Tags

Type your tag names separated by a space and hit enter

The unexpected structural role of glutamate synthase [4Fe-4S](+1,+2) clusters as demonstrated by site-directed mutagenesis of conserved C residues at the N-terminus of the enzyme beta subunit.
Arch Biochem Biophys. 2005 Apr 15; 436(2):355-66.AB

Abstract

Azospirillum brasilense glutamate synthase (GltS) is a complex iron-sulfur flavoprotein whose catalytically active alphabeta protomer (alpha subunit, 162kDa; beta subunit, 52.3 kDa) contains one FAD, one FMN, one [3Fe-4S](0,+1), and two [4Fe-4S](+1,+2) clusters. The structure of the alpha subunit has been determined providing information on the mechanism of ammonia transfer from L-glutamine to 2-oxoglutarate through a 30 A-long intramolecular tunnel. On the contrary, details of the electron transfer pathway from NADPH to the postulated 2-iminoglutarate intermediate through the enzyme flavin co-factors and [Fe-S] clusters are largely indirect. To identify the location and role of each one of the GltS [4Fe-4S] clusters, we individually substituted the four cysteinyl residues forming the first of two conserved C-rich regions at the N-terminus of GltS beta subunit with alanyl residues. The engineered genes encoding the beta subunit variants (and derivatives carrying C-terminal His6-tags) were co-expressed with the wild-type alpha subunit gene. In all cases the C/A substitutions prevented alpha and beta subunits association to yield the GltS alphabeta protomer. This result is consistent with the fact that these residues are responsible for the formation of glutamate synthase [4Fe-4S](+1,+2) clusters within the N-terminal region of the beta subunit, and that these clusters are implicated not only in electron transfer between the GltS flavins, but also in alphabeta heterodimer formation by structuring an N-terminal [Fe-S] beta subunit interface subdomain, as suggested by the three-dimensional structure of dihydropyrimidine dehydrogenase, an enzyme containing an N-terminal beta subunit-like domain.

Authors+Show Affiliations

Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15797248

Citation

Agnelli, Paola, et al. "The Unexpected Structural Role of Glutamate Synthase [4Fe-4S](+1,+2) Clusters as Demonstrated By Site-directed Mutagenesis of Conserved C Residues at the N-terminus of the Enzyme Beta Subunit." Archives of Biochemistry and Biophysics, vol. 436, no. 2, 2005, pp. 355-66.
Agnelli P, Dossena L, Colombi P, et al. The unexpected structural role of glutamate synthase [4Fe-4S](+1,+2) clusters as demonstrated by site-directed mutagenesis of conserved C residues at the N-terminus of the enzyme beta subunit. Arch Biochem Biophys. 2005;436(2):355-66.
Agnelli, P., Dossena, L., Colombi, P., Mulazzi, S., Morandi, P., Tedeschi, G., Negri, A., Curti, B., & Vanoni, M. A. (2005). The unexpected structural role of glutamate synthase [4Fe-4S](+1,+2) clusters as demonstrated by site-directed mutagenesis of conserved C residues at the N-terminus of the enzyme beta subunit. Archives of Biochemistry and Biophysics, 436(2), 355-66.
Agnelli P, et al. The Unexpected Structural Role of Glutamate Synthase [4Fe-4S](+1,+2) Clusters as Demonstrated By Site-directed Mutagenesis of Conserved C Residues at the N-terminus of the Enzyme Beta Subunit. Arch Biochem Biophys. 2005 Apr 15;436(2):355-66. PubMed PMID: 15797248.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The unexpected structural role of glutamate synthase [4Fe-4S](+1,+2) clusters as demonstrated by site-directed mutagenesis of conserved C residues at the N-terminus of the enzyme beta subunit. AU - Agnelli,Paola, AU - Dossena,Laura, AU - Colombi,Paolo, AU - Mulazzi,Sara, AU - Morandi,Paola, AU - Tedeschi,Gabriella, AU - Negri,Armando, AU - Curti,Bruno, AU - Vanoni,Maria A, PY - 2005/01/09/received PY - 2005/02/02/revised PY - 2005/3/31/pubmed PY - 2005/5/25/medline PY - 2005/3/31/entrez SP - 355 EP - 66 JF - Archives of biochemistry and biophysics JO - Arch Biochem Biophys VL - 436 IS - 2 N2 - Azospirillum brasilense glutamate synthase (GltS) is a complex iron-sulfur flavoprotein whose catalytically active alphabeta protomer (alpha subunit, 162kDa; beta subunit, 52.3 kDa) contains one FAD, one FMN, one [3Fe-4S](0,+1), and two [4Fe-4S](+1,+2) clusters. The structure of the alpha subunit has been determined providing information on the mechanism of ammonia transfer from L-glutamine to 2-oxoglutarate through a 30 A-long intramolecular tunnel. On the contrary, details of the electron transfer pathway from NADPH to the postulated 2-iminoglutarate intermediate through the enzyme flavin co-factors and [Fe-S] clusters are largely indirect. To identify the location and role of each one of the GltS [4Fe-4S] clusters, we individually substituted the four cysteinyl residues forming the first of two conserved C-rich regions at the N-terminus of GltS beta subunit with alanyl residues. The engineered genes encoding the beta subunit variants (and derivatives carrying C-terminal His6-tags) were co-expressed with the wild-type alpha subunit gene. In all cases the C/A substitutions prevented alpha and beta subunits association to yield the GltS alphabeta protomer. This result is consistent with the fact that these residues are responsible for the formation of glutamate synthase [4Fe-4S](+1,+2) clusters within the N-terminal region of the beta subunit, and that these clusters are implicated not only in electron transfer between the GltS flavins, but also in alphabeta heterodimer formation by structuring an N-terminal [Fe-S] beta subunit interface subdomain, as suggested by the three-dimensional structure of dihydropyrimidine dehydrogenase, an enzyme containing an N-terminal beta subunit-like domain. SN - 0003-9861 UR - https://www.unboundmedicine.com/medline/citation/15797248/The_unexpected_structural_role_of_glutamate_synthase_[4Fe_4S]_+1+2__clusters_as_demonstrated_by_site_directed_mutagenesis_of_conserved_C_residues_at_the_N_terminus_of_the_enzyme_beta_subunit_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-9861(05)00063-9 DB - PRIME DP - Unbound Medicine ER -