Kegg Pathway: Lysine degradation

Int J Syst Evol Microbiol. 2008 Sep; 58(Pt 9): 2003-12
Ganesan A, Chaussonnerie S, Tarrade A, Dauga C, Bouchez T, Pelletier E, Le Paslier D, Sghir A

A novel anaerobic, mesophilic, amino-acid-utilizing bacterium, strain 158(T), was isolated from an anaerobic digester of a wastewater treatment plant. Cells of strain 158(T) were non-motile, rod-shaped (2.0-3.0 x 0.8-1.0 mum) and stained Gram-negative. Optimal growth occurred at 37 degrees C and pH 7.0 in an anaerobic basal medium containing 1 % Casamino acids. Strain 158(T) fermented arginine, histidine, Lysine and serine and showed growth on yeast extract, brain-heart infusion (BHI) medium and tryptone, but not on carbohydrates, organic acids or alcohols. The end products of degradation were: acetate, butyrate, H(2) and CO(2) from arginine; acetate, propionate, butyrate, H(2) and CO(2) from Lysine; and acetate, propionate, butyrate, valerate, H(2) and CO(2) from histidine, serine, BHI medium, Casamino acids and tryptone. The DNA G+C content was 55.8 mol%. The 16S rRNA gene sequence of strain 158(T) showed only 92.6 % sequence similarity with that of Synergistes jonesii, the only described species of the 'Synergistes' group. The major cellular fatty acids were iso-C(15 : 0) (16.63 %), iso-C(15 : 0) 3-OH (12.41 %) and C(17 : 1)omega6c (9.46 %) and the polar fatty acids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylamine; these fatty acid profiles did not resemble those of any recognized bacterial species. Due to the considerable differences in genotypic, phenotypic and phylogenetic characteristics between strain 158(T) and those of its nearest relative, it is proposed that strain 158(T) represents a novel species in a new genus, Cloacibacillus evryensis gen. nov., sp. nov., in the phylum 'Synergistetes'. The type strain is 158(T) (=DSM 19522(T)=JCM 14828(T)).

Proteins. 2008 Sep 2;
Zambelli B, Turano P, Musiani F, Neyroz P, Ciurli S

The biosynthesis of the active metal-bound form of the nickel-dependent enzyme urease involves the formation of a Lysine-carbamate functional group concomitantly with the delivery of two Ni(2+) ions into the precast active site of the apoenzyme and with GTP hydrolysis. In the urease system, this role is performed by UreG, an accessory protein belonging to the group of homologous P-loop GTPases, often required to complete the biosynthesis of nickel-enzymes. This study is focused on UreG from Helicobacter pylori (HpUreG), a bacterium responsible for gastric ulcers and cancer, infecting large part of the human population, and for which urease is a fundamental virulence factor. The soluble HpUreG was expressed in E. coli and purified to homogeneity. On-line size exclusion chromatography and light scattering indicated that apo-HpUreG exists as a monomer in solution. Circular dichroism, which demonstrated the presence of a well-defined secondary structure, and NMR spectroscopy, which revealed a large number of residues that appear structured on the basis of their backbone amide proton chemical shift dispersion, indicated that, at variance with other UreG proteins so far characterized, this protein is significantly folded in solution. The amino acid sequence of HpUreG is 29% identical to that of HypB from Methanocaldococcus jannaschii, a dimeric zinc-binding GTPase involved in the in vivo assembly of [Ni,Fe]-hydrogenase. A homology-based molecular model of HpUreG was calculated, which allowed us to identify structural and functional features of the protein. Isothermal titration microcalorimetry demonstrated that HpUreG specifically binds 0.5 equivalents of Zn(2+) per monomer (K(d) = 0.33 +/- 0.03 muM), whereas it has 20-fold lower affinity for Ni(2+) (K(d) = 10 +/- 1 muM). Zinc ion binding (but not Ni(2+) binding) causes protein dimerization, as confirmed using light scattering measurements. The structural rearrangement occurring upon Zn(2+)-binding and consequent dimerization was evaluated using circular dichroism and fluorescence spectroscopy. Fully conserved histidine and cysteine residues were identified and their role in zinc binding was verified by site-directed mutagenesis and microcalorimetry. The results are analyzed and discussed with respect to analogous examples of GTPases in nickel metabolism. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

