Recombinant Picrophilus torridus 1,4-alpha-glucan branching enzyme GlgB (glgB), partial

What is Picrophilus torridus and why is it significant for enzyme studies?

Picrophilus torridus is an extremophilic organism belonging to the euryarchaeal phylum that thrives in extraordinarily acidic environments. It grows optimally at 60°C and pH 0.7, making it one of the most acidophilic thermophiles known to date . The internal pH of Picrophilus has been determined to be 4.6, which represents the lowest cytoplasmic pH known in any organism . This extreme adaptation makes P. torridus an exceptional model for studying enzymes that function under harsh conditions.

The significance of P. torridus for enzyme studies stems from its ability to maintain enzymatic activity in environments that would typically denature proteins. Enzymes from this organism, including GlgB, have evolved unique structural and functional properties that enable them to operate under conditions of high acidity and elevated temperatures, offering valuable insights into protein stability mechanisms and potential biotechnological applications .

What is the function of the GlgB enzyme in glycogen/starch metabolism?

The GlgB enzyme (1,4-alpha-glucan branching enzyme) plays a crucial role in glycogen and starch metabolism by catalyzing the formation of α-(1→6) branch points in α-(1→4)-linked glucan chains. Specifically, GlgB:

• Converts linear α-(1→4)-linked amylose chains to form α-(1→4,6) branching points

• Contributes to the three-dimensional structure of glycogen and starch

• Impacts the physicochemical properties of these polysaccharides, including solubility, viscosity, and digestibility

• May play a role in bacterial persistence and durability through its effects on glycogen structure

In P. torridus and other organisms, GlgB works in concert with other enzymes in glycogen metabolism pathways, such as glycogen synthase (GlgA) and ADP-glucose pyrophosphorylase (GlgC) .

What are the optimal conditions for GlgB activity from extremophiles like P. torridus?

While the search results don't specifically detail the optimal conditions for P. torridus GlgB activity, we can infer some characteristics based on information about other enzymes from this organism:

• Temperature optimum: Likely around 55-60°C, similar to the organism's growth temperature optimum

• pH optimum: May be significantly higher than the environmental pH, as seen with other P. torridus enzymes such as glucose dehydrogenase (GdhA), which shows optimal activity at pH 6.5 despite the organism's extremely acidic habitat

• Stability: Likely exhibits remarkable stability at low pH and elevated temperatures compared to mesophilic counterparts

The discrepancy between the organism's internal pH (4.6) and the optimal pH for enzyme activity suggests adaptation mechanisms that allow these enzymes to function effectively despite extreme environmental conditions .

How can the GlgB gene from P. torridus be cloned and expressed in E. coli?

Based on methodologies used for similar enzymes from P. torridus, a researcher could follow this protocol:

• Gene identification and primer design: Identify the glgB gene sequence in the P. torridus genome (similar to how ORF Pto0332 was identified for GADH) . Design primers with appropriate restriction sites for the expression vector.

• PCR amplification: Amplify the glgB gene using PCR with high-fidelity DNA polymerase to minimize errors.

• Cloning strategy: Clone the PCR product into an expression vector such as pET17b or pET19b using restriction enzymes (e.g., NdeI, BamHI, EcoRV, or XhoI) .

• Host selection: Transform the construct into E. coli BL21 codon plus (RIL) to address potential codon usage bias issues .

• Expression optimization: Expression conditions might include:

  • Induction with IPTG (0.5 mM) when cells reach OD600 of 0.6

  • Initial incubation at 30°C for 2h followed by 16°C for 17h

  • Potential addition of 2% hexadecane to improve expression, as demonstrated for other P. torridus enzymes

• Protein purification: Purify using affinity chromatography (Ni-NTA for His-tagged constructs) followed by size-exclusion chromatography if needed .

This approach has proven successful for other P. torridus enzymes like glucose dehydrogenase (GdhA) and γ-glutamyl transpeptidase .

What methods can be used to characterize the branching activity of GlgB?

Several complementary methods can be employed to characterize GlgB branching activity:

• Amylose content quantification: Measure the reduction in amylose content after GlgB treatment using iodine-binding assays .

• Reducing ends determination: Quantify reducing ends after debranching treated substrates with isoamylase and pullulanase. This directly measures branch point formation, as shown in this data from a metagenome-derived GlgB study:

Statistically significant difference

• Mass spectrometry: Use isotopic labeling of substrates (e.g., with 13C) to determine if branching occurs via intrachain or interchain transfer mechanisms .

• High-performance size-exclusion chromatography (HPSEC): Analyze molecular size distribution changes in starch samples after GlgB treatment .

• In vitro digestibility assays: Assess how GlgB-introduced branches affect substrate digestibility by pancreatic amylases and brush border enzymes .

