title: 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.
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.affiliation: Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Agnelli
authors.forename: Laura
authors.initials: L
authors.lastname: Dossena
authors.forename: Paolo
authors.initials: P
authors.lastname: Colombi
authors.forename: Sara
authors.initials: S
authors.lastname: Mulazzi
authors.forename: Paola
authors.initials: P
authors.lastname: Morandi
authors.forename: Gabriella
authors.initials: G
authors.lastname: Tedeschi
authors.forename: Armando
authors.initials: A
authors.lastname: Negri
authors.forename: Bruno
authors.initials: B
authors.lastname: Curti
authors.forename: Maria A
authors.initials: MA
authors.lastname: Vanoni
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000641:Ammonia / Q000737:chemistry; D000818:Animals; D015806:Azospirillum brasilense / Q000201:enzymology; D002417:Cattle; D002845:Chromatography; D042943:Dihydrouracil Dehydrogenase (NADP) / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004579:Electron Transport; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D005415:Flavins / Q000737:chemistry; D005970:Glutamate Synthase / Q000737:chemistry* / Q000378:metabolism; D005977:Glutarates / Q000737:chemistry; D007098:Imino Acids / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D007656:Ketoglutaric Acids / Q000737:chemistry; D007700:Kinetics; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
chemical_list: D005415:Flavins; D005977:Glutarates; D007098:Imino Acids; D007506:Iron-Sulfur Proteins; D007656:Ketoglutaric Acids; D009841:Oligonucleotides; C054902:iminoglutarate; D009249:NADP; D000641:Ammonia; D007501:Iron; D042943:Dihydrouracil Dehydrogenase (NADP); D005970:Glutamate Synthase; D000409:Alanine