Crystal structure of glutamine amidotransferase from <i>Thermotoga maritima</i>

Korolev, Sergey; Skarina, T.; Evdokimova, E.; Beasley, Steven; Edwards, A.M.; Joachimiak, Andrzej; Savchenko, Alexei

doi:10.1002/prot.10161

Cited by 15 publications

(16 citation statements)

References 13 publications

(9 reference statements)

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“…Thus, oxyanion hole formation is most likely a key regulatory step, analogous to that described for Ser and Cys proteases (24,25). The conformation seen in the autonomous Pdx2 structure would then represent an enzymatic ''ground state,'' similar to that observed for Plasmodium falciparum Pdx2 (14), as has also been observed in Thermotoga maritima ImGP (26), where the carbonyl oxygen points toward the substratebinding site.…”

Section: Structural Changes In the Pdx1 Synthase Subunit Upon Complexmentioning

confidence: 63%

Structure of a bacterial pyridoxal 5′-phosphate synthase complex

Strohmeier

Raschle

Mazurkiewicz

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

113

272

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Vitamin B6 is an essential metabolic cofactor that has more functions in humans than any other single nutrient. Its de novo biosynthesis occurs through two mutually exclusive pathways that are absent in animals. The predominant pathway found in most prokaryotes, fungi, and plants has only recently been discovered. It is distinguished by a glutamine amidotransferase, which is remarkable in that it alone can synthesize the cofactor form, pyridoxal 5-phosphate (PLP), directly from a triose and a pentose saccharide and glutamine. Here we report the 3D structure of the PLP synthase complex with substrate glutamine bound as well as those of the individual synthase and glutaminase subunits Pdx1 and Pdx2, respectively. The complex is made up of 24 protein units assembled like a cogwheel, a dodecameric Pdx1 to which 12 Pdx2 subunits attach. In contrast to the architecture of previously determined glutamine amidotransferases, macromolecular assembly is directed by an N-terminal ␣-helix on the synthase. Interaction with the synthase subunit leads to glutaminase activation, resulting in formation of an oxyanion hole, a prerequisite for catalysis. Mutagenesis permitted identification of the remote glutaminase and synthase catalytic centers and led us to propose a mechanism whereby ammonia shuttles between these active sites through a methionine-rich hydrophobic tunnel.3D structure ͉ ammonia tunnel ͉ glutamine amidotransferase ͉ oxyanion ͉ vitamin B6

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Section: Structural Changes In the Pdx1 Synthase Subunit Upon Complexmentioning

confidence: 63%

Structure of a bacterial pyridoxal 5′-phosphate synthase complex

Strohmeier

Raschle

Mazurkiewicz

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

113

272

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“…2) Oxyanion hole formation is a general principle among enzymes with catalytic triads stabilizing the transient negative charge during the hydrolytic reaction. Within the triad GATase family, the oxyanion hole has been conclusively described for ImGPS (43,52). In general, the oxyanion hole is formed by two amide nitrogens, one from the residues following the catalytic nucleophile and the second from an adjacent ␤-strand, referred to as the "oxyanion strand."…”

