What a difference a methylene makes: replacing Glu with Asp or Aad in the Lys-urea-Glu pharmacophore of PSMA-targeting radioligands to reduce kidney and salivary gland uptake.

The aim of this study was to investigate the effect of replacing Glu in the Lys-urea-Glu PSMA-targeting pharmacophore of [68Ga]Ga-HTK03041 with a close analog on the uptake of kidneys, salivary glands and PSMA-expressing tumor xenografts. Methods: HTK03161, HTK03149 and HTK03189A/B were obtained by replacing Glu in HTK03041 with Asp, Aad (L-2-aminoadipic acid) and Api (2-aminopimelic acid), respectively. PSMA binding affinities were measured by competition binding assays. PET imaging and biodistribution studies of 68Ga-labeled ligands were performed in LNCaP tumor-bearing mice. The best candidate HTK03149 was selected and radiolabeled with 177Lu, and SPECT imaging and biodistribution studies were performed in LNCaP tumor-bearing mice. Radiation dosimetry calculation was conducted using the OLINDA software. Radioligand therapy study was performed in LNCaP tumor-bearing mice treated with [177Lu]Lu-HTK03149 (9.3-148 MBq), [177Lu]Lu-PSMA-617 (37 MBq) or natLu-HTK03149 (500 pmol). Results: PSMA binding affinities (Ki) of Ga-HTK03161, Ga-HTK03149, Ga-HTK03189A and Lu-HTK03149 were 3.88±0.66, 6.99±0.80, 550±35.7 and 1.57±0.42 nM, respectively. PET imaging showed that all 68Ga-labeled HTK03161, HTK03149 and HTK03189A/B were excreted mainly via the renal pathway and had minimal uptake in all organs/tissues including kidneys and salivary glands. Tumor xenografts were clearly visualized in PET images of [68Ga]Ga-HTK03161 and [68Ga]Ga-HTK03149 but were barely visualized using [68Ga]Ga-HTK03189A/B. Tumor uptake values for [68Ga]Ga-HTK03161, [68Ga]Ga-HTK03149, [68Ga]Ga-HTK0189A and [68Ga]Ga-HTK03189B were 12.7±1.91, 19.1±6.37, 2.10±0.28 and 0.67±0.15 %IA/g, respectively at 1h post-injection, and their average kidney and salivary gland uptake values were 2.13-4.41 and 0.20-0.23 %IA/g, respectively. Longitudinal SPECT imaging studies showed that [177Lu]Lu-HTK03149 was excreted mainly through the renal pathway with high uptake in LNCaP tumors and minimal uptake in all normal organs/tissues. The tumor uptake of [177Lu]Lu-HTK03149 peaked at 4h post-injection (20.9±2.99 %IA/g) and the uptake was sustained over time. Compared to [177Lu]Lu-PSMA-617, [177Lu]Lu-HTK03149 had 145% increase in tumor absorbed dose but 70% less in kidney absorbed dose, leading to an 7.1-fold increase in tumor-to-kidney absorbed dose ratio. Radioligand therapy studies showed that only half of the [177Lu]Lu-PSMA-617 injected dosage was needed for [177Lu]Lu-HTK03149 to achieve the same median survival. Conclusion: Replacing Glu in the PSMA-targeting Lys-urea-Glu pharmacophore of [68Ga]Ga-HTK03041 with Asp and Aad generates [68Ga]Ga-HTK03161 and [68Ga]Ga-HTK03149, respectively, and the new derivatives retain high uptake in LNCaP tumors and have minimal uptake in normal organs/tissues including kidneys and salivary glands. [177Lu]Lu-HTK03149 also retain high uptake in LNCaP tumors and has minimal uptake in normal organs/tissues, and is, therefore, promising for clinical translation to treat prostate cancer.

Acetylation, Methylation and Allysine Modification Profile of Viral and Host Proteins during Influenza A Virus Infection.

