Expression of catalytically active barley glutamyl tRNAGlu reductase in Escherichia coli as a fusion protein with glutathione S-transferase.

Abstract

ABSTRACT8-Aminolevulinate in plants, algae, cyanobacteria, and several other bacteria such as Escherichia coli and Bacillus subtilis is synthesized from glutamate by means of a tRNAGIU mediated pathway. The enzyme glutamyl tRNAGIU reductase catalyzes the second step in this pathway, the reduction of tRNA bound glutamate to give glutamate 1-semialdehyde. The hemA gene from barley encoding the glutamyl tRNAGIU reductase was expressed in E. coli cells joined at its amino terminal end to Schistosoma japonicum glutathione Stransferase (GST). GST-glutamyl tRNAGlU reductase fusion protein and the reductase released from it by thrombin digestion catalyzed the reduction of glutamyl tRNAGIlu to glutamate 1-semialdehyde. The specific activity of the fusion protein was 120 pmol ,ug-l min-1. The fusion protein used tRNAGIU from barley chloroplasts preferentially to E. coli tRNAGIU and its activity was inhibited by hemin. It migrated as an 82-kDa polypeptide with SDS/PAGE and eluted with an apparent molecular mass of 450 kDa from Superose 12. After removal of the GST by thrombin, the protein migrated as an -60-kDa polypeptide with SDS/PAGE, whereas gel filtration on Superose 12 yielded an apparent molecule mass of 250 kDa. Isolated fusion protein contained heme, which could be reduced by NADPH and oxidized by air.

Crystal structure of Arabidopsis glutamyl-tRNA reductase in complex with its stimulator protein

“…The active conformation of GluTR observed in the GluTR-GluBP complex suggests a stimulatory role of GluBP in GluTR regulation. To test this, we measured the effect of GluBP on GluTR activity by determining ALA formation by using a coupled enzyme assay containing GluTR and GSAM as described previously (24)(25)(26). Addition of GluBP to the assay mixture increases ALA formation approximately threefold vs. the level in the absence of GluBP (Fig.…”

“…GluTR activity was measured in a reconstitution assay with purified recombinant E. coli glutamyl-tRNA synthetase and GSAM (24)(25)(26). The 250-μL assay mixture contained 50 μg glutamyl-tRNA synthetase, 156 μg GluTR, 195 μg GSAM, 1 mM NADPH, 200 μM glutamate, and 360 μg E. coli total tRNA, in a buffer consisting of 50 mM K-Hepes, pH 7.9, 1 mM DTT, and 25 mM MgCl 2 .…”

Tetrapyrrole Metabolism inArabidopsis thaliana

“…Two mechanisms have been reported to regulate GluTR activity. In the first example, heme directly binds GluTR and inhibits its activity (Vothknecht et al 1996(Vothknecht et al , 1998. GluTR is inhibited by heme at a site distinct from the catalytic center of the enzyme (Pontoppidan and Kannangara 1994; Vothknecht et al 1996), and thus the control of tetrapyrrole biosynthesis in higher plants is exclusively attributed to heme (Beale 1999).…”

“…In the first example, heme directly binds GluTR and inhibits its activity (Vothknecht et al 1996(Vothknecht et al , 1998. GluTR is inhibited by heme at a site distinct from the catalytic center of the enzyme (Pontoppidan and Kannangara 1994; Vothknecht et al 1996), and thus the control of tetrapyrrole biosynthesis in higher plants is exclusively attributed to heme (Beale 1999). In the Arabidopsis hy1 mutant and the tomato aurea and yellow-green-2 mutants, impariment in the activity of HO or PΦB synthase led to repression of GluTR activity (Goslings et al 2004;Terry and Kendrick 1999).…”

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

“…To achieve this end, specific sequences are included between the tag(s) and native protein and then cleaved with site-specific proteases. Common proteases include enterokinase [172], factor Xa [8], SUMO protease [48], tobacco etch virus (TEV) protease [173], thrombin [174,175], and 3C [176], outlined in Supporting information, Table S4. Proteases may cleave fusion proteins at unintended sites [151] and the buffer conditions that promote protease activity and specificity may not be suitable for fusion protein and product solubility [177].…”