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Literature summary extracted from

  • Thoden, J.B.; Firestine, S.; Nixon, A.; Benkovic, S.J.; Holden, H.M.
    Molecular structure of Escherichia coli PurT-encoded glycinamide ribonucleotide transformylase (2000), Biochemistry, 39, 8791-8802 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
6.3.1.21 gene purT, recombinant overexpression of the enzyme in Escherichia coli strain BL21(DE3) Escherichia coli

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
6.3.1.21 purified enzyme complexed with the nonhydrolyzable ATP analogue AMP-PNP and complexed with AMP-PNP and glycinamide ribonucleotide, hanging drop vapor diffusion method, mixing of 20 mg/ml protein in 15 mM HEPES, pH 7.5, 5 mM AMP-PNP, 10 mM MgCl2, and 250 mM NaCl, in an 1:1 ratio with reservoir solution containing 18-22% w/v methyl ether poly(ethylene glycol) 5000, 100 mM MOPS, pH 6.7, and 100 mM MgCl2, 4°C, 2-3 weeks, X-ray diffraction structure determination analysis at 1.9 A resolution. For preparation of the complex with GAR, crystals grown in the presence of AMP-PNP are transferred to a solution containing 20% w/v methyl ether poly(ethylene glycol) 5000, 50 mM MgCl2, 350 mM NaCl, 2.5 mM AMP-PNP, 100 mM MOPS (pH 6.7), and 1 mM GAR, the crystals are allowed to soak for 12 h, X-ray diffraction structure determination analysis at 1.75 A resolution. Structure modelling, detailed overview Escherichia coli

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.3.1.21 Mg2+ required Escherichia coli

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
6.3.1.21 ATP + formate + N1-(5-phospho-beta-D-ribosyl)glycinamide Escherichia coli
-
 ADP + phosphate + N2-formyl-N1-(5-phospho-beta-D-ribosyl)glycinamide
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
6.3.1.21 Escherichia coli P33221
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
6.3.1.21 recombinant enzyme from Escherichia coli strain BL21(DE3) by streptomycin sulfate precipitation, anion exchange chromatography, ultrafiltration, dialysis, and gel filtration, followed by dialysis and ultrafiltration Escherichia coli

Reaction

EC Number Reaction Comment Organism Reaction ID
6.3.1.21 ATP + formate + N1-(5-phospho-beta-D-ribosyl)glycinamide = ADP + phosphate + N2-formyl-N1-(5-phospho-beta-D-ribosyl)glycinamide possible catalytic mechanism for PurT transformylase. In this mechanism, the breakage of the beta-gamma phosphate bond results in a shift of the Mg2+-formyl phosphate thereby bringing the carbonyl carbon of the formyl phosphate near the amine of glycinamide ribonucleotide (GAR). Asp268 functions to deprotonate the amine of GAR, which then reacts with the carbonyl group of formyl phosphate to release phosphate and formyl-GAR Escherichia coli
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.3.1.21 ATP + formate + N1-(5-phospho-beta-D-ribosyl)glycinamide
-
 Escherichia coli ADP + phosphate + N2-formyl-N1-(5-phospho-beta-D-ribosyl)glycinamide
-
 ?

Subunits

EC Number Subunits Comment Organism
6.3.1.21 dimer the A-domain is formed by segment Thr2-Ala122 and is dominated by a five-stranded parallel beta-pleated sheet flanked on either side by two alpha-helices. The smallest of the three structural motifs of the PurT transformylase, the B-domain is formed by Glu123-Gly196 and contains a four-stranded antiparallel beta-sheet with the strands ranging in length from three to five amino acid residues. The most complicated of the domains, the C-motif extends from Val197 to Gly392 and is composed primarily of an eight-stranded antiparallel beta-pleated sheet formed by Phe202-Ser210, Val215-Gln225, Tyr230-Gln235, Gly262-Val270, Val275-Ser281, Ala321-Ile326, Gln349-Leu352, and Gly365-Thr370. There are two additional regions of antiparallel beta-sheet formed by Gln329-Ser332 and Ile358-Ser361 and Thr336-Asp338 and Val388-Gly392, respectively. The four alpha-helices located in the C-domain range in length from four to eighteen amino acid residues. There are seven classical reverse turns in the C-domain that link these various beta-strands and alpha-helices together (three type I, one type I', one type II, one type II', and one type III). The dimeric interface is formed from regions provided by both the A- and C-domains. Enzyme structure analysis, detailed overview Escherichia coli

Synonyms

EC Number Synonyms Comment Organism
6.3.1.21 glycinamide ribonucleotide transformylase
-
 Escherichia coli
6.3.1.21 PurT
-
 Escherichia coli
6.3.1.21 PurT transformylase
-
 Escherichia coli
6.3.1.21 PurT-encoded glycinamide ribonucleotide transformylase
-
 Escherichia coli

Cofactor

EC Number Cofactor Comment Organism Structure
6.3.1.21 ATP
-
 Escherichia coli
6.3.1.21 additional information key amino acid side chains involved in binding the nonhydrolyzable ATP analogue AMP-PNP to the enzyme include Arg114, Lys155, Glu195, Glu203, and Glu267. Strikingly, the amino group of GAR that is formylated during the reaction lies at 2.8 A from one of the gamma-phosphoryl oxygens of the AMP-PNP Escherichia coli

General Information

EC Number General Information Comment Organism
6.3.1.21 evolution the PurT transformylase belongs to the ATP-grasp superfamily of proteins. The common theme among members of this superfamily is a catalytic reaction mechanism that requires ATP and proceeds through an acyl phosphate intermediate. All of the enzymes belonging to the ATP-grasp superfamily are composed of three structural motifs, termed the A-, B-, and C-domains, and in each case, the ATP is wedged between the B- and C-domains. Superposition of the Rossmann folds found in UDP-galactose 4-epimerase and PurT transformylase, overview Escherichia coli
6.3.1.21 additional information specifically in PurT transformylase, the glycinamide ribonucleotide (GAR) substrate is anchored to the protein via Glu82, Asp286, Lys355, Arg362, and Arg363. Structure analysis of the active site for PurT transformylase, overview Escherichia coli
6.3.1.21 physiological function in Escherichia coli, the PurT-encoded glycinamide ribonucleotide transformylase, or PurT transformylase, catalyzes an alternative formylation of glycinamide ribonucleotide (GAR) in the de novo pathway for purine biosynthesis Escherichia coli