Protein modifications dynamically occur and regulate biological processes in all organisms. Towards understanding the significance of protein modifications in influenza virus infection, we performed a global mass spectrometry screen followed by bioinformatics analyses of acetylation, methylation and allysine modification in human lung epithelial cells in response to influenza A virus infection. We discovered 8 out of 10 major viral proteins and 245 out of 2280 host proteins detected to be differentially modified by three modifications in infected cells. Some of the identified proteins were modified on multiple amino acids residues and by more than one modification; the latter occurred either on different or same residues. Most of the modified residues in viral proteins were conserved across >40 subtypes of influenza A virus, and influenza B or C viruses and located on the protein surface. Importantly, many of those residues have already been determined to be critical for the influenza A virus. Similarly, many modified residues in host proteins were conserved across influenza A virus hosts like humans, birds, and pigs. Finally, host proteins undergoing the three modifications clustered in common functional networks of metabolic, cytoskeletal, and RNA processes, all of which are known to be exploited by the influenza A virus.

Identification of a conserved N-terminal domain in the first module of ACV synthetases.

The l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine synthetase (ACVS) is a trimodular nonribosomal peptide synthetase (NRPS) that provides the peptide precursor for the synthesis of ß-lactams. The enzyme has been extensively characterized in terms of tripeptide formation and substrate specificity. The first module is highly specific and is the only NRPS unit known to recruit and activate the substrate l-α-aminoadipic acid, which is coupled to the α-amino group of l-cysteine through an unusual peptide bond, involving its δ-carboxyl group. Here we carried out an in-depth investigation on the architecture of the first module of the ACVS enzymes from the fungus Penicillium rubens and the bacterium Nocardia lactamdurans. Bioinformatic analyses revealed the presence of a previously unidentified domain at the N-terminus which is structurally related to condensation domains, but smaller in size. Deletion variants of both enzymes were generated to investigate the potential impact on penicillin biosynthesis in vivo and in vitro. The data indicate that the N-terminal domain is important for catalysis.

Simultaneous quantification of alpha-aminoadipic semialdehyde, piperideine-6-carboxylate, pipecolic acid and alpha-aminoadipic acid in pyridoxine-dependent epilepsy.

The measurements of lysine metabolites provide valuable information for the rapid diagnosis of pyridoxine-dependent epilepsy (PDE). Here, we aimed to develop a sensitive method to simultaneously quantify multiple lysine metabolites in PDE, including α-aminoadipic semialdehyde (a-AASA), piperideine-6-carboxylate (P6C), pipecolic acid (PA) and α-aminoadipic acid (α-AAA) in plasma, serum, dried blood spots (DBS), urine and dried urine spots (DUS). Fifteen patients with molecularly confirmed PDE were detected using liquid chromatography-mass spectrometry (LC-MS/MS) method. Compared to the control groups, the concentrations of a-AASA, P6C and the sum of a-AASA and P6C (AASA-P6C) in all types of samples from PDE patients were markedly elevated. The PA and a-AAA concentrations ranges overlapped partially between PDE patients and control groups. The concentrations of all the analytes in plasma and serum, as well as in urine and DUS were highly correlated. Our study provided more options for the diverse sample collection in the biochemical tests according to practical requirements. With treatment modality of newly triple therapy investigated, biomarker study might play important role not only on diagnosis but also on treatment monitoring and fine tuning the diet. The persistently elevated analytes with good correlation between plasma and DBS, as well as urine and DUS made neonatal screening using DBS and DUS possible.

Deactivation of the autotrophic sulfate assimilation pathway substantially reduces high-level ß-lactam antibiotic biosynthesis and arthrospore formation in a production strain from Acremonium chrysogenum.