These methodologies provide comprehensive insights into both the quantity and quality of branching activity.

How can site-directed mutagenesis be used to study functional residues of GlgB?

Site-directed mutagenesis is a powerful approach for investigating structure-function relationships in GlgB. The general methodology includes:

• Target identification: Identify conserved residues through sequence alignment of GlgB from different species or through structural analysis of homologous proteins.

• Primer design: Design primers containing the desired mutation, typically using QuikChange or similar methods (as demonstrated for M. tuberculosis GlgB, where Ser470 was mutated to Pro) .

• Mutagenesis procedure:

  • Perform PCR with high-fidelity polymerase using the mutagenic primers

  • Digest parental DNA with DpnI

  • Transform into competent E. coli

  • Verify mutations by sequencing

• Functional characterization: Compare the activity of wild-type and mutant enzymes using the methods described in section 2.2.

• Structural analysis: Assess how mutations affect protein folding, stability, and substrate binding through techniques like circular dichroism, thermal shift assays, or crystallography.

This approach has successfully elucidated functional residues in similar enzymes, such as the demonstration that changing Tyr327 to Asn327 in P. torridus γ-glutamyl transpeptidase introduced significant transpeptidase activity .

How does the N-terminal domain of GlgB affect branching patterns and substrate specificity?

The N-terminal domain of GlgB plays a critical role in determining branching patterns and substrate specificity:

• Domain organization: GlgB proteins can be classified into different groups based on N-terminal domain structure:

  • Those with only the N1 domain

  • Those with both N1 and N2 domains

  • Those with an additional N0 domain (~100 amino acids) ahead of the N1 domain

• Functional impact: The N-terminus has been experimentally confirmed to control glycogen branching degree and average chain length (ACL) .

• Evolutionary significance: N-terminal variation appears to be a key factor in the adaptation of GlgB to different ecological niches and may influence bacterial durability through its effects on glycogen structure .

• Substrate interaction: The N-terminal domain likely contributes to substrate recognition and binding, influencing whether the enzyme preferentially acts on amylose or amylopectin. For example, some GlgB enzymes show a higher preference for amylose as a substrate, while others prefer branched amylopectin .

Further research incorporating comparative analysis of GlgB N-termini from different species, particularly extremophiles like P. torridus, could yield valuable insights into how these domains have evolved to accommodate different substrates and environmental conditions.

What is the mechanism of GlgB branching - intrachain or interchain transfer?

The mechanism of GlgB branching can vary depending on the source organism. For M. tuberculosis GlgB, detailed investigations using 13C-labeled substrates have demonstrated that branching occurs strictly via intrachain transfer :

• Experimental approach:

  • Generation of 13C-labeled and unlabeled populations of maltooligosaccharides

  • Mixing of these populations and exposure to GlgB

  • Analysis of products to determine label distribution

• Possible outcomes:

  • Intrachain transfer: Masses of products identical to substrates

  • Interchain transfer: Redistribution of label in products

  • Both mechanisms: Partial redistribution of label

• Results: For M. tuberculosis GlgB, no redistribution of 13C labels was observed, confirming that branching occurs exclusively through intrachain transfer .

For P. torridus GlgB specifically, similar experiments would need to be conducted to determine whether it follows the same mechanistic pattern or if it can also perform interchain transfers. The extreme environmental adaptations of P. torridus might influence the preferred mechanism of its GlgB.

How does GlgB activity impact starch digestibility and potential research applications?

GlgB-mediated branching significantly affects starch digestibility and has potential research applications:

• Impact on digestibility: GlgB treatment reduces in vitro starch digestibility, as demonstrated by decreased glucose release after digestion with pancreatic amylases and brush border enzymes:

Statistically significant difference

• Mechanism: GlgB introduces branch points that create structures resistant to digestive enzymes, potentially forming resistant starch .

• Research applications:

  • Functional food development with reduced glycemic index

  • Improved bread quality (increased volume, decreased crumb firmness)

  • Retardation of starch retrogradation in food products

  • Novel biomaterials with tailored physical properties

• Advantages of extremophilic GlgB: Enzymes from extremophiles like P. torridus may offer superior stability during food processing conditions compared to mesophilic counterparts, potentially expanding the range of applications .

Future research could focus on comparing the branching patterns and resulting starch modifications produced by GlgB enzymes from different sources, including extremophiles, to identify those with optimal properties for specific applications.

How does GlgB integrate with other enzymes in glycogen/starch metabolism pathways?

