Structural basis for putrescine activation of human S-adenosylmethionine decarboxylase

Shridhar Bale, Maria M. Lopez, George I. Makhatadze, Qingming Fang, Anthony Pegg, Steven E. Ealick

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Putrescine (1,4-diaminobutane) activates the autoprocessing and decarboxylation reactions of human S-adenosylmethionine decarboxylase (AdoMetDC), a critical enzyme in the polyamine biosynthetic pathway. In human AdoMetDC, putrescine binds in a buried pocket containing acidic residues Asp174, Glu178, and Glu256. The pocket is away from the active site but near the dimer interface; however, a series of hydrophilic residues connect the putrescine binding site and the active site. Mutation of these acidic residues modulates the effects of putrescine. D174N, E178Q, and E256Q mutants were expressed and dialyzed to remove putrescine and studied biochemically using X-ray crystallography, UV-CD spectroscopy, analytical ultracentrifugation, and ITC binding studies. The results show that the binding of putrescine to the wild type dimeric protein is cooperative. The D174N mutant does not bind putrescine, and the E178Q and E256Q mutants bind putrescine weakly with no cooperativity. The crystal structure of the mutants with and without putrescine and their complexes with S-adenosylmethionine methyl ester were obtained. Binding of putrescine results in a reorganization of four aromatic residues (Phe285, Phe315, Tyr318, and Phe320) and a conformational change in the loop 312-320. The loop shields putrescine from the external solvent, enhancing its electrostatic and hydrogen bonding effects. The E256Q mutant with putrescine added shows an alternate conformation of His243, Glu11, Lys80, and Ser229, the residues that link the active site and the putrescine binding site, suggesting that putrescine activates the enzyme through electrostatic effects and acts as a switch to correctly orient key catalytic residues.

Original languageEnglish (US)
Pages (from-to)13404-13417
Number of pages14
JournalBiochemistry
Volume47
Issue number50
DOIs
StatePublished - Dec 16 2008

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Adenosylmethionine Decarboxylase
Putrescine
Chemical activation
Catalytic Domain
Static Electricity
Electrostatics
Binding Sites
S-Adenosylmethionine
Decarboxylation

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Bale, S., Lopez, M. M., Makhatadze, G. I., Fang, Q., Pegg, A., & Ealick, S. E. (2008). Structural basis for putrescine activation of human S-adenosylmethionine decarboxylase. Biochemistry, 47(50), 13404-13417. https://doi.org/10.1021/bi801732m
Bale, Shridhar ; Lopez, Maria M. ; Makhatadze, George I. ; Fang, Qingming ; Pegg, Anthony ; Ealick, Steven E. / Structural basis for putrescine activation of human S-adenosylmethionine decarboxylase. In: Biochemistry. 2008 ; Vol. 47, No. 50. pp. 13404-13417.
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abstract = "Putrescine (1,4-diaminobutane) activates the autoprocessing and decarboxylation reactions of human S-adenosylmethionine decarboxylase (AdoMetDC), a critical enzyme in the polyamine biosynthetic pathway. In human AdoMetDC, putrescine binds in a buried pocket containing acidic residues Asp174, Glu178, and Glu256. The pocket is away from the active site but near the dimer interface; however, a series of hydrophilic residues connect the putrescine binding site and the active site. Mutation of these acidic residues modulates the effects of putrescine. D174N, E178Q, and E256Q mutants were expressed and dialyzed to remove putrescine and studied biochemically using X-ray crystallography, UV-CD spectroscopy, analytical ultracentrifugation, and ITC binding studies. The results show that the binding of putrescine to the wild type dimeric protein is cooperative. The D174N mutant does not bind putrescine, and the E178Q and E256Q mutants bind putrescine weakly with no cooperativity. The crystal structure of the mutants with and without putrescine and their complexes with S-adenosylmethionine methyl ester were obtained. Binding of putrescine results in a reorganization of four aromatic residues (Phe285, Phe315, Tyr318, and Phe320) and a conformational change in the loop 312-320. The loop shields putrescine from the external solvent, enhancing its electrostatic and hydrogen bonding effects. The E256Q mutant with putrescine added shows an alternate conformation of His243, Glu11, Lys80, and Ser229, the residues that link the active site and the putrescine binding site, suggesting that putrescine activates the enzyme through electrostatic effects and acts as a switch to correctly orient key catalytic residues.",
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Bale, S, Lopez, MM, Makhatadze, GI, Fang, Q, Pegg, A & Ealick, SE 2008, 'Structural basis for putrescine activation of human S-adenosylmethionine decarboxylase', Biochemistry, vol. 47, no. 50, pp. 13404-13417. https://doi.org/10.1021/bi801732m