The filamentous ascomycete Acremonium chrysogenum is the only industrial producer of the ß-lactam antibiotic cephalosporin C. Synthesis of all ß-lactam antibiotics starts with the three amino acids l-α-aminoadipic acid, l-cysteine and l-valine condensing to form the δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine tripeptide. The availability of building blocks is essential in every biosynthetic process and is therefore one of the most important parameters required for optimal biosynthetic production. Synthesis of l-cysteine is feasible by various biosynthetic pathways in all euascomycetes, and sequencing of the Acr. chrysogenum genome has shown that a full set of sulfur-metabolizing genes is present. In principle, two pathways are effective: an autotrophic one, where the sulfur atom is taken from assimilated sulfide to synthesize either l-cysteine or l-homocysteine, and a reverse transsulfuration pathway, where l-methionine is the sulfur donor. Previous research with production strains has focused on reverse transsulfuration, and concluded that both l-methionine and reverse transsulfuration are essential for high-level cephalosporin C synthesis. Here, we conducted molecular genetic analysis with A3/2, another production strain, to investigate the autotrophic pathway. Strains lacking either cysteine synthase or homocysteine synthase, enzymes of the autotrophic pathway, are still autotrophic for sulfur. However, deletion of both genes results in sulfur amino acid auxotrophic mutants exhibiting delayed biomass production and drastically reduced cephalosporin C synthesis. Furthermore, both single- and double-deletion strains are more sensitive to oxidative stress and form fewer arthrospores. Our findings provide evidence that autotrophic sulfur assimilation is essential for growth and cephalosporin C biosynthesis in production strain A3/2 from Acr. chrysogenum.

(1) H NMR spectroscopy of glioblastoma stem-like cells identifies alpha-aminoadipate as a marker of tumor aggressiveness.

Patients suffering from glioblastoma multiforme (GBM) face a poor prognosis with median survival of about 14 months. High recurrence rate and failure of conventional treatments are attributed to the presence of GBM cells with stem-like properties (GSCs). Metabolite profiles of 42 GSC lines established from the tumor tissue of adult GBM patients were screened with (1) H NMR spectroscopy and compared with human neural progenitor cells from human adult olfactory bulb (OB-NPCs) and from the developing human brain (HNPCs). A first subset (n=12) of GSCs exhibited a dramatic accumulation of the metabolite α-aminoadipate (αAAD), product of the oxidation of α-aminoadipic semialdehyde catalyzed by the ALDH7A1 aldehyde dehydrogenase (ALDH) family in lysine catabolism. αAAD was low/not detectable in a second GSC subset (n=13) with the same neural metabolic profile as well as in a third GSC subset (n=17) characterized by intense lipid signals. Likewise, αAAD was not detected in the spectra of OB-NPCs or HNPCs. Inhibition of mitochondrial ATP synthase by oligomycin treatment revealed that the lysine degradative pathway leading to αAAD formation proceeds through saccharopine, as usually observed in developing brain. Survival curves indicated that high αAAD levels in GSCs significantly correlated with poor patient survival, similarly to prostate and non-small-cell-lung cancers, where activity of ALDH7A1 correlates with tumor aggressiveness.

Long-term consumption of dried bonito dashi (a traditional Japanese fish stock) reduces anxiety and modifies central amino acid levels in rats.

Dried bonito dashi, a traditional Japanese fish stock, enhances palatability of various dishes because of its specific flavor. Daily intake of dashi has also been shown to improve mood status such as tension-anxiety in humans. This study aimed at investigating beneficial effects of dashi ingestion on anxiety/depression-like behaviors and changes in amino acid levels in the brain and plasma in rats. Male Wistar rats were given either dried bonito dashi or water for long-term (29 days; Experiment 1) or single oral administration (Experiment 2). Anxiety and depression-like behaviors were tested using the open field and forced swimming tests, respectively. Concentrations of amino acids were measured in the hippocampus, hypothalamus, cerebellum, and jugular vein. During the long-term (29 days) consumption, rats given 2% dashi frequently entered the center zone and spent more time compared with the water controls in the open field test. However, the dashi was ineffective on depression-like behavior. In the hippocampus, concentrations of hydroxyproline, anserine, and valine were increased by dashi while those of asparagine and phenylalanine were decreased. In the hypothalamus, the methionine concentration was decreased. In a single oral administration experiment, the dashi (1%, 2% or 10%) showed no effects on behaviors. Significance was observed only in the concentrations of α-aminoadipic acid, cystathionine, and ornithine in the hippocampus. Dried bonito dashi is a functional food having anxiolytic-like effects. Daily ingestion of the dashi, even at lower concentrations found in the cuisine, reduces anxiety and alters amino acid levels in the brain.