Cell. 2008 Aug 22; 134(4): 668-78
Newton K, Matsumoto ML, Wertz IE, Kirkpatrick DS, Lill JR, Tan J, Dugger D, Gordon N, Sidhu SS, Fellouse FA, Komuves L, French DM, Ferrando RE, Lam C, Compaan D, Yu C, Bosanac I, Hymowitz SG, Kelley RF, Dixit VM

Posttranslational modification of proteins with polyubiquitin occurs in diverse signaling pathways and is tightly regulated to ensure cellular homeostasis. Studies employing ubiquitin mutants suggest that the fate of polyubiquitinated proteins is determined by which Lysine within ubiquitin is linked to the C terminus of an adjacent ubiquitin. We have developed linkage-specific antibodies that recognize polyubiquitin chains joined through Lysine 63 (K63) or 48 (K48). A cocrystal structure of an anti-K63 linkage Fab bound to K63-linked diubiquitin provides insight into the molecular basis for specificity. We use these antibodies to demonstrate that RIP1, which is essential for tumor necrosis factor-induced NF-kappaB activation, and IRAK1, which participates in signaling by interleukin-1beta and Toll-like receptors, both undergo polyubiquitin editing in stimulated cells. Both kinase adaptors initially acquire K63-linked polyubiquitin, while at later times K48-linked polyubiquitin targets them for proteasomal degradation. Polyubiquitin editing may therefore be a general mechanism for attenuating innate immune signaling.

Mol Cell Proteomics. 2008 Aug 23;
Zhang J, Sprung R, Pei J, Tan X, Kim S, Zhu H, Liu CF, Grishin NV, Zhao Y

Lysine acetylation and its regulatory enzymes are known to have pivotal roles in mammalian cellular physiology. However, the extent and function of this modification in prokaryotic cells remain largely unexplored, thereby presenting a hurdle to further functional study of this modification in prokaryotic systems. Here we report the first global screening of Lysine acetylation, identifying 138 modification sites in 91 proteins from E. coli. None of the proteins has been previously associated with this modification. Among the identified proteins are transcriptional regulators, as well as others with diverse functions. Interestingly, more than 70% of the acetylated proteins are metabolic enzymes and translation regulators, suggesting an intimate link of this modification to energy metabolism. The new dataset suggests that Lysine acetylation could be abundant in prokaryotic cells. In addition, these results also imply that functions of Lysine acetylation beyond regulation of gene expression are evolutionarily conserved from bacteria to mammals. Furthermore, we demonstrate that bacterial Lysine acetylation is regulated in response to stress stimuli.

Science. 2008 Aug 22; 321(5892): 1086-8
Ferrari R, Pellegrini M, Horwitz GA, Xie W, Berk AJ, Kurdistani SK

Adenovirus e1a induces quiescent human cells to replicate. We found that e1a causes global relocalization of the RB (retinoblastoma) proteins (RB, p130, and p107) and p300/CBP histone acetyltransferases on promoters, the effect of which is to restrict the acetylation of histone 3 Lysine-18 (H3K18ac) to a limited set of genes, thereby stimulating cell cycling and inhibiting antiviral responses and cellular differentiation. Soon after expression, e1a binds transiently to promoters of cell cycle and growth genes, causing enrichment of p300/CBP, PCAF (p300/CBP-associated factor), and H3K18ac; depletion of RB proteins; and transcriptional activation. e1a also associates transiently with promoters of antiviral genes, causing enrichment for RB, p130, and H4K16ac; increased nucleosome density; and transcriptional repression. At later times, e1a and p107 bind mainly to promoters of development and differentiation genes, repressing transcription. The temporal order of e1a binding requires its interactions with p300/CBP and RB proteins. Our data uncover a defined epigenetic reprogramming leading to cellular transformation.

Science. 2008 Aug 22; 321(5892): 1084-5
Horwitz GA, Zhang K, McBrian MA, Grunstein M, Kurdistani SK, Berk AJ

Adenovirus small early region 1a (e1a) protein drives cells into S phase by binding RB family proteins and the closely related histone acetyl transferases p300 and CBP. The interaction with RB proteins displaces them from DNA-bound E2F transcription factors, reversing their repression of cell cycle genes. However, it has been unclear how the e1a interaction with p300 and CBP promotes passage through the cell cycle. We show that this interaction causes a threefold reduction in total cellular histone H3 Lysine 18 acetylation (H3K18ac). CBP and p300 are required for acetylation at this site because their knockdown causes specific hypoacetylation at H3K18. SV40 T antigen also induces H3K18 hypoacetylation. Because global hypoacetylation at this site is observed in prostate carcinomas with poor prognosis, this suggests that processes resulting in global H3K18 hypoacetylation may be linked to oncogenic transformation.