Structural basis for putrescine activation of human S-adenosylmethionine decarboxylase. / Bale, Shridhar; Lopez, Maria M.; Makhatadze, George I.; Fang, Qingming; Pegg, Anthony; Ealick, Steven E.

In: Biochemistry, Vol. 47, No. 50, 16.12.2008, p. 13404-13417.

Research output: Contribution to journalArticle

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T1 - Structural basis for putrescine activation of human S-adenosylmethionine decarboxylase

AU - Bale, Shridhar

AU - Lopez, Maria M.

AU - Makhatadze, George I.

AU - Fang, Qingming

AU - Pegg, Anthony

AU - Ealick, Steven E.

PY - 2008/12/16

Y1 - 2008/12/16

N2 - Putrescine (1,4-diaminobutane) activates the autoprocessing and decarboxylation reactions of human S-adenosylmethionine decarboxylase (AdoMetDC), a critical enzyme in the polyamine biosynthetic pathway. In human AdoMetDC, putrescine binds in a buried pocket containing acidic residues Asp174, Glu178, and Glu256. The pocket is away from the active site but near the dimer interface; however, a series of hydrophilic residues connect the putrescine binding site and the active site. Mutation of these acidic residues modulates the effects of putrescine. D174N, E178Q, and E256Q mutants were expressed and dialyzed to remove putrescine and studied biochemically using X-ray crystallography, UV-CD spectroscopy, analytical ultracentrifugation, and ITC binding studies. The results show that the binding of putrescine to the wild type dimeric protein is cooperative. The D174N mutant does not bind putrescine, and the E178Q and E256Q mutants bind putrescine weakly with no cooperativity. The crystal structure of the mutants with and without putrescine and their complexes with S-adenosylmethionine methyl ester were obtained. Binding of putrescine results in a reorganization of four aromatic residues (Phe285, Phe315, Tyr318, and Phe320) and a conformational change in the loop 312-320. The loop shields putrescine from the external solvent, enhancing its electrostatic and hydrogen bonding effects. The E256Q mutant with putrescine added shows an alternate conformation of His243, Glu11, Lys80, and Ser229, the residues that link the active site and the putrescine binding site, suggesting that putrescine activates the enzyme through electrostatic effects and acts as a switch to correctly orient key catalytic residues.

AB - Putrescine (1,4-diaminobutane) activates the autoprocessing and decarboxylation reactions of human S-adenosylmethionine decarboxylase (AdoMetDC), a critical enzyme in the polyamine biosynthetic pathway. In human AdoMetDC, putrescine binds in a buried pocket containing acidic residues Asp174, Glu178, and Glu256. The pocket is away from the active site but near the dimer interface; however, a series of hydrophilic residues connect the putrescine binding site and the active site. Mutation of these acidic residues modulates the effects of putrescine. D174N, E178Q, and E256Q mutants were expressed and dialyzed to remove putrescine and studied biochemically using X-ray crystallography, UV-CD spectroscopy, analytical ultracentrifugation, and ITC binding studies. The results show that the binding of putrescine to the wild type dimeric protein is cooperative. The D174N mutant does not bind putrescine, and the E178Q and E256Q mutants bind putrescine weakly with no cooperativity. The crystal structure of the mutants with and without putrescine and their complexes with S-adenosylmethionine methyl ester were obtained. Binding of putrescine results in a reorganization of four aromatic residues (Phe285, Phe315, Tyr318, and Phe320) and a conformational change in the loop 312-320. The loop shields putrescine from the external solvent, enhancing its electrostatic and hydrogen bonding effects. The E256Q mutant with putrescine added shows an alternate conformation of His243, Glu11, Lys80, and Ser229, the residues that link the active site and the putrescine binding site, suggesting that putrescine activates the enzyme through electrostatic effects and acts as a switch to correctly orient key catalytic residues.

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