Pipecolic acid enhances resistance to bacterial infection and primes salicylic acid and nicotine accumulation in tobacco.

Distinct amino acid metabolic pathways constitute integral parts of the plant immune system. We have recently identified pipecolic acid (Pip), a lysine-derived non-protein amino acid, as a critical regulator of systemic acquired resistance (SAR) and basal immunity to bacterial infection in Arabidopsis thaliana. In Arabidopsis, Pip acts as an endogenous mediator of defense amplification and priming. For instance, Pip conditions plants for effective biosynthesis of the phenolic defense signal salicylic acid (SA), accumulation of the phytoalexin camalexin, and expression of defense-related genes. Here, we show that tobacco plants respond to leaf infection by the compatible bacterial pathogen Pseudomonas syringae pv tabaci (Pstb) with a significant accumulation of several amino acids, including Lys, branched-chain, aromatic, and amide group amino acids. Moreover, Pstb strongly triggers, alongside the biosynthesis of SA and increases in the defensive alkaloid nicotine, the production of the Lys catabolites Pip and α-aminoadipic acid. Exogenous application of Pip to tobacco plants provides significant protection to infection by adapted Pstb or by non-adapted, hypersensitive cell death-inducing P. syringae pv maculicola. Pip thereby primes tobacco for rapid and strong accumulation of SA and nicotine following bacterial infection. Thus, our study indicates that the role of Pip as an amplifier of immune responses is conserved between members of the rosid and asterid groups of eudicot plants and suggests a broad practical applicability for Pip as a natural enhancer of plant disease resistance.

Comparison of the secondary metabolites in two scales of cephalosporin C (CPC) fermentation and two different post-treatment processes.

Cephalosporin C (CPC) is the precursor of a class of antibiotics that were more effective than traditional penicillins. CPC production is performed mainly through fermentation by Acremonium chrysogenum, whose secondary metabolism was sensitive to the environmental changes. In the present work, secondary metabolites were measured by ion-pair reversed-phase liquid chromatography tandemed with hybrid quadrupole time-of-flight mass spectrometry, and the disparity of them from two scales of CPC fermentations (pilot and industrial) and also two different post-treatment processes (oxalic acid and formaldehyde added and control) were investigated. When fermentation size was enlarged from pilot scale (50 l) to industrial scale (156,000 l), the remarkable disparities of concentrations and changing trends of the secondary metabolites in A. chrysogenum were observed, which indicated that the productivity of CPC biosynthesis was higher in the large scale of fermentation. Three environmental factors were measured, and the potential reasons that might cause the differences were analyzed. In the post-treatment process after industrial fermentation, the changes of these secondary metabolites in the tank where oxalic acid and formaldehyde were added were much less than the control tank where none was added. This indicated that the quality of the final product was more stable after the oxalic acid and formaldehyde were added in the post-treatment process. These findings provided new insight into industrial CPC production.

L-glutamate enhances methylmercury toxicity by synergistically increasing oxidative stress.