Biochem Pharmacol. 2008 Jul 31;
Scialpi F, Malatesta M, Peschiaroli A, Rossi M, Melino G, Bernassola F

Itch, an E3 protein ubiquitin ligase (E3), which belongs to the homologous to E6-AP carboxy terminus (HECT)-type subfamily, catalyzes its own ubiquitylation. The precise nature of Itch-mediated self-modification and its biological outcome are not completely understood. Here, we show that Itch auto-ubiquitylation is an intermolecular reaction generating Lys63-linkages, rather than the Lys48-linked polyubiquitin chains that target proteins for proteasomal degradation. As a result, Itch is a relatively high stable protein, whose levels are not significantly affected by treatment by either proteasome or lysosome inhibitors. Furthermore, we demonstrate that the decay rate of a catalytic inactive Itch mutant, which is devoided of self-ubiquitylating activity, is barely indistinguishable from the one of the wild-type protein. These data definitely establish a nondegradative role for Lys63-linked Itch self-ubiquitylation.

Nature. 2008 Aug 20;
Song MS, Salmena L, Carracedo A, Egia A, Lo-Coco F, Teruya-Feldstein J, Pandolfi PP

Nuclear exclusion of the PTEN (phosphatase and tensin homologue deleted in chromosome 10) tumour suppressor has been associated with cancer progression. However, the mechanisms leading to this aberrant PTEN localization in human cancers are currently unknown. We have previously reported that ubiquitinylation of PTEN at specific Lysine residues regulates its nuclear-cytoplasmic partitioning. Here we show that functional promyelocytic leukaemia protein (PML) nuclear bodies co-ordinate PTEN localization by opposing the action of a previously unknown PTEN-deubiquitinylating enzyme, herpesvirus-associated ubiquitin-specific protease (HAUSP, also known as USP7), and that the integrity of this molecular framework is required for PTEN to be able to enter the nucleus. We find that PTEN is aberrantly localized in acute promyelocytic leukaemia, in which PML function is disrupted by the PML-RARalpha fusion oncoprotein. Remarkably, treatment with drugs that trigger PML-RARalpha degradation, such as all-trans retinoic acid or arsenic trioxide, restore nuclear PTEN. We demonstrate that PML opposes the activity of HAUSP towards PTEN through a mechanism involving the adaptor protein DAXX (death domain-associated protein). In support of this paradigm, we show that HAUSP is overexpressed in human prostate cancer and is associated with PTEN nuclear exclusion. Thus, our results delineate a previously unknown PML-DAXX-HAUSP molecular network controlling PTEN deubiquitinylation and trafficking, which is perturbed by oncogenic cues in human cancer, in turn defining a new deubiquitinylation-dependent model for PTEN subcellular compartmentalization.

Mol Biol Cell. 2008 Aug 20;
Ikeda H, Kerppola TK

Monitoring Editor: William P. Tansey Ubiquitination regulates many cellular functions including protein localization and degradation. Each function is specified by unique determinants in the conjugate. Ubiquitinated Jun is localized to lysosomes for degradation. Here, we characterized determinants of Jun ubiquitination and lysosomal localization using ubiquitin-mediated fluorescence complementation (UbFC) in living cells and analysis of the stoichiometry of ubiquitin linked to Jun extracted from cells. The delta region of Jun and isoleucine-44 in ubiquitin were required for lysosomal localization of the conjugate. Ubiquitin containing only Lysine-27, but no other single-Lysine ubiquitin, mediated Jun ubiquitination, albeit at lower stoichiometry than wild-type ubiquitin. These conjugates were predominantly nuclear, but coexpression of Lysine-27 and Lysine-less ubiquitins enhanced the mean stoichiometry of Jun ubiquitination and lysosomal localization of the conjugate. HRS and TSG101 colocalized with ubiquitinated Jun. Knockdown of HRS or TSG101 inhibited lysosomal localization of ubiquitinated Jun and reduced Jun turnover. Ubiquitination of other Fos and Jun family proteins had distinct effects on their localization. Our results indicate that Jun is poly-ubiquitinated by E3 ligases that produce Lysine-27 linked chains. Lysosomal localization of the conjugate requires determinants in Jun and in ubiquitin that are recognized in part by TSG101 and HRS, facilitating selective translocation and degradation of ubiquitinated Jun.

