J. Phys. Chem. B, 108 (41), 16250 -16254, 2004. 10.1021/jp048788l S1089-5647(04)08788-7
Web Release Date: September 17, 2004
Copyright (c) 2004 American Chemical Society

J. Phys. Chem. B, 108 (41), 16255 -16260, 2004. 10.1021/jp0487861 S1089-5647(04)08786-3
Web Release Date: September 17, 2004
Copyright (c) 2004 American Chemical Society

Molecular dynamics simulation of the interaction between the complex iron-sulfur flavoprotein glutamate synthase and its substrates

Vincenza M. Coiro1,2, Alfredo Di Nola1, Maria A. Vanoni3, Massimiliano Aschi4, Alessandro Coda5, Bruno Curti3 and Danilo Roccatano6

1 Dipartimento di Chimica, Universita` di Roma "La Sapienza," 00185 Roma, Italy

2 Istituto di Cristallografia, CNR, Sezione di Roma, 00016 Monterotondo Stazione, Roma, Italy

3 Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita' degli Studi di Milano, 20133 Milano, Italy

4 Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universita` de L'Aquila, 67010 L'Aquila, Italy

5 Dipartimento di Genetica e Microbiologia, Universita` di Pavia, 27100 Pavia, Italy

6 School of Engineering and Science, International University Bremen, 28759 Bremen, Germany

FULL TEXT

(RECEIVED May 13, 2004; FINAL REVISION August 4, 2004; ACCEPTED August 4, 2004)

Abstract

Glutamate synthase (GltS) is a complex iron-sulfur flavoprotein that catalyzes the reductive transfer of L-glutamine amide group to the C2 carbon of 2-oxoglutarate yielding two molecules of L-glutamate. Molecular dynamics calculations in explicit solvent were carried out to gain insight into the conformational flexibility of GltS and into the role played by the enzyme substrates in regulating the catalytic cycle. We have modelled the free (unliganded) form of Azospirillum brasilense GltS {alpha} subunit and the structure of the reduced enzyme in complex with the L-glutamine and 2-oxoglutarate substrates starting from the crystallographically determined coordinates of the GltS {alpha} subunit in complex with L-methionine sulphone and 2-oxoglutarate. The present 4-ns molecular dynamics calculations reveal that the GltS glutaminase site may exist in a catalytically inactive conformation unable to bind glutamine, and in a catalytically competent conformation, which is stabilized by the glutamine substrate. Substrates binding also induce (1) closure of the loop formed by residues 263-271 with partial shielding of the glutaminase site from solvent, and (2) widening of the ammonia tunnel entrance at the glutaminase end to allow for ammonia diffusion toward the synthase site. The Q-loop of glutamate synthase, which acts as an active site lid in other amidotransferases, seems to maintain an open conformation. Finally, binding of L-methionine sulfone, a glutamine analog that mimics the tetrahedral transient species occurring during its hydrolysis, causes a coordinated rigid-body motion of segments of the glutaminase domain that results in the inactive conformation observed in the crystal structure of GltS {alpha} subunit.

Keywords: glutamate synthase; molecular dynamics; amidotransferase; ammonia channeling; flavoprotein; iron-sulfur clusters

Abbreviations: GltS, glutamate synthase . NADPH, nicotinamide adenine dinucleotide phosphate . NADH, nicotinamide adenine dinucleotide . FAD, flavin adenine dinucleotide . FMN, flavin mononucleotide . NAD(P)H-GltS, reduced nicotinamide adenine dinucleotide (phosphate)-dependent glutamate synthase . Fd, ferredoxin . Fd-GltS, Fd-dependent glutamate synthase . {alpha}-GltS, {alpha} subunit of NADPH-dependent glutamate synthase . {alpha}-GltSfree, {alpha}-GltS in the substrate-free state with the FMN cofactor in the oxidized state . {alpha}-GltSbound, {alpha}-GltS in complex with its substrates L-glutamine and 2-oxoglutarate with the FMN cofactor in the reduced state . MetS, L-methionine sulphone . ONL, 5-oxo-L-norleucine . 2-OG, 2-oxoglutarate . 2-IG, 2-iminoglutarate . MD, molecular dynamics . ED, essential dynamics . RMSD, root-mean-square deviation . RMSF, root-mean-square fluctuation