Recombinant Treponema denticola Glycine--tRNA ligase (glyQS)
Product Specs
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Target Background
KEGG: tde:TDE0020
STRING: 243275.TDE0020
Q&A
Q1: What is the primary biochemical role of glyQS in T. denticola?
A1: Glycine--tRNA ligase (glyQS) catalyzes the attachment of glycine to tRNA<sup>Gly</sup>, a critical step in protein biosynthesis. This class II aminoacyl-tRNA synthetase (aaRS) ensures correct charging of tRNA<sup>Gly</sup> with glycine, enabling translation fidelity. Structurally, glyQS employs a negatively charged active-site pocket to exclude non-glycine substrates, relying on conserved residues (e.g., glutamate, arginine) for substrate recognition .
Q2: How is glyQS expressed and purified for research?
A2: Recombinant glyQS is typically expressed in E. coli using shuttle plasmids (e.g., pKMR4PE derivatives). Purification involves affinity chromatography (e.g., His-tag systems) followed by size-exclusion chromatography to achieve >85% purity . Key considerations include:
Q3: How does glyQS interact with tRNA<sup>Gly</sup> at the atomic level?
A3: Structural studies (e.g., X-ray crystallography) reveal glyQS binds tRNA<sup>Gly</sup> via a conserved class II aaRS fold. Key interactions include:
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Substrate recognition: A negatively charged pocket (e.g., Glu residues) excludes amino acids with side chains, ensuring glycine specificity .
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Catalytic mechanism: ATP hydrolysis generates glycyl-adenylate, which transfers glycine to tRNA<sup>Gly</sup>’s 3′-OH group. The reaction follows a two-step mechanism involving adenylate formation and transfer .
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EF-Tu interaction: Elongation factor Tu (EF-Tu) binds glyQS to protect mischarged tRNA<sup>Gly</sup> from deacylation by D-aminoacyl-tRNA deacylase (DTD), ensuring translation fidelity .
Q4: What challenges exist in studying glyQS’s role in T. denticola pathogenesis?
A4: While glyQS is essential for protein synthesis, its direct role in pathogenesis remains unclear. Challenges include:
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Functional redundancy: T. denticola may utilize alternative pathways for glycine metabolism.
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Host interaction complexity: GlyQS’s role in evading host immunity (e.g., complement system) requires co-study with virulence factors like FhbB .
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Experimental limitations: Knockout mutants are difficult to generate due to T. denticola’s anaerobic growth requirements and antibiotic resistance .
Q5: How does glyQS contribute to glutathione metabolism in T. denticola?
A5: GlyQS indirectly supports glutathione catabolism by maintaining glycine availability. T. denticola metabolizes glutathione via a three-step pathway (GGT → CGase → cystalysin), producing H<sub>2</sub>S, which damages host tissues . While glyQS itself does not directly degrade glutathione, glycine deprivation (e.g., via glyQS inhibition) would impair downstream metabolism.
Q6: Why do studies report conflicting data on glyQS’s substrate specificity?
A6: Discrepancies arise from methodological variations:
For example, T. denticola glyQS shows higher affinity for glycine (K<sub>m</sub> = 8.2 µM<sup>-1</sup>min<sup>-1</sup>) compared to leucine (K<sub>m</sub> = 1.1 µM<sup>-1</sup>min<sup>-1</sup>), confirming strict substrate preference .
Q7: How can recombinant glyQS be used to study protein misfolding?
A7: GlyQS serves as a model for studying chiral proofreading in aaRSs. Researchers can:
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Engineer mutants: Introduce substitutions in the active-site pocket (e.g., Glu → Ala) to disrupt glycine binding.
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Monitor mischarging: Use DTD to deacylate non-cognate aminoacyl-tRNA<sup>Gly</sup> (e.g., D-alanine-tRNA<sup>Gly</sup>) and measure residual activity .
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Assess EF-Tu protection: Co-incubate glyQS with EF-Tu to evaluate rescue of mischarged tRNA<sup>Gly</sup> .
Q8: What are best practices for validating glyQS’s enzymatic activity?
A8: Validate glyQS activity using:
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Radioactive assays: Measure <sup>3</sup>H-glycine incorporation into tRNA<sup>Gly</sup>.
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HPLC-based quantification: Detect glycyl-adenylate intermediates or charged tRNA<sup>Gly</sup> via UV absorbance .
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Competition studies: Use analogs (e.g., ethanolamine) to inhibit glycine binding and confirm specificity .
Q9: What structural features distinguish glyQS from other class II aaRSs?
A9: GlyQS exhibits unique adaptations for glycine recognition:
These features ensure glycine specificity despite its small size .
Q10: How does glyQS’s thermostability impact biochemical studies?
A10: GlyQS’s high thermostability (e.g., Thermus thermophilus homologs) allows:
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High-temperature assays: Study catalytic mechanisms under extreme conditions.
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Crystallization: Facilitate X-ray crystallography for structural insights .
Q11: What unanswered questions remain in glyQS research?
A11: Key gaps include:
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In vivo regulation: How glyQS expression is modulated during T. denticola infection.
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Host interaction: Whether glyQS affects host cell signaling pathways (e.g., via glycine metabolism).
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Therapeutic targeting: Potential for inhibiting glyQS to disrupt T. denticola pathogenesis.
