Recombinant Erwinia tasmaniensis Serine hydroxymethyltransferase (glyA)
Description
Enzyme Function and Biochemical Properties
SHMT plays a dual role in:
-
Glycine synthesis: Converts serine to glycine, essential for protein and purine biosynthesis .
-
One-carbon metabolism: Generates MTHF, a cofactor for thymidylate and methionine synthesis .
In E. tasmaniensis, SHMT activity is inferred from its conserved genetic presence in related species. For example, E. coli SHMT (GlyA) exhibits a substrate affinity of 0.1–0.3 mM for MTHF and an aldol cleavage activity of 1.3 µmol·min⁻¹·mg⁻¹ with L-threonine as a substrate .
Recombinant Production and Purification
Recombinant SHMT is typically produced via heterologous expression in E. coli. Key steps include:
-
Gene cloning: The glyA gene is amplified and inserted into expression vectors (e.g., pQE30 or pMCSG49) .
-
Induction: Expression is induced with isopropyl-β-D-thiogalactoside (IPTG) .
-
Affinity purification: His-tagged enzymes are purified using Ni²⁺-nitrilotriacetic acid (Ni-NTA) chromatography, achieving >85% purity .
For example, Streptococcus thermophilus GlyA was overexpressed in E. coli M15, yielding 83% activity recovery after purification .
Substrate Specificity and Catalytic Activity
SHMT exhibits broad substrate versatility:
| Substrate | Activity (µmol·min⁻¹·mg⁻¹) | Organism | Source |
|---|---|---|---|
| L-serine | 32.5 | E. coli | |
| L-threonine | 1.3 | C. glutamicum | |
| L-allo-threonine | 0.5 | S. thermophilus |
E. tasmaniensis SHMT likely shares similar catalytic traits, given structural conservation across bacterial SHMTs .
Applications and Biotechnological Relevance
-
Amino acid production: Engineered SHMT variants enhance glycine yields in Corynebacterium glutamicum .
-
Antimicrobial targets: SHMT inhibitors (e.g., SHIN1) show promise against Staphylococcus aureus by disrupting folate metabolism .
-
Enantioselective synthesis: SHMTs catalyze stereospecific reactions for β-hydroxy-α-amino acid production .
Research Gaps and Future Directions
Existing data on E. tasmaniensis GlyA remain sparse. Key priorities include:
-
Functional characterization: Substrate kinetics and structural analysis.
-
Metabolic engineering: Leveraging SHMT for glycine overproduction in industrial strains.
-
Therapeutic exploration: Assessing SHMT inhibitors for E. tasmaniensis-related infections.
Product Specs
Target Background
KEGG: eta:ETA_10130
STRING: 465817.ETA_10130
