Pyridoxal Phosphate Enzymes: Mechanistic, Structural, and Evolutionary Considerations

Eliot, Andrew C.; Kirsch, Jack F.

doi:10.1146/annurev.biochem.73.011303.074021

Cited by 802 publications

(833 citation statements)

References 104 publications

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“…We did not observe electron density for the N-and C-terminal extremities of LpSpl 56-605 , including the above-mentioned residues 56-106 and residues 533-605. The basic architecture of LpSpl was typical of type 1 PLP-dependent enzymes (28), and the presence of an N-terminal region clusters LpSpl with the other characterized SPL enzymes ( Fig. 1 B and C; SI Appendix, S2 A and B).…”

Section: Resultsmentioning

confidence: 91%

Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy

Rolando

Escoll

Nora

et al. 2016

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

116

130

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Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen's Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis.Legionella pneumophila | sphingosine-1-phosphate lyase | autophagy | sphingolipids | virulence T he Gram-negative intracellular bacterium Legionella pneumophila is an opportunistic human pathogen responsible for Legionnaires' disease. The bacteria are naturally found in freshwater systems where they replicate within protozoan hosts (1). It is thought that the adaptation to replication within amoebas has equipped L. pneumophila with the factors required to replicate successfully within human macrophages following opportunistic infection (2). Through genome sequencing, we have discovered that L. pneumophila encodes a high number and variety of proteins similar in sequence to eukaryotic proteins that are never or rarely found in other prokaryotic genomes (3). Subsequent phylogenetic analyses have suggested that many of these proteins were acquired by horizontal gene transfer (3, 4). One of these proteins exhibits a high degree of similarity to eukaryotic sphingosine-1 phosphate lyase (SPL). The L. pneumophila SPL homolog (LpSpl encoded by gene lpp2128, lpg2176, or legS2) is conserved in all L. pneumophila strains sequenced to date, but absent from Legionella longbeachae (SI Appendix, Table S1). Phylogenetic analysis of SPL sequences showed that the L. pneumophila spl gene was most likely acquired early during evolution by horizontal gene transfer from a protozoan organism (4, 5). With the increase in genome sequences available...

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Section: Resultsmentioning

confidence: 91%

Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy

Rolando

Escoll

Nora

et al. 2016

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

116

130

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“…The amino group originally transferred to AeKAT-PLP to form AeKAT-PMP is then transferred to keto acid 2, forming amino acid 2 and regenerating the AeKAT-PLP form. Finally, amino acid 2 is ejected, and the cycle is complete (23). The fact that cysteine is found in only one subunit of the biological dimer in the cocrystallized crystal structure is interesting.…”

Section: Discussionmentioning

confidence: 99%

Structural Insight into the Mechanism of Substrate Specificity of Aedes Kynurenine Aminotransferase^,

et al. 2008

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Aedes aegypti kynurenine aminotransferase (AeKAT) is a multifunctional aminotransferase. It catalyzes the transamination of a number of amino acids and uses many biologically relevant α-keto acids as amino group acceptors. AeKAT also is a cysteine S-conjugate β-lyase. The most important function of AeKAT is the biosynthesis of kynurenic acid, a natural antagonist of NMDA and α7-nicotinic acetylcholine receptors. Here, we report the crystal structures of AeKAT in complex with its best amino acid substrates, glutamine and cysteine. Glutamine is found in both subunits of the biological dimer, and cysteine is found in one of the two subunits. Both substrates form external aldemines with pyridoxal 5-phosphate in the structures. This is the first instance in which one pyridoxal 5-phosphate enzyme has been crystallized with cysteine or glutamine forming external aldimine complexes, cysteinyl aldimine and glutaminyl aldimine. All the units with substrate are in the closed conformation form, and the unit without substrate is in the open form, which suggests that the binding of substrate induces the conformation change of AeKAT. By comparing the active site residues of the AeKAT-cysteine structure with those of the human KAT I-phenylalanine structure, we determined that Tyr286 in AeKAT is changed to Phe278 in human KAT I, which may explain why AeKAT transaminates hydrophilic amino acids more efficiently than human KAT I does.The sequence of Aedes aegypti kynurenine aminotransferase (AeKAT) 1 is 47% identical with that of human KAT I and 51.9% identical with that of human KAT III (1). Human, rat, and mouse KAT I enzymes have been extensively studied (2,3). Mammalian KAT I, also called glutamine transaminase K (GTK), is a multifunctional aminotransferase that acts on a number of amino acids (glutamine, cysteine, phenylalanine, kynurenine, methionine, etc.) and uses many biologically relevant keto acids as amino group acceptors (3,4). AeKAT and a glutamine:phenylpyruvate aminotransferase from Thermus thermophilus, homologues of mammalian KAT I, have also been systematically characterized (5,6). One of the enzymatic products of KAT I is kynurenic acid (KYNA), which is the only known endogenous antagonist of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors (7-9). KYNA is also the antagonist of the α7-nicotinic acetylcholine receptor (10-12). Very † This work was supported by Grant AI 44399 from the National Institutes of Health. ‡ The atomic coordinates and structure factors (entries 2r5c and 2r5e) have been deposited in the Protein Data Bank. Many other functions have been proposed for mammalian KAT I enzymes, including maintaining low levels of phenylpyruvate, closing the methionine salvage pathway, regulating α-keto acid levels, sparing the essential amino acids, and maintaining a continual equilibrium among the amino acids (3,17). However, quantitatively, the most important amine donor for KAT I enzymes in vivo is glutamine. The product of glutamine transamination (α-ketoglutaramate) is rapidly remov...