J Biol Chem. 2008 Aug 18;
Lineberry N, Su L, Soares L, Fathman CG

The ubiquitin E3 ligase GRAIL (Rnf128) is a type 1 transmembrane protein that induces T cell anergy through the ubiquitination activity of its cytosolic RING finger. GRAIL also contains an equally large luminal region consisting primarily of an uncharacterized protease-associated (PA) domain. Using two-hybrid technology to screen for proteins that bound the PA domain identified CD151, a member of the tetraspanin family of membrane proteins. GRAIL bound to the luminal/extracellular portion of both CD151 and the related tetraspanin CD81 using its PA domain, which promoted ubiquitination of cytosolic Lysine residues. GRAIL exhibited specificity for Lysines only within the tetraspanin amino terminus, even in the presence of other cytosolic Lysine residues in the substrate. GRAIL mediated ubiquitination promoted proteasomal degradation and cell surface downregulation of tetraspanins via K48 linkages. As a result, the juxtaposition of PA and RING finger domains across a lipid bilayer facilitates the capture of transmembrane substrates for subsequent ubiquitination. These findings identify for the first time a single subunit E3 ligase containing a substrate-binding domain spatially restricted by a membrane from its E2 recruitment domain, as well as an E3 ligase for members of the tetraspanin family.

FASEB J. 2008 Aug 15;
Jakubowski H, Boers GH, Strauss KA

Severely elevated plasma homocysteine (Hcy) levels observed in genetic disorders of Hcy metabolism are associated with pathologies in multiple organs and lead to premature death due to vascular complications. In addition to elevating plasma Hcy, mutations in cystathionine beta-synthase (CBS) or methylenetetrahydrofolate reductase (MTHFR) gene lead to markedly elevated levels of circulating Hcy-thiolactone. The thiooester chemistry of Hcy-thiolactone underlies its ability to form isopeptide bonds with protein Lysine residues (N-Hcy-protein), which may impair or alter the protein's function. However, it was not known whether genetic deficiencies in Hcy metabolism affect N-Hcy-protein levels in humans. Here we show that plasma N-Hcy-protein levels are significantly elevated in CBS- and MTHFR-deficient patients. We also show that CBS-deficient patients have significantly elevated plasma levels of prothrombotic N-Hcy-fibrinogen. These results provide a possible explanation for increased atherothrombosis observed in CBS-deficient patients.-Jakubowski, H., Boers, G. H. J., Strauss, K. A. Mutations in cystathionine beta-synthase or methylenetetrahydrofolate reductase gene increase N-homocysteinylated protein levels in humans.

Metabolism. 2008 Sep; 57(9): 1211-20
Dhar A, Desai K, Kazachmov M, Yu P, Wu L

Methylglyoxal (MG), a metabolic by-product, reacts with certain proteins to yield irreversible advanced glycation end products (AGEs) and increases oxidative stress that causes the pathophysiological changes in diabetes, hypertension, and aging. Although MG production from glucose has been well documented, the contribution of other intermediates of different metabolic pathways to MG formation is far less known. Our aim was to determine and compare the formation of MG, MG-induced AGE, N(epsilon)-carboxyethyl-Lysine (CEL), inducible nitric oxide synthase (iNOS), nitric oxide, and peroxynitrite from different metabolic precursors in cultured rat aortic vascular smooth muscle cells (VSMCs). High-performance liquid chromatography was used to determine MG levels, whereas nitrite + nitrate, indicators of nitric oxide production, and peroxynitrite levels were measured with specific assay kits. The CEL and iNOS were detected using immunocytochemistry. There was a concentration-dependent increase in MG levels in VSMCs after 3-hour incubation with 5, 15, and 25 mmol/L of D-glucose, fructose, or aminoacetone. Aminoacetone produced a 7-fold increase in MG levels above the basal value followed by fructose (3.9-fold), D-glucose (3.5-fold), acetol (2.8-fold), and sucrose (2.3-fold) after a 3-hour incubation with 25 mmol/L of each precursor. L-Glucose, 3-O-methylglucose, and mannitol had no effect on MG production. All precursors, except l-glucose, 3-O-methylglucose and mannitol, increased CEL. Aminoacetone, D-glucose, and fructose significantly increased iNOS, nitrite/nitrate, and peroxynitrite levels. In conclusion, aminoacetone is the most potent precursor of MG production in VSMCs, followed by fructose and d-glucose. This could have important implications in relation to high dietary fructose and protein intake.