GlgB functions as part of a coordinated enzymatic network in glycogen/starch metabolism:

• In the GlgC-GlgA pathway: GlgB works in conjunction with:

  • ADP-glucose pyrophosphorylase (GlgC) - synthesizes ADP-glucose from glucose-1-phosphate

  • Glycogen synthase (GlgA) - uses ADP-glucose to extend α-1,4-glucan chains

  • GlgB - creates branch points in the growing chains

  • Glycogen phosphorylase (GlgP) - involved in glycogen degradation

  • Glucan hydrolase/transferase (GlgX) - assists in glycogen breakdown

• In the GlgE pathway: Found in some bacteria like M. tuberculosis, where:

  • GlgE uses maltose 1-phosphate as the donor substrate

  • GlgB creates branch points

  • Together, these enzymes can form high-molecular-weight α(1→6)-branched α(1→4)-glucan particles resembling native glycogen without the need for a primer

• Operon organization: In some organisms, glycogen metabolism genes are organized in an operon structure, as seen in Rhodobacter sphaeroides, where a 6-kb DNA fragment contains the glgC, glgA genes, and partial sequences of glgP, glgX, and glgB .

Understanding how GlgB from extremophiles like P. torridus integrates with other enzymes in its native metabolic context could provide insights into how these pathways have adapted to extreme conditions.

What is known about glucose metabolism in P. torridus and how might GlgB fit into this pathway?

P. torridus employs a non-phosphorylative variant of the Entner-Doudoroff (ED) pathway for glucose metabolism:

• Pathway enzymes: Studies have confirmed the presence of all enzyme activities required for this pathway in P. torridus, including:

  • Glucose dehydrogenase (GdhA)

  • Gluconate dehydratase

  • 2-keto-3-deoxygluconate aldolase

  • Glyceraldehyde dehydrogenase (GADH)

  • Glycerate kinase (2-phosphoglycerate forming)

  • Enolase

  • Pyruvate kinase

• Initial step: Glucose oxidation is catalyzed by glucose/galactose dehydrogenase (GdhA), which has been cloned, expressed, and characterized .

• GlgB's potential role: While not directly part of the ED pathway, GlgB likely participates in:

  • Storage of excess glucose as glycogen/α-glucans during nutrient abundance

  • Mobilization of these reserves during nutrient limitation

  • Potentially providing an advantage in stress resistance and persistence

• Metabolic challenges: The extreme acidity in P. torridus presents unique challenges for metabolism, such as the instability of NADPH (a product of the GdhA reaction) at the cytoplasmic pH of 4.6, with a half-life of merely 2.4 minutes at 60°C .

Understanding how GlgB functions within this metabolic context could provide insights into strategies for energy storage and utilization under extreme conditions.

What is the phylogenetic relationship between P. torridus GlgB and other branching enzymes?

While specific phylogenetic analysis of P. torridus GlgB is not detailed in the search results, general principles of GlgB evolution can be applied:

• Domain-based classification: GlgB enzymes can be categorized based on N-terminal domain structure, which has evolutionary significance :

  • Single N1 domain

  • N1+N2 domains

  • N0+N1+N2 domains

• Taxonomic distribution: Different GlgB types show specific distribution patterns across bacterial species, suggesting evolutionary adaptations to different ecological niches .

• Archaeal context: As a euryarchaeon, P. torridus GlgB would be expected to show sequence and structural features distinct from bacterial homologs, reflecting the early divergence of Archaea and Bacteria.

• Extremophile adaptations: Comparative analysis would likely reveal specific sequence features in P. torridus GlgB that enable function under extreme conditions, similar to adaptations seen in other P. torridus enzymes .

A comprehensive phylogenetic analysis comparing P. torridus GlgB to homologs from other extremophiles and mesophiles could provide valuable insights into the evolution of enzyme function under extreme conditions.

How do the membrane adaptations of P. torridus affect enzyme function in extreme environments?

P. torridus has evolved remarkable membrane adaptations that influence enzyme function in its extreme environment:

• Membrane lipid composition: P. torridus membranes contain specialized lipids:

  • Glycerol dialkyl glycerol tetraethers (GDGTs)

  • Glycerol trialkyl glycerol tetraethers (GTGTs)

  • These form monolayer arrangements rather than the typical bilayer

• Environmental responsive adjustments:

  • The abundance of core GDGTs per cell decreases with increasing pH, with the highest abundance at pH 0.3 (14.8 ± 5.5 fg cell^-1)

  • Both core and polar GDGTs decrease with increasing temperature

  • The GDGT ring index (a measure of cyclopentyl rings) correlates positively with temperature

• Implications for enzyme function:

  • These membrane adaptations likely help maintain appropriate intracellular conditions for enzyme function

  • The monolayer arrangement may provide exceptional impermeability to protons, protecting enzymes from the extreme external acidity

  • The altered membrane environment may influence protein folding, stability, and activity

Understanding the interplay between membrane adaptations and enzyme function in P. torridus provides a more complete picture of how these organisms have evolved to thrive in extreme environments and could inform strategies for engineering enzymes with enhanced stability.