Section: Discussionmentioning

confidence: 99%

Vitamin B6 Biosynthesis by the Malaria Parasite Plasmodium falciparum

Gengenbacher

Fitzpatrick

Raschle

et al. 2006

Journal of Biological Chemistry

116

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Vitamin B6 is one of nature's most versatile cofactors. Most organisms synthesize vitamin B6 via a recently discovered pathway employing the proteins Pdx1 and Pdx2. Here we present an in-depth characterization of the respective orthologs from the malaria parasite, Plasmodium falciparum. Expression profiling of Pdx1 and -2 shows that blood-stage parasites indeed possess a functional vitamin B6 de novo biosynthesis. Recombinant Pdx1 and Pdx2 form a complex that functions as a glutamine amidotransferase with Pdx2 as the glutaminase and Pdx1 as pyridoxal-5-phosphate synthase domain. Complex formation is required for catalytic activity of either domain. Pdx1 forms a chimeric bi-enzyme with the bacterial YaaE, a Pdx2 ortholog, both in vivo and in vitro, although this chimera does not attain full catalytic activity, emphasizing that species-specific structural features govern the interaction between the protein partners of the PLP synthase complexes in different organisms. To gain insight into the activation mechanism of the parasite bi-enzyme complex, the three-dimensional structure of Pdx2 was determined at 1.62 Å . The obstruction of the oxyanion hole indicates that Pdx2 is in a resting state and that activation occurs upon Pdx1-Pdx2 complex formation.Plasmodium falciparum is the causative agent of severe malaria. Each year up to two million human deaths and enormous economic losses are attributed to this parasite. Drug resistance in P. falciparum has been aggravating the problem in many parts of the world during the last two decades, which considering the lack of a protective vaccine, is the major obstacle to combat the disease. Hence, new antimalarials are urgently needed. Requirements for nutrients and vitamins have previously been discussed as possible novel targets (1). Indeed the P. falciparum genome contains genes that encode enzymes necessary for the syntheses of the vitamin precursor chorismate (2-4), vitamin B6 (5, 6), and the vitaminlike cofactor lipoic acid (7).Vitamin B6 is renowned in the medical field as being involved in more bodily functions than any other single nutrient. It is required for the maintenance of physical as well as mental health. The term "vitamin B6" collectively refers to the vitamers pyridoxal, pyridoxine, and pyridoxamine, and their respective phosphate esters. The metabolically active form is pyridoxal 5Ј-phosphate (PLP), 6 an essential co-enzyme in numerous pathways such as amino acid metabolism and the biosynthesis of antibiotic compounds. In contrast to mammals, which have to take up vitamin B6 from their diet, bacteria, fungi, plants, and the protozoan P. falciparum have the ability to synthesize the vitamin de novo.Analyses of a number of available genomes has demonstrated that most organisms, including all archaea, fungi, plants, and protozoa and most eubacteria use a class I glutamine amidotransferase (GATase) composed of two domains, a glutaminase and its associated acceptor/ synthase domain to generate vitamin B6 (8 -13). Structural knowledge on class I GATases in genera...

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“…206 Also, the crystal structure of apo HisH from T. maritima does not show an elevated B -factor, consistent with the observation that in the absence of bound PRFAR, the Gln site in HisH has negligible catalytic activity. 225 Thus, if HisH remains inactive without PRFAR-induced millisecond motions, it does not follow a “pre-existing equilibrium”-type allosteric model. However, the possibility remains that IGPS catalysis involves conformational changes that have yet to be detected by modern solution NMR methods and requires further investigation.…”

Section: Imidazole Glycerol Phosphate Synthasementioning

confidence: 99%

Solution NMR Spectroscopy for the Study of Enzyme Allostery

2016

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Allostery is a ubiquitous biological regulatory process in which distant binding sites within a protein or enzyme are functionally and thermodynamically coupled. Allosteric interactions play essential roles in many enzymological mechanisms, often facilitating formation of enzyme-substrate complexes and/or product release. Thus, elucidating the forces that drive allostery is critical to understanding the complex transformations of biomolecules. Currently, a number of models exist to describe allosteric behavior, taking into account energetics as well as conformational rearrangements and fluctuations. In the following review, we discuss the use of solution NMR techniques designed to probe allosteric mechanisms in enzymes. NMR spectroscopy is unequaled in its ability to detect structural and dynamical changes in biomolecules, and the case studies presented herein demonstrate the range of insights to be gained from this valuable method. We also provide a detailed technical discussion of several specialized NMR experiments that are ideally suited for the study of enzymatic allostery.

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Crystal structure of glutamine amidotransferase from Thermotoga maritima

Cited by 15 publications

References 13 publications

Structure of a bacterial pyridoxal 5′-phosphate synthase complex

Structure of a bacterial pyridoxal 5′-phosphate synthase complex

Vitamin B6 Biosynthesis by the Malaria Parasite Plasmodium falciparum

Solution NMR Spectroscopy for the Study of Enzyme Allostery

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