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“…25 However, as is typical of TS structures, the observed hydrogen-bonding pattern in MtTS suggests that N1 is not protonated. Ser299 accepts a hydrogen bond from Gly327-N, and is therefore likely to donate a hydrogen bond to Thr326, which in turn donates a hydrogen bond to N1.…”

Section: Mechanismmentioning

confidence: 83%

Structural, Biochemical, and In Vivo Investigations of the Threonine Synthase from Mycobacterium tuberculosis

Covarrubias¹,

Högbom²,

Bergfors³

et al. 2008

Journal of Molecular Biology

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PostprintThis is the accepted version of a paper published in Journal of Molecular Biology. This paper has been peer-reviewed but does not include the final publisher proof-corrections or journal pagination. Citation for the original published paper (version of record):Covarrubias, A., Högbom, M., Bergfors, T., Carroll, P., Mannerstedt, K. et al. (2008) Structural, biochemical and in vivo investigations of the threonine synthase from Mycobacterium tuberculosis. Journal of Molecular * Manuscript 2 SummaryThreonine biosynthesis is a general feature of prokaryotes, eukaryotic microorganisms and higher plants. Since mammals lack the appropriate synthetic machinery, instead obtaining the amino acid through their diet, the pathway is a potential focus for the development of novel antibiotics, anti-fungal agents and herbicides. Threonine synthase, a pyridoxal 5-phosphate-dependent enzyme, catalyses the final step in the pathway, in which L-homoserine phosphate and water are converted into threonine and inorganic phosphate. In the present publication we report structural and functional studies of Mycobacterium tuberculosis threonine synthase, the product of the rv1295 (thrC) gene. The structure gives new insights into the catalytic mechanism of threonine synthases in general, specifically by suggesting the direct involvement of the phosphate moiety of the cofactor, rather than the inorganic phosphate product, in transferring a proton from C4' to C in the formation of the -unsaturated aldimine. It further provides a basis for understanding why this enzyme has a higher pH optimum than has been reported elsewhere for threonine synthases, and gives rise to the prediction that the equivalent enzyme from Thermus thermophilus will exhibit similar behavior. A deletion of the relevant gene generated a strain of M. tuberculosis that requires threonine for growth; such auxotrophic strains are frequently attenuated in vivo, indicating that threonine synthase is a potential drug target in this organism.

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Pyridoxal Phosphate Enzymes: Mechanistic, Structural, and Evolutionary Considerations

Cited by 802 publications

References 104 publications

Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy

Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy

Structural Insight into the Mechanism of Substrate Specificity of Aedes Kynurenine Aminotransferase^,

Structural, Biochemical, and In Vivo Investigations of the Threonine Synthase from Mycobacterium tuberculosis

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Pyridoxal Phosphate Enzymes: Mechanistic, Structural, and Evolutionary Considerations

Cited by 802 publications

References 104 publications

Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy

Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy

Structural Insight into the Mechanism of Substrate Specificity of Aedes Kynurenine Aminotransferase,

Structural, Biochemical, and In Vivo Investigations of the Threonine Synthase from Mycobacterium tuberculosis

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Structural Insight into the Mechanism of Substrate Specificity of Aedes Kynurenine Aminotransferase^,