De novo design of proteins provides an attractive approach to uncover the essential features required for their functions. Previously, we described the design and crystal structure determination of a di-Zn(II) complex of “due-ferri-1” (DF1), a protein patterned after the diiron−dimanganese class of redox-active proteins [Lombardi, A.; Summa, C.; Geremia, S.; Randaccio, L.; Pavone, V.; DeGrado, W. F. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 6298−6305]. The overall structure of DF1, which contains a carboxylate-bridged dinuclear metal site, agrees well with the intended design. However, access to this dimetal site is blocked by a pair of hydrophobic leucine residues (L13 and L13‘), which prevent facile entry of metal ions and small molecules. We have now taken the next step in the eventual construction of a catalytically active metalloenzyme by engineering an active site cavity into DF1 through the replacement of these two leucine residues with smaller residues. The crystal structure of the dimanganous form of L13A-DF1 indeed shows a substrate access channel to the dimetal center. In the crystal structure, water molecules and a ligating dimethyl sulfoxide molecule, which forms a monatomic bridge between the metal ions, occupy the cavity. Furthermore, the diferric form of a derivative of L13A-DF1, DF2, is shown to bind azide, acetate, and small aromatic molecules.

Toward the De novo Design of a Catalytically Active Helix-Bundle: a Substrate Accessible Carboxylate-bridged Dinuclear Metal Center / DI COSTANZO, L.; Wade, H.; Geremia, S.; Randaccio, L.; Pavone, V.; Degrado, W. F.; Lombardi, A.. - In: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. - ISSN 0002-7863. - STAMPA. - 123:51(2001), pp. 12749-12757. [10.1021/ja010506x]

Toward the De novo Design of a Catalytically Active Helix-Bundle: a Substrate Accessible Carboxylate-bridged Dinuclear Metal Center

L. DI COSTANZO;V. PAVONE;A. LOMBARDI
2001

Abstract

De novo design of proteins provides an attractive approach to uncover the essential features required for their functions. Previously, we described the design and crystal structure determination of a di-Zn(II) complex of “due-ferri-1” (DF1), a protein patterned after the diiron−dimanganese class of redox-active proteins [Lombardi, A.; Summa, C.; Geremia, S.; Randaccio, L.; Pavone, V.; DeGrado, W. F. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 6298−6305]. The overall structure of DF1, which contains a carboxylate-bridged dinuclear metal site, agrees well with the intended design. However, access to this dimetal site is blocked by a pair of hydrophobic leucine residues (L13 and L13‘), which prevent facile entry of metal ions and small molecules. We have now taken the next step in the eventual construction of a catalytically active metalloenzyme by engineering an active site cavity into DF1 through the replacement of these two leucine residues with smaller residues. The crystal structure of the dimanganous form of L13A-DF1 indeed shows a substrate access channel to the dimetal center. In the crystal structure, water molecules and a ligating dimethyl sulfoxide molecule, which forms a monatomic bridge between the metal ions, occupy the cavity. Furthermore, the diferric form of a derivative of L13A-DF1, DF2, is shown to bind azide, acetate, and small aromatic molecules.
2001
Toward the De novo Design of a Catalytically Active Helix-Bundle: a Substrate Accessible Carboxylate-bridged Dinuclear Metal Center / DI COSTANZO, L.; Wade, H.; Geremia, S.; Randaccio, L.; Pavone, V.; Degrado, W. F.; Lombardi, A.. - In: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. - ISSN 0002-7863. - STAMPA. - 123:51(2001), pp. 12749-12757. [10.1021/ja010506x]
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/147721
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 95
  • ???jsp.display-item.citation.isi??? 74
social impact