Abstract
The initial reaction of tetrapyrrole formation in archaea is catalyzed by a NADPH-dependent glutamyl-tRNA reductase (GluTR). The hemA gene encoding GluTR was cloned from the extremely thermophilic archaeon Methanopyrus kandleri and overexpressed in Escherichia coli. Purified recombinant GluTR is a tetrameric enzyme with a native M(r) = 190,000 +/- 10,000. Using a newly established enzyme assay, a specific activity of 0.75 nmol h(-1) mg(-1) at 56 degrees C with E. coli glutamyl-tRNA as substrate was measured. A temperature optimum of 90 degrees C and a pH optimum of 8.1 were determined. Neither heme cofactor, nor flavin, nor metal ions were required for GluTR catalysis. Heavy metal compounds, Zn(2+), and heme inhibited the enzyme. GluTR inhibition by the newly synthesized inhibitor glutamycin, whose structure is similar to the 3' end of the glutamyl-tRNA substrate, revealed the importance of an intact chemical bond between glutamate and tRNA(Glu) for substrate recognition. The absolute requirement for NADPH in the reaction of GluTR was demonstrated using four NADPH analogues. Chemical modification and site-directed mutagenesis studies indicated that a single cysteinyl residue and a single histidinyl residue were important for catalysis. It was concluded that during GluTR catalysis the highly reactive sulfhydryl group of Cys-48 acts as a nucleophile attacking the alpha-carbonyl group of tRNA-bound glutamate with the formation of an enzyme-localized thioester intermediate and the concomitant release of tRNA(Glu). In the presence of NADPH, direct hydride transfer to enzyme-bound glutamate, possibly facilitated by His-84, leads to glutamate-1-semialdehyde formation. In the absence of NADPH, a newly discovered esterase activity of GluTR hydrolyzes the highly reactive thioester of tRNA(Glu) to release glutamate.
Highlights
ALA1 is the general precursor molecule for the biosynthesis of tetrapyrroles like chlorophylls, hemes, and coenzyme B12 [1]
Investigation of glutamyltRNA reductase (GluTR) catalysis has been hampered by its low cellular concentration, difficulties with the overexpression of the respective gene from various species in Escherichia coli, and the tendency of the enzyme to aggregate and precipitate at high protein concentration [3, 4]
We describe the utilization of recombinant GluTR from the extreme thermophilic archaeon Methanopyrus kandleri for the determination of the native molecular mass and the elucidation of essential structural features of the substrate and the cofactor NADPH for enzyme recognition
Summary
The ALA-auxotrophic E. coli hemA strain GE1387 was transformed with a M. kandleri genomic library prepared in the vector pBluescript SKϩ, and complementing clones were identified as described before for the cloning of Methanobacterium thermoautotrophicum hemA [11]. The following oligonucleotides, with newly introduced codons underlined, were employed to generate the mutants indicated: for C6S GGAGGACCTGGTGAGCGTCGGTATCAC- The protein solution (10 mg/ml) was loaded with a flow rate of 1.5 ml/min onto a 50-ml Red Sepharose CL-6B column (diameter 3 cm), which had previously been equilibrated with 20 mM Na-HEPES, pH 8.1, including 1 mM -mercaptoethanol (buffer A). Fractions containing GluTR were pooled, dialyzed against buffer A, and loaded at a concentration of 5 mg of protein per ml of column volume onto a MonoQ HR 10/10 column previously equilibrated with buffer A. Fractions containing GluTR were pooled, dialyzed against buffer A, and concentrated to 7 mg/ml by ultrafiltration as described above. The native molecular mass of recombinant GluTR in the absence of tRNA substrate was determined as described previously [12]
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