J Cell Biol. 2008 Aug 11; 182(3): 481-95
Cakouros D, Mills K, Denton D, Paterson A, Daish T, Kumar S

The sequential modifications of histones form the basis of the histone code that translates into either gene activation or repression. Nuclear receptors recruit a cohort of histone-modifying enzymes in response to ligand binding and regulate proliferation, differentiation, and cell death. In Drosophila melanogaster, the steroid hormone ecdysone binds its heterodimeric receptor ecdysone receptor/ultraspiracle to spatiotemporally regulate the transcription of several genes. In this study, we identify a novel cofactor, Drosophila Lysine ketoglutarate reductase (dLKR)/saccharopine dehydrogenase (SDH), that is involved in ecdysone-mediated transcription. dLKR/SDH binds histones H3 and H4 and suppresses ecdysone-mediated transcription of cell death genes by inhibiting histone H3R17me2 mediated by the Drosophila arginine methyl transferase CARMER. Our data suggest that the dynamic recruitment of dLKR/SDH to ecdysone-regulated gene promoters controls the timing of hormone-induced gene expression. In the absence of dLKR/SDH, histone methylation occurs prematurely, resulting in enhanced gene activation. Consistent with these observations, the loss of dLKR/SDH in Drosophila enhances hormone-regulated gene expression, affecting the developmental timing of gene activation.

Mol Cell. 2008 Aug 8; 31(3): 347-59
Ding N, Zhou H, Esteve PO, Chin HG, Kim S, Xu X, Joseph SM, Friez MJ, Schwartz CE, Pradhan S, Boyer TG

Mediator occupies a central role in RNA polymerase II transcription as a sensor, integrator, and processor of regulatory signals that converge on protein-coding gene promoters. Compared to its role in gene activation, little is known regarding the molecular mechanisms and biological implications of Mediator as a transducer of repressive signals. Here we describe a protein interaction network required for extraneuronal gene silencing comprising Mediator, G9a histone methyltransferase, and the RE1 silencing transcription factor (REST; also known as neuron restrictive silencer factor, NRSF). We show that the MED12 interface in Mediator links REST with G9a-dependent histone H3K9 dimethylation to suppress neuronal genes in nonneuronal cells. Notably, missense mutations in MED12 causing the X-linked mental retardation (XLMR) disorders FG syndrome and Lujan syndrome disrupt its REST corepressor function. These findings implicate Mediator in epigenetic restriction of neuronal gene expression to the nervous system and suggest a pathologic basis for MED12-associated XLMR involving impaired REST-dependent neuronal gene regulation.

Mol Cell. 2008 Aug 8; 31(3): 305-6
Malik S, Roeder RG

Adding to the concept that Mediator acts as an "integrative hub,"Ding et al. (2008) report in this issue of Molecular Cell that, by facilitating the recruitment of the G9a methyl transferase, Mediator can also promote epigenetic silencing of selected genes.

Biomacromolecules. 2008 Aug 9;
Berski S, van Bergeijk J, Schwarzer D, Stark Y, Kasper C, Scheper T, Grothe C, Gerardy-Schahn R, Kirschning A, Dräger G

Restorative medicine has a constant need for improved scaffold materials. Degradable biopolymers often suffer from uncontrolled chemical or enzymatic hydrolysis by the host. The need for a second surgery on the other hand is a major drawback for nondegradable scaffold materials. In this paper we report the design and synthesis of a novel polysialic acid-based hydrogel with promising properties. Hydrogel synthesis was optimized and enzymatic degradation was studied using a phage-born endosialidase. After addition of endosialidase, hydrogels readily degraded depending on the amount of initially used cross-linker within 2 to 11 days. This polysialic acid hydrogel is not cytotoxic, completely stable under physiological conditions, and could be evaluated as growth support for PC12 cells. Here, additional coating with collagen I, poly- l-Lysine or matrigel is mandatory to improve the properties of the material.

