Biochemistry of plants N-heterocyclic non-protein amino acids.

Plants catalyze the biosynthesis of a large number of non-protein amino acids, which are usually toxic for other organisms. In this review, the chemistry and metabolism of N-heterocyclic non-protein amino acids from plants are described. These N-heterocyclic non-protein amino acids are composed of ß-substituted alanines and include mimosine, ß-pyrazol-1-yl-L-alanine, willardiine, isowillardiine, and lathyrine. These ß-substituted alanines consisted of an N-heterocyclic moiety and an alanyl side chain. This review explains how these individual moieties are derived from their precursors and how they are used as the substrate for biosynthesizing the respective N-heterocyclic non-protein amino acids. In addition, known catabolism and possible role of these non-protein amino acids in the actual host is explained.

Serine restriction alters sphingolipid diversity to constrain tumour growth.

Serine, glycine and other nonessential amino acids are critical for tumour progression, and strategies to limit their availability are emerging as potential therapies for cancer1-3. However, the molecular mechanisms driving this response remain unclear and the effects on lipid metabolism are relatively unexplored. Serine palmitoyltransferase (SPT) catalyses the de novo biosynthesis of sphingolipids but also produces noncanonical 1-deoxysphingolipids when using alanine as a substrate4,5. Deoxysphingolipids accumulate in the context of mutations in SPTLC1 or SPTLC26,7-or in conditions of low serine availability8,9-to drive neuropathy, and deoxysphinganine has previously been investigated as an anti-cancer agent10. Here we exploit amino acid metabolism and the promiscuity of SPT to modulate the endogenous synthesis of toxic deoxysphingolipids and slow tumour progression. Anchorage-independent growth reprogrammes a metabolic network involving serine, alanine and pyruvate that drives the endogenous synthesis and accumulation of deoxysphingolipids. Targeting the mitochondrial pyruvate carrier promotes alanine oxidation to mitigate deoxysphingolipid synthesis and improve spheroid growth, similar to phenotypes observed with the direct inhibition of SPT or ceramide synthesis. Restriction of dietary serine and glycine potently induces the accumulation of deoxysphingolipids while decreasing tumour growth in xenograft models in mice. Pharmacological inhibition of SPT rescues xenograft growth in mice fed diets restricted in serine and glycine, and the reduction of circulating serine by inhibition of phosphoglycerate dehydrogenase (PHGDH) leads to the accumulation of deoxysphingolipids and mitigates tumour growth. The promiscuity of SPT therefore links serine and mitochondrial alanine metabolism to membrane lipid diversity, which further sensitizes tumours to metabolic stress.

Whole genome mining reveals a diverse repertoire of lanthionine synthetases and lanthipeptides among the genus Paenibacillus.

AIM: Lanthionine or methyllanthionine-containing lanthipeptides belongs to ribosomally synthesized and post-translationally modified peptides (RiPPs) family. Recent revolution in sequencing has made available huge genome sequence dataset of micro-organisms. In this study, we performed genome mining of the complete and partial genome sequences of 479 bacteria of the genus Paenibacillus to determine the diversity and distribution of lanthipeptide gene clusters. METHODS AND RESULTS: All genome sequences were annotated by RAST and subsequently analysed by BAGEL and antiSMASH. A total of 221 lanthipeptide gene clusters were identified in 127 strains of the genus Paenibacillus. One hundred and fifty gene clusters were found associated with the production of class I lanthipeptides while 58 and 13 gene clusters were related to class II and class IV lanthipeptide production respectively. Frequency of strains whose genomes encode putative lanthipeptide precursors was 26·5%. CONCLUSIONS: The results of lanthionine synthetases analysis suggested that diversity of lanