Azospirillum brasilense glutamate synthase (GltS) is a complex iron–sulfur flavoprotein whose catalytically active αβ protomer (α subunit, 162 kDa; β 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 α subunit has been determined providing information on the mechanism of ammonia transfer from l -glutamine to 2-oxoglutarate through a 30 Å-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 β subunit with alanyl residues. The engineered genes encoding the β subunit variants (and derivatives carrying C-terminal His6 -tags) were co-expressed with the wild-type α subunit gene. In all cases the C/A substitutions prevented α and β subunits association to yield the GltS αβ 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 β subunit, and that these clusters are implicated not only in electron transfer between the GltS flavins, but also in αβ heterodimer formation by structuring an N-terminal [Fe–S] β subunit interface subdomain, as suggested by the three-dimensional structure of dihydropyrimidine dehydrogenase, an enzyme containing an N-terminal β subunit-like domain.
Titolo: | 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. |
Autori: | AGNELLI, PAOLA CECILIA (Primo) DOSSENA, LAURA (Secondo) CURTI, BRUNO (Penultimo) VANONI, MARIA ANTONIETTA (Ultimo) |
Parole Chiave: | Amidotransferase; Ammonia assimilation; Assembly of iron-sulfur clusters; Dihydropyrimidine dehydrogenase; Flavoprotein; Glutamate synthase; Iron-sulfur clusters; Protein engineering; Site-directed mutagenesis |
Settore Scientifico Disciplinare: | Settore BIO/10 - Biochimica |
Data di pubblicazione: | 15-apr-2005 |
Rivista: | |
Tipologia: | Article (author) |
Digital Object Identifier (DOI): | 10.1016/j.abb.2005.02.009 |
Appare nelle tipologie: | 01 - Articolo su periodico |