Genetics. 2008 Aug; 179(4): 1933-44
Raisner RM, Madhani HD

Sirtuins are conserved proteins implicated in myriad key processes including gene control, aging, cell survival, metabolism, and DNA repair. In Saccharomyces cerevisiae, the sirtuin Silent information regulator 2 (Sir2) promotes silent chromatin formation, suppresses recombination between repeats, and inhibits senescence. We performed a genomewide screen for factors that negatively regulate Sir activity at a reporter gene placed immediately outside a silenced region. After linkage analysis, assessment of Sir dependency, and knockout tag verification, 40 loci were identified, including 20 that have not been previously described to regulate Sir. In addition to chromatin-associated factors known to prevent ectopic silencing (Bdf1, SAS-I complex, Rpd3L complex, Ku), we identified the Rtt109 DNA repair-associated histone H3 Lysine 56 acetyltransferase as an anti-silencing factor. Our findings indicate that Rtt109 functions independently of its proposed effectors, the Rtt101 cullin, Mms1, and Mms22, and demonstrate unexpected interplay between H3K56 and H4K16 acetylation. The screen also identified subunits of mediator (Soh1, Srb2, and Srb5) and mRNA metabolism factors (Kem1, Ssd1), thus raising the possibility that weak silencing affects some aspect of mRNA structure. Finally, several factors connected to metabolism were identified. These include the PAS-domain metabolic sensor kinase Psk2, the mitochondrial homocysteine detoxification enzyme Lap3, and the Fe-S cluster protein maturase Isa2. We speculate that PAS kinase may integrate metabolic signals to control sirtuin activity.

PLoS Genet. 2008 Aug; 4(8): e1000150
Rosso L, Marques AC, Reichert AS, Kaessmann H

Many new gene copies emerged by gene duplication in hominoids, but little is known with respect to their functional evolution. Glutamate dehydrogenase (GLUD) is an enzyme central to the glutamate and energy metabolism of the cell. In addition to the single, GLUD-encoding gene present in all mammals (GLUD1), humans and apes acquired a second GLUD gene (GLUD2) through retroduplication of GLUD1, which codes for an enzyme with unique, potentially brain-adapted properties. Here we show that whereas the GLUD1 parental protein localizes to mitochondria and the cytoplasm, GLUD2 is specifically targeted to mitochondria. Using evolutionary analysis and resurrected ancestral protein variants, we demonstrate that the enhanced mitochondrial targeting specificity of GLUD2 is due to a single positively selected glutamic acid-to-Lysine substitution, which was fixed in the N-terminal mitochondrial targeting sequence (MTS) of GLUD2 soon after the duplication event in the hominoid ancestor approximately 18-25 million years ago. This MTS substitution arose in parallel with two crucial adaptive amino acid changes in the enzyme and likely contributed to the functional adaptation of GLUD2 to the glutamate metabolism of the hominoid brain and other tissues. We suggest that rapid, selectively driven subcellular adaptation, as exemplified by GLUD2, represents a common route underlying the emergence of new gene functions.

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Aug 1; 64(Pt 8): 700-6
Alexopoulos E, Kanjee U, Snider J, Houry WA, Pai EF

The decameric inducible Lysine decarboxylase (LdcI) from Escherichia coli has been crystallized in space groups C2 and C222(1); the Ta6Br12(2+) cluster was used to derivatize the C2 crystals. The method of single isomorphous replacement with anomalous scattering (SIRAS) as implemented in SHELXD was used to solve the Ta6Br12(2+)-derivatized structure to 5 A resolution. Many of the Ta6Br12(2+)-binding sites had twofold and fivefold noncrystallographic symmetry. Taking advantage of this feature, phase modification was performed in DM. The electron-density map of LdcI displays many features in agreement with the low-resolution negative-stain electron-density map [Snider et al. (2006), J. Biol. Chem. 281, 1532-1546].

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Aug 1; 64(Pt 8): 686-91
Elling RA, Fucini RV, Hanan EJ, Barr KJ, Zhu J, Paulvannan K, Yang W, Romanowski MJ

Polo-like kinase 1 (Plk1) is a member of the Polo-like kinase family of serine/threonine kinases involved in the regulation of cell-cycle progression and cytokinesis and is an attractive target for the development of anticancer therapeutics. The catalytic domain of this enzyme shares significant primary amino-acid homology and structural similarity with another mitotic kinase, Aurora A. While screening an Aurora A library of ATP-competitive compounds, a urea-containing inhibitor with low affinity for mouse Aurora A but with submicromolar potency for human and zebrafish Plk1 (hPlk1 and zPlk1, respectively) was identified. A crystal structure of the zebrafish Plk1 kinase domain-inhibitor complex reveals that the small molecule occupies the purine pocket and extends past the catalytic Lysine into the adaptive region of the active site. Analysis of the structures of this protein-inhibitor complex and of similar small molecules cocrystallized with other kinases facilitates understanding of the specificity of the inhibitor for Plk1 and documents for the first time that Plk1 can accommodate extended ATP-competitive compounds that project toward the adaptive pocket and help the enzyme order its activation segment.