Involvement of the C terminus in intramolecular nitrogen channeling in glucosamine 6-phosphate synthase: evidence from a 1.6 å crystal structure of the isomerase domain

AbstractBackground: Glucosamine 6-phosphate synthase (GlmS) catalyses the first step in hexosamine metabolism, converting fructose-6P (6 phosphate) into glucosamine-6P using glutamine as a nitrogen source. GlmS is a bienzyme complex consisting of two domains that catalyse glutamine hydrolysis and sugar-phosphate isomerisation, respectively. Knowledge of the three-dimensional structure of GlmS is essential for understanding the general principles of catalysis by ketol isomerases and the mechanism of nitrogen transfer in glutamine amidotransferases.Results: The crystal structure of the isomerase domain of the Escherichia coli GlmS with the reaction product, glucosamine-6P, has been determined at 1.57 å resolution. It is comprised of two topologically identical subdomains, each of which is dominated by a nucleotide-binding motif of a flavodoxin type. The catalytic site is assembled by dimerisation of the protein.Conclusions: The isomerase active site of GlmS seems to be the result of evolution through gene duplication and subsequent dimerisation. Isomerisation of fructose-6P is likely to involve the formation of a Schiff base with Lys603 of the enzyme, the ring-opening step catalysed by His504, and the proton transfer from C1 to C2 of the substrate effected by Glu488. The highly conserved C-terminal fragment of the chain may play a key role in substrate binding, catalysis and communication with the glutaminase domain. The corresponding sequence pattern DXPXXLAK[SC]VT (in single-letter amino-acid code, where X is any amino acid and letters in brackets indicate that either serine or cysteine may take this position) may be considered as a fingerprint of GlmS

Preliminary Crystallographic Study of Cyclohexadienyl Dehydratase from Pseudomonas aeruginosa

Single crystals of cyclohexadienyl dehydratase from Pseudomonas aeruginosa have been obtained by vapour diffusion from ammonium sulphate solution (Ph 6·0) at 4°C. The crystals belong to the tetragonal space group $P4_32_12$ or $P4_12_12$ with a = b = 105·5 Å and c = 165·0 Å. The asymmetric unit contains at least one dimeric protein molecule with M$_f$ = 72 kDa. The crystals diffract to 3 Å resolution and are suitable for an X-ray analysis.Keyword

Crystallization and Preliminary X-ray Analysis of the Two Domains of Glucosamine-6-phosphate Synthase from Escherichia coli

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Refined Crystal Structure of the Catalytic Domain of Dihydrolipoyl Transacetylase (E2p) from Azotobacter vinelandii at 2·6 Å Resolution

Dihydrolipoyl transacetylase (E2p) is both structurally and functionally the central enzyme of the pyruvate dehydrogenase multienzyme complex. The crystal structure of the catalytic domain, i.e. residues 382 to 637, of Azotobacter vinelandii E2p (E2pCD) was solved by multiple isomorphous replacement and refined by energy minimization procedures. The final model contains 2182 protein atoms and 37 ordered water molecules. The R-factor is 18·7% for 10,344 reflections between 10·0 and 2·6 Å resolution. The root-mean-square shift deviation from the ideal values is 0·017 Å for bond lengths and 3·3° for bond angles. The N-terminal residues 382 to 394 are disordered and not visible in the electron density map, otherwise all residues have well-defined density. The catalytic domain forms an oligomer of 24 subunits, having octahedral 432 symmetry. In the E2pCD crystals, the 24 subunits are related by the crystallographic symmetry. The cubic arrangement of subunits gives rise to a large hollow cube with edges of 120 Å. The faces of the cube have pores of diameter of 30 Å. The true building block of the cube is the E2p trimer, eight of which occupy the corners of the cube. Two levels of intermolecular contacts can be distinguished: (1) the extensive interactions between 3-fold related subunits leading to a tightly associated trimer; and (2) the interactions along the 2-fold axis leading to the assembly of the trimers into the cubic 24-mer. Each subunit has a topology similar to chloramphenicol acetyltransferase (CAT) and comprises a central β-sheet surrounded by five α-helices. The comparison of the two proteins indicates a large rotation of the N-terminal residues 395 to 426 of E2pCD, which reshapes the substrate binding site and extends the interaction between threefold related subunits. The catalytic centre consists of a 30 Å long channel extending from the "inner" side of the trimmer to the "outer" side, where inner and outer refer to the position in the 24-meric cubic core of the pyruvate dehydrogenase complex and correspond with CoA and lipoamide binding sites, respectively. The active site is formed by the residues with the lowest mobility as indicated by the atomic B -factors. Five proline residues surround the active site. The side-chain of His610, which, by analogy with CAT, is most likely involved in catalysis, is stabilized in its unusual conformation by the salt-bridge between Asp609 and Arg611, and by contacts with the side-chains of Val435, Tyr608, Leu425′ and Ile571′, the latter two residues being located on a threefold related subunit. At the N terminus of the protein, residues 395 to 402 form an extended arm. Since they are part of the linker connecting the catalytic to the E1/E3 binding domain, their conformation is suggestive that the linker might consist of segments of rather inflexible extended polypeptide chain connected to each other by more flexible "hinge" residues

Substrate binding is required for assembly of the active conformation of the catalytic site in Ntn amidotransferases: evidence from the 1.8 Å crystal structure of the glutaminase domain of glucosamine 6-phosphate synthase

Amidotransferases use the amide nitrogen of glutamine in a number of important biosynthetic reactions. They are composed of a glutaminase domain, which catalyzes the hydrolysis of glutamine to glutamate and ammonia, and a synthetase domain, catalyzing amination of the substrate. To gain insight into the mechanism of nitrogen transfer, we examined the structure of the glutaminase domain of glucosamine 6-phosphate synthase (GLMS)