Recombinant Idiomarina loihiensis 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (gpmI), partial
Description
Introduction
Idiomarina loihiensis is a deep-sea γ-proteobacterium originally isolated from a hydrothermal vent at a depth of 1,300 meters on the Lōʻihi submarine volcano, Hawaii . This bacterium exhibits remarkable adaptability, thriving in a wide temperature range (4°C to 46°C) and salinity levels (0.5% to 20% NaCl) . The genome of I. loihiensis consists of 2,839,318 base pairs, encoding 2,640 proteins, four rRNA operons, and 56 tRNA genes .
Genomic and Metabolic Features
The I. loihiensis genome reveals a preference for amino acid catabolism over sugar fermentation for carbon and energy acquisition . It encodes a complete set of enzymes for the biosynthesis of most amino acids, except for leucine, isoleucine, valine, threonine, and methionine . In vivo experiments have confirmed that I. loihiensis is auxotrophic for valine and threonine . The genome also contains a cluster of 32 genes responsible for the synthesis of exopolysaccharides and capsular polysaccharides, facilitating the formation of biofilms .
Phosphoglycerate Mutase (GpmI)
Idiomarina loihiensis possesses a 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (gpmI) . Phosphoglycerate mutases (PGMs) are essential enzymes in the glycolytic pathway, catalyzing the transfer of a phosphate group between the second and third carbon atoms of glycerate, specifically the interconversion of 3-phosphoglycerate (3PG) and 2-phosphoglycerate (2PG) . These enzymes are vital for energy production and carbon metabolism in all living organisms.
Recombinant GpmI
Recombinant GpmI refers to the phosphoglycerate mutase enzyme that has been produced using recombinant DNA technology. This involves isolating the gene encoding GpmI from Idiomarina loihiensis, cloning it into a suitable expression vector, and expressing it in a host organism such as E. coli . The recombinant protein can then be purified and studied in vitro to understand its biochemical properties, structure, and function.
Potential Functions and Adaptations
The presence of a 2,3-BPG-independent phosphoglycerate mutase in Idiomarina loihiensis may reflect an adaptation to its deep-sea hydrothermal vent environment. Further research is needed to elucidate the specific advantages conferred by this enzymatic variation, but it could relate to:
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Metabolic Flexibility: Allowing the organism to maintain glycolytic flux under varying conditions of phosphate availability.
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Regulation: Providing an alternative regulatory mechanism compared to 2,3-BPG-dependent PGMs.
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Environmental Adaptation: Optimizing enzymatic function under the unique pressure, temperature, and chemical conditions of deep-sea vents.
Research Findings and Data
While specific research findings and data tables directly focusing on the "Recombinant Idiomarina loihiensis 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (gpmI), partial" are not available, studies on Idiomarina loihiensis and other phosphoglycerate mutases provide valuable context .
Table 1: General Characteristics of Idiomarina loihiensis**
| Characteristic | Description |
|---|---|
| Isolation Source | Hydrothermal vent at Lōʻihi Seamount, Hawaii |
| Depth | 1,300 meters |
| Temperature Range | 4°C to 46°C |
| Salinity Range | 0.5% to 20% NaCl |
| Genome Size | 2,839,318 base pairs |
| Number of Encoded Proteins | 2,640 |
| Metabolism | Primarily amino acid catabolism |
| Phosphoglycerate Mutase | 2,3-bisphosphoglycerate-independent |
Table 2: Amino Acid Biosynthesis in Idiomarina loihiensis**
| Amino Acid | Biosynthesis Capability |
|---|---|
| Leucine | Incomplete |
| Isoleucine | Incomplete |
| Valine | Incomplete |
| Threonine | Incomplete |
| Methionine | Incomplete |
| Other | Complete |
Product Specs
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Target Background
Catalyzes the interconversion of 2-phosphoglycerate and 3-phosphoglycerate.
KEGG: ilo:IL0233
STRING: 283942.IL0233
Q&A
Basic Research Questions
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What is the genomic context and basic characteristics of phosphoglycerate mutase in Idiomarina loihiensis?
Idiomarina loihiensis possesses a phosphoglycerate mutase gene (gpmI) within its glycolytic operon that contains gapR, gapA, pgk, tpiA, and eno . The complete genome sequence of I. loihiensis reveals a single chromosome of 2,839,318 base pairs with a 47% GC content . Phosphoglycerate mutase catalyzes the reversible isomerization of 3-phosphoglycerate and 2-phosphoglycerate, a critical step in glycolysis . In I. loihiensis, this enzyme appears to be essential for energy metabolism, particularly as this bacterium relies primarily on amino acid catabolism rather than sugar fermentation for carbon and energy .
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How does 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) differ from 2,3-bisphosphoglycerate-dependent forms (dPGAM)?
The two types of phosphoglycerate mutases differ fundamentally in their catalytic mechanisms. The 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) does not require 2,3-bisphosphoglycerate as a cofactor, instead utilizing a metal ion (often manganese) in its active site for catalysis . In contrast, dPGAM requires 2,3-bisphosphoglycerate as a cofactor and operates through a phosphoenzyme intermediate . Structurally, iPGAMs are typically monomeric or dimeric proteins with a molecular weight of approximately 55-60 kDa, while dPGAMs are usually tetrameric with subunits of about 25-30 kDa. The iPGAM from I. loihiensis represents an adaptation potentially related to the extreme conditions of its hydrothermal vent habitat .
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Why study phosphoglycerate mutase from Idiomarina loihiensis specifically?
I. loihiensis represents an excellent model organism for studying metabolic adaptations to extreme environments, particularly deep-sea hydrothermal vents. This bacterium was isolated from a depth of 1,300m on the Lōihi submarine volcano, Hawaii . It survives in a wide range of temperatures (4°C to 46°C) and salinities (0.5% to 20% NaCl) , making its enzymes potentially valuable for understanding adaptive mechanisms. The phosphoglycerate mutase from this organism may possess unique properties reflecting adaptations to high pressure, variable temperatures, and the metal-rich environment of hydrothermal vents , offering insights into enzyme evolution and potential biotechnological applications.
Experimental Design and Methodology
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What expression systems are most effective for producing recombinant I. loihiensis gpmI?
Based on studies with similar enzymes, the most effective expression system for recombinant I. loihiensis gpmI typically involves E. coli BL21(DE3) containing the pET expression system with a hexahistidine tag for purification. Key methodological considerations include:
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Using low-temperature induction (16-18°C) to enhance solubility
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Supplementing growth media with manganese (50-100 μM MnCl₂) if the enzyme is metal-dependent
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Including 300-500 mM NaCl in buffers to maintain protein stability (reflecting the halophilic nature of I. loihiensis)
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Using a buffer system containing 50 mM Tris-HCl (pH 8.0) with 10% glycerol to improve stability
For optimal results, expression should be induced with 0.1-0.5 mM IPTG when cultures reach an OD₆₀₀ of 0.6-0.8, followed by overnight expression at 16°C .
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What purification strategy yields the highest specific activity for recombinant I. loihiensis gpmI?
A multi-step purification approach typically yields the highest specific activity:
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Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin
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Intermediate purification: Ion exchange chromatography (IEX) using Q-Sepharose
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Polishing step: Size exclusion chromatography (SEC) using Superdex 200
Buffer conditions should include 50 mM Tris-HCl (pH 8.0), 150-300 mM NaCl, 10% glycerol, and 1 mM DTT. For metal-dependent variants, include 1-2 mM MnCl₂ in all buffers to maintain enzyme activity. This approach has been shown to achieve approximately 6-fold increase in specific activity with final yields of approximately 30-35% , similar to results obtained with glyceraldehyde-3-phosphate dehydrogenase from I. loihiensis.
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How can the enzymatic activity of recombinant I. loihiensis gpmI be measured accurately?
The enzymatic activity can be measured using a coupled enzyme assay system:
Forward reaction (3PGA → 2PGA):
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Couple with enolase, pyruvate kinase, and lactate dehydrogenase
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Monitor NADH oxidation at 340 nm (ε = 6,220 M⁻¹cm⁻¹)
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Reaction mixture: 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 50 mM KCl, 1 mM DTT, 0.2 mM NADH, 1 mM ADP, 1 mM 3-phosphoglycerate, 2.5 units enolase, 2.5 units pyruvate kinase, 5 units lactate dehydrogenase
Reverse reaction (2PGA → 3PGA):
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Couple with phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase
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Monitor NADH formation at 340 nm
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Reaction mixture: 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 50 mM KCl, 1 mM DTT, 0.2 mM NAD⁺, 1 mM ATP, 1 mM 2-phosphoglycerate, 2.5 units phosphoglycerate kinase, 5 units glyceraldehyde-3-phosphate dehydrogenase
One unit of enzyme activity is defined as the amount that catalyzes the formation of 1 μmol of product per minute under the assay conditions .
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Research Applications and Future Directions
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How might recombinant I. loihiensis gpmI be applied in biotechnology research?
Recombinant I. loihiensis gpmI offers several potential biotechnological applications:
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Biocatalysis: Development of thermostable and salt-tolerant enzymes for industrial processes
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Biosensing: Creation of enzyme-based biosensors for 2- or 3-phosphoglycerate detection
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Structural biology: Model system for studying enzyme adaptation to extreme environments
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Metabolic engineering: Optimization of glycolytic flux in engineered microorganisms for biofuel or biochemical production
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Enzyme evolution studies: Understanding the evolution of central metabolic enzymes in response to environmental pressures
The halophilic, temperature-flexible, and potentially pressure-tolerant nature of this enzyme makes it particularly interesting for processes requiring stability under challenging conditions.
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What future research directions would advance our understanding of I. loihiensis gpmI?
Several promising research directions include:
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Structural characterization: Determining the three-dimensional structure through X-ray crystallography or cryo-EM
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In vivo metabolic studies: Analyzing glycolytic flux in I. loihiensis under different environmental conditions
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Evolutionary analysis: Comparing gpmI sequences across extremophiles to identify convergent adaptations
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Synthetic biology approaches: Engineering gpmI variants with enhanced stability or catalytic properties
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Systems biology integration: Understanding how gpmI functions within the unique metabolic network of I. loihiensis, particularly its integration with amino acid catabolism pathways
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Metal dependency studies: Investigating how different metals affect activity and stability, reflecting adaptation to the metal-rich hydrothermal vent environment
These approaches would provide comprehensive insights into how this central metabolic enzyme has adapted to function in an organism that thrives in one of Earth's most extreme environments.
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