Methylmercury (MeHg) is a well-known environmental toxicant. With its lipophilic nature and high reactivity to sulfhydryl groups, it is widely distributed and accumulated in the body to damage cells. Oxidative stress is proposed as a major mechanism underlying the cytotoxic action of MeHg. In the present study, we found that L-glutamate (L-Glu) concentration-dependently increased MeHg cytotoxicity in HeLa S3 cells. The enhancement of the toxicity was accompanied by enhanced apoptosis, increased production of reactive oxygen species, and decreased glutathione level. An anti-oxidant N-acetylcysteine largely alleviated the cytotoxicity, suggesting enhanced oxidative stress behind L-Glu-elicited increase of MeHg toxicity. The effect was specific to L-Glu and L-alpha-aminoadipate, whereas D-Glu, L-aspartate, and D-aspartate were not effective. In addition, the cystine uptake by the cells was mostly mediated by a L-Glu/L-alpha-aminoadipate-sensitive amino acid transport system x(-)(C). All these results suggest that the inhibition of system x(-)(C) by L-Glu underlies the enhancement of MeHg cytotoxicity. The enhancement was highly synergistic because MeHg and L-Glu alone had little toxic effect in the conditions used. This synergism was confirmed in neural cells (neuroblastoma cell lines). It is proposed that similar mechanisms may underlie the neural toxicity of MeHg, particularly in the locality of lesions characteristic of MeHg toxicity.

Lysine is catabolized to 2-aminoadipic acid in Penicillium chrysogenum by an omega-aminotransferase and to saccharopine by a lysine 2-ketoglutarate reductase. Characterization of the omega-aminotransferase.

The biosynthesis and catabolism of lysine in Penicillium chrysogenum is of great interest because these pathways provide 2-aminoadipic acid, a precursor of the tripeptide delta-L-2-aminoadipyl-L-cysteinyl-D-valine that is an intermediate in penicillin biosynthesis. In vivo conversion of labelled L-lysine into two different intermediates was demonstrated by HPLC analysis of the intracellular amino acid pool. L-lysine is catabolized to 2-aminoadipic acid by an omega-aminotransferase and to saccharopine by a lysine-2-ketoglutarate reductase. In lysine-containing medium both activities were expressed at high levels, but the omega-aminotransferase activity, in particular, decreased sharply when ammonium was used as the nitrogen source. The omega-aminotransferase was partially purified, and found to accept L-lysine, L-ornithine and, to a lesser extent, N-acetyl-L-lysine as amino-group donors. 2-Ketoglutarate, 2-ketoadipate and, to a lesser extent, pyruvate served as amino group acceptors. This pattern suggests that this enzyme, previously designated as a lysine-6-aminotransferase, is actually an omega-aminotransferase. When 2-ketoadipate is used as substrate, the reaction product is 2-aminoadipic acid, which contributes to the pool of this intermediate available for penicillin biosynthesis. The N-terminal end of the purified 45-kDa omega-aminotransferase was sequenced and was found to be similar to the corresponding segment of the OAT1 protein of Emericella (Aspergillus) nidulans. This information was used to clone the gene encoding this enzyme.

Inactivation of the lys7 gene, encoding saccharopine reductase in Penicillium chrysogenum, leads to accumulation of the secondary metabolite precursors piperideine-6-carboxylic acid and pipecolic acid from alpha-aminoadipic acid.

Pipecolic acid serves as a precursor of the biosynthesis of the alkaloids slaframine and swainsonine (an antitumor agent) in some fungi. It is not known whether other fungi are able to synthesize pipecolic acid. Penicillium chrysogenum has a very active alpha-aminoadipic acid pathway that is used for the synthesis of this precursor of penicillin. The lys7 gene, encoding saccharopine reductase in P. chrysogenum, was target inactivated by the double-recombination method. Analysis of a disrupted strain (named P. chrysogenum SR1-) showed the presence of a mutant lys7 gene lacking about 1,000 bp in the 3'-end region. P. chrysogenum SR1- lacked saccharopine reductase activity, which was recovered after transformation of this mutant with the intact lys7 gene in an autonomously replicating plasmid. P. chrysogenum SR1- was a lysine auxotroph and accumulated piperideine-6-carboxylic acid. When mutant P. chrysogenum SR1- was grown with L-lysine as the sole nitrogen source and supplemented with DL-alpha-aminoadipic acid, a high level of pipecolic acid accumulated intracellularly. A comparison of strain SR1- with a lys2-defective mutant provided evidence showing that P. chrysogenum synthesizes pipecolic acid from alpha-aminoadipic acid and not from L-lysine catabolism.

Crystal structure of a lysine biosynthesis enzyme, LysX, from Thermus thermophilus HB8.

The thermophilic bacterium Thermus thermophilus synthesizes lysine through the alpha-aminoadipate pathway, which uses alpha-aminoadipate as a biosynthetic intermediate of lysine. LysX is the essential enzyme in this pathway, and is believed to catalyze the acylation of alpha-aminoadipate. We have determined the crystal structures of LysX and its complex with ADP at 2.0A and 2.38A resolutions, respectively. LysX is composed of three alpha+beta domains, each composed of a four to five-stranded beta-sheet core flanked by alpha-helices. The C-terminal and central domains form an ATP-grasp fold, which is responsible for ATP binding. LysX has two flexible loop regions, which are expected to play an important role in substrate binding and protection. In spite of the low level of sequence identity, the overall fold of LysX is surprisingly similar to that of other ATP-grasp fold proteins, such as D-Ala:D-Ala ligase, PurT-encoded glycinamide ribonucleotide transformylase, glutathione synthetase, and synapsin I. In particular, they share a similar spatial arrangement of the amino acid residues around the ATP-binding site. This observation strongly suggests that LysX is an ATP-utilizing enzyme that shares a common evolutionary ancestor with other ATP-grasp fold proteins possessing a carboxylate-amine/thiol ligase activity.

L-delta-(alpha-Aminoadipoyl)-L-cysteine-D-valine synthetase: production of dipeptides containing valine residue at its C-terminus.

L-delta-(alpha-Aminoadipoyl)-L-cysteine-D-valine synthetase (ACVS) has been recently studied as a model enzyme for peptide synthetases. It was found that in the absence of alpha-aminoadipic acid but in the presence of several cysteine analogues it was incorporated into several analogue dipeptides upon incubation of the potential cysteine analogues with ACVS. [(14)C]Cysteine was incorporated into the[(14)C]cysteinyl-valine analogue dipeptides. Notably, [(14)C]valine incorporation in the presence of N-acylated cysteine analogues was observed. The alpha-aminoadipic acid activation site is influential, inhibitory or promotive, on the production of these putative dipeptide products. The production of dipeptide analogues, containing valine or analogues at the C-terminus, leads to the speculation that the biosynthetic direction of ACV could be from the C-terminus to the N-terminus.

Visualising the activity of the cystine-glutamate antiporter in glial cells using antibodies to aminoadipic acid, a selectively transported substrate.

The cystine-glutamate antiporter is a transport system that facilitates the uptake of cystine, concomitant with the release of glutamate. The cystine accumulated by this transporter is generally considered for use in the formation of the cysteine-containing antioxidant glutathione, which is abundant in many glial cells. This study used the simple strategy of generating an antibody to aminoadipic acid, a selective substrate for the cystine-glutamate antiporter. Stereospecific accumulation of aminoadipic acid into specific cell types in rat brain slice preparations was detected immunocytochemically. Strong accumulation was detected in astroglial cells in all brain regions studied including those in white matter tracts. Strong accumulation into radial glial cells, including the retinal Müller cells and the Bergmann glial cells was also observed. Glial accumulation was observed not only in cells within the blood brain barrier, but also outside such; anterior pituitary folliculostellate cell and intermediate lobe pituitary glial cells exhibited strong accumulation of aminoadipic acid. Interestingly, some glial cells such as the posterior pituitary glial cells (pituicytes) exhibited very little if any accumulation of aminoadipic acid. Within the brain labelling was not uniform. Particularly strong labelling was noted in some regions, such as the glial cells surrounding the CA1 pyramidal cells. By contrast, neurons never exhibited uptake of aminoadipic acid. Because cystine uptake is associated with glutamate release, it is suggested that this antiporter might contribute to release of glutamate from glial cells under some pathophysiological conditions.

The catabolic function of the alpha-aminoadipic acid pathway in plants is associated with unidirectional activity of lysine-oxoglutarate reductase, but not saccharopine dehydrogenase.

