Molecular structure of Escherichia coli purT-encoded glycinamide ribonucleotide transformylase

J. B. Thoden, S. Firestine, A. Nixon, Stephen Benkovic, H. M. Holden

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

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. On the basis of amino acid sequence analyses, it is known that 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. Here we describe two high-resolution X-ray crystallographic structures of PurT transformylase from E. coli: one form complexed with the nonhydrolyzable ATP analogue AMPPNP and the second with bound AMPPNP and GAR. The latter structure is of special significance because it represents the first ternary complex to be determined for a member of the ATP-grasp superfamily involved in purine biosynthesis and as such provides new information about the active site region involved in ribonucleotide binding. Specifically in PurT transformylase, the GAR substrate is anchored to the protein via Glu 82, Asp 286, Lys 355, Arg 362, and Arg 363. Key amino acid side chains involved in binding the AMPPNP to the enzyme include Arg 114, Lys 155, Glu 195, Glu 203, and Glu 267. Strikingly, the amino group of GAR that is formylated during the reaction lies at 2.8 Å from one of the γ-phosphoryl oxygens of the AMPPNP.

Original languageEnglish (US)
Pages (from-to)8791-8802
Number of pages12
JournalBiochemistry
Volume39
Issue number30
DOIs
StatePublished - Aug 1 2000

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Phosphoribosylglycinamide Formyltransferase
Molecular Structure
Adenylyl Imidodiphosphate
Escherichia coli
Molecular structure
Adenosine Triphosphate
Hand Strength
Hydroxymethyl and Formyl Transferases
Biosynthesis
Ribonucleotides
Amino Acids
Protein Sequence Analysis
Enzymes
Catalytic Domain
Proteins
Phosphates
X-Rays
Oxygen
X rays
Substrates

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Thoden, J. B., Firestine, S., Nixon, A., Benkovic, S., & Holden, H. M. (2000). Molecular structure of Escherichia coli purT-encoded glycinamide ribonucleotide transformylase. Biochemistry, 39(30), 8791-8802. https://doi.org/10.1021/bi000926j
Thoden, J. B. ; Firestine, S. ; Nixon, A. ; Benkovic, Stephen ; Holden, H. M. / Molecular structure of Escherichia coli purT-encoded glycinamide ribonucleotide transformylase. In: Biochemistry. 2000 ; Vol. 39, No. 30. pp. 8791-8802.
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abstract = "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. On the basis of amino acid sequence analyses, it is known that 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. Here we describe two high-resolution X-ray crystallographic structures of PurT transformylase from E. coli: one form complexed with the nonhydrolyzable ATP analogue AMPPNP and the second with bound AMPPNP and GAR. The latter structure is of special significance because it represents the first ternary complex to be determined for a member of the ATP-grasp superfamily involved in purine biosynthesis and as such provides new information about the active site region involved in ribonucleotide binding. Specifically in PurT transformylase, the GAR substrate is anchored to the protein via Glu 82, Asp 286, Lys 355, Arg 362, and Arg 363. Key amino acid side chains involved in binding the AMPPNP to the enzyme include Arg 114, Lys 155, Glu 195, Glu 203, and Glu 267. Strikingly, the amino group of GAR that is formylated during the reaction lies at 2.8 {\AA} from one of the γ-phosphoryl oxygens of the AMPPNP.",
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Thoden, JB, Firestine, S, Nixon, A, Benkovic, S & Holden, HM 2000, 'Molecular structure of Escherichia coli purT-encoded glycinamide ribonucleotide transformylase', Biochemistry, vol. 39, no. 30, pp. 8791-8802. https://doi.org/10.1021/bi000926j

Molecular structure of Escherichia coli purT-encoded glycinamide ribonucleotide transformylase. / Thoden, J. B.; Firestine, S.; Nixon, A.; Benkovic, Stephen; Holden, H. M.

In: Biochemistry, Vol. 39, No. 30, 01.08.2000, p. 8791-8802.

Research output: Contribution to journalArticle

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T1 - Molecular structure of Escherichia coli purT-encoded glycinamide ribonucleotide transformylase

AU - Thoden, J. B.

AU - Firestine, S.

AU - Nixon, A.

AU - Benkovic, Stephen

AU - Holden, H. M.

PY - 2000/8/1

Y1 - 2000/8/1

N2 - 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. On the basis of amino acid sequence analyses, it is known that 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. Here we describe two high-resolution X-ray crystallographic structures of PurT transformylase from E. coli: one form complexed with the nonhydrolyzable ATP analogue AMPPNP and the second with bound AMPPNP and GAR. The latter structure is of special significance because it represents the first ternary complex to be determined for a member of the ATP-grasp superfamily involved in purine biosynthesis and as such provides new information about the active site region involved in ribonucleotide binding. Specifically in PurT transformylase, the GAR substrate is anchored to the protein via Glu 82, Asp 286, Lys 355, Arg 362, and Arg 363. Key amino acid side chains involved in binding the AMPPNP to the enzyme include Arg 114, Lys 155, Glu 195, Glu 203, and Glu 267. Strikingly, the amino group of GAR that is formylated during the reaction lies at 2.8 Å from one of the γ-phosphoryl oxygens of the AMPPNP.

AB - 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. On the basis of amino acid sequence analyses, it is known that 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. Here we describe two high-resolution X-ray crystallographic structures of PurT transformylase from E. coli: one form complexed with the nonhydrolyzable ATP analogue AMPPNP and the second with bound AMPPNP and GAR. The latter structure is of special significance because it represents the first ternary complex to be determined for a member of the ATP-grasp superfamily involved in purine biosynthesis and as such provides new information about the active site region involved in ribonucleotide binding. Specifically in PurT transformylase, the GAR substrate is anchored to the protein via Glu 82, Asp 286, Lys 355, Arg 362, and Arg 363. Key amino acid side chains involved in binding the AMPPNP to the enzyme include Arg 114, Lys 155, Glu 195, Glu 203, and Glu 267. Strikingly, the amino group of GAR that is formylated during the reaction lies at 2.8 Å from one of the γ-phosphoryl oxygens of the AMPPNP.

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