controlled exposure to O₂

• Verification of metal content:

  • Inductively coupled plasma mass spectrometry (ICP-MS) to quantify metal incorporation

  • Spectroscopic techniques to verify the oxidation states and environment of incorporated metals

The specific metal incorporation protocol will significantly impact subsequent functional and structural studies, making this a critical methodological consideration.