Whereas plants and animals use the alpha-aminoadipic acid pathway to catabolize lysine, yeast and fungi use the very same pathway to synthesize lysine. These two groups of organisms also possess structurally distinct forms of two enzymes in this pathway, namely lysine-oxoglutarate reductase (lysine-ketoglutarate reductase; LKR) and saccharopine dehydrogenase (SDH): in plants and animals these enzymes are linked on to a single bifunctional polypeptide, while in yeast and fungi they exist as separate entities. In addition, yeast LKR and SDH possess bi-directional activities, and their anabolic function is regulated by complex transcriptional and post-transcriptional controls, which apparently ascertain differential accumulation of intermediate metabolites; in plants, the regulation of the catabolic function of these two enzymes is not known. To elucidate the regulation of the catabolic function of plant bifunctional LKR/SDH enzymes, we have used yeast as an expression system to test whether a plant LKR/SDH also possesses bi-directional LKR and SDH activities, similar to the yeast enzymes. The Arabidopsis enzyme complemented a yeast SDH, but not LKR, null mutant. Identical results were obtained when deletion mutants encoding only the LKR or SDH domains of this bifunctional polypeptide were expressed individually in the yeast cells. Moreover, activity assays showed that the Arabidopsis LKR possessed catabolic, but not anabolic, activity, and its uni-directional activity stems from its structure rather than its linkage to SDH. Our results suggest that the uni-directional activity of LKR plays an important role in regulating the catabolic function of the alpha-amino adipic acid pathway in plants.

Lysine biosynthesis in Saccharomyces cerevisiae: mechanism of alpha-aminoadipate reductase (Lys2) involves posttranslational phosphopantetheinylation by Lys5.

A key step in fungal biosynthesis of lysine, enzymatic reduction of alpha-aminoadipate at C6 to the semialdehyde, requires two gene products in Saccharomyces cerevisiae, Lys2 and Lys5. Here, we show that the 31-kDa Lys5 is a specific posttranslational modification catalyst, using coenzyme A (CoASH) as a cosubstrate to phosphopantetheinylate Ser880 of the 155-kDa Lys2 and activate it for catalysis. Lys2 was subcloned from S. cerevisiae and expressed in and purified from Escherichia coli as a full-length 155-kDa enzyme, as a 105-kDa adenylation/peptidyl carrier protein (A/PCP) fragment (residues 1-924), and as a 14-kDa PCP fragment (residues 809-924). The apo-PCP fragment was covalently modified to phosphopantetheinylated holo-PCP by pure Lys5 and CoASH with a Km of 1 microM and kcat of 3 min-1 for both the PCP and CoASH substrates. The adenylation domain of the A/PCP fragment activated S-carboxymethyl-L-cysteine (kcat/Km = 840 mM-1 min-1) at 16% the efficiency of L-alpha-aminoadipate in [32P]PPi/ATP exchange assays. The holo form of the A/PCP 105-kDa fragment of Lys2 covalently aminoacylated itself with [35S]S-carboxymethyl-L-cysteine. Addition of NADPH discharged the covalent acyl-S-PCP Lys2, consistent with a reductive cleavage of the acyl-S-enzyme intermediate. These results identify the Lys5/Lys2 pair as a two-component system in which Lys5 covalently primes Lys2, allowing alpha-aminoadipate reductase activity by holo-Lys2 with catalytic cycles of autoaminoacylation and reductive cleavage. This is a novel mechanism for a fungal enzyme essential for amino acid metabolism.

Characterisation of Saccharomyces cerevisiae ARO8 and ARO9 genes encoding aromatic aminotransferases I and II reveals a new aminotransferase subfamily.

The ARO8 and ARO9 genes of Saccharomyces cerevisiae were isolated by complementation of the phenylalanine/tyrosine auxotrophy of an aro8 and aro9 double-mutant strain that is defective in aromatic aminotransferase I (aro8) and II (aro9). The genes were sequenced, and deletion mutants were constructed and analysed. The expression of ARO8 and ARO9 was studied. The deduced amino acid sequences of Aro8p and Aro9p suggest that the former is a 500-residue, 56168-Da polypeptide and the latter a 513-residue, 58516-Da polypeptide. They correspond, respectively, to Ygl202p and Yhr137p, two putative proteins of unknown function revealed by systematic sequencing of the yeast genome. We show that aromatic aminotransferases I and II are homologous proteins, members of aminotransferase subgroup I, and, together with three other proteins, they constitute within the subgroup a new subfamily of enzymes specialised for aromatic amino acid and alpha-aminoadipate transamination. ARO8 expression is subject to the general control of amino acid biosynthesis. ARO9 expression is induced when aromatic amino acids are present in the growth medium and also in aro8 mutants grown on minimal ammonia medium. An autonomously replicating sequence (ARS) element is located between the ARO8 gene and YGL201c which encodes a protein of the minichromosome maintenance family.

Purification and characterization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Penicillium chrysogenum.

delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) from Penicillium chrysogenum was purified to homogeneity by a combination of (NH4)2SO4 precipitation, protamine sulphate treatment, ion-exchange chromatography, gel filtration and hydrophobic interaction chromatography. The molecular mass of ACVS was estimated with native gradient gel electrophoresis and SDS/PAGE. The native enzyme consisted of a single polymer chain with an estimated molecular mass of 470 kDa. The denatured enzyme had an estimated molecular mass of 440 kDa. The influence of different reaction parameters such as substrates, cofactors and pH on the activity of the purified ACVS was investigated. The Km values for the three precursor substrates L-alpha-aminoadipic acid, L-cysteine and L-valine were determined as 45, 80 and 80 microM respectively, and the optimal assay concentration of ATP was found to be 5 mM (with 20 mM MgCl2). The dimer of the reaction product bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (bisACV) gave feedback inhibition of the purified ACVS; the inhibition parameter KbisACV was determined as 1.4 mM. Furthermore dithiothreitol was shown to inhibit the purified ACVS. From the addition of a glucose pulse to a steady-state glucose-limited continuous culture of P. chrysogenum it was found that there is glucose repression of the synthesis of ACVS and that there must be a constant turnover of ACVS owing to synthesis and degradation.

L-delta-(alpha-Aminoadipoyl)-L-cysteinyl-D-valine synthetase: thioesterification of valine is not obligatory for peptide bond formation.

L-delta-(alpha-Aminoadipoyl)-L-cysteinyl-D-valine (ACV) synthetase is probably the simplest known peptide synthetase in terms of the number of reactions catalyzed. In the "thiol-template" proposal for nonribosomal peptide synthesis, a key step is transfer of aminoacyl groups derived from the substrates to enzyme-bound thiols prior to peptide bond formation. No incorporation of 18O was seen in AMP isolated from the reaction mixture when di[18O]valine was incubated with relatively large amounts of active synthetase and MgATP. We therefore utilized di[18O]valine as a substrate for the biosynthesis of the diastereomeric dipeptides L-O-(methylserinyl)-L-valine and L-O-(methylserinyl)-D-valine [Shiau, C.-Y., Baldwin, J. E., Byford, M. F., Sobey, W. J., & Schofield, C. J. (1995) FEBS Lett. 358, 97-100]. In the L-O-(methylserinyl)-L-valine product, no significant loss of 18O was observed. However, in the L-O-(methylserinyl)-D-valine product, a significant loss of one or both 18O labels was observed. Thus, both peptide bond formation and the epimerization of the valine residue can both occur before formation of any thioester bond to the valine carboxylate in the biosynthesis of these dipeptides. The usual qualitative test for thioesterification of substrates to the synthetase, lability of enzyme-bound radiolabeled amino acid to performic acid, proved inconclusive in our hands. These results require a new mechanism for the enzymic synthesis of L-O-(methylserinyl)-L-valine and L-O-(methylserinyl)-D-valine and imply that a revised mechanism for ACV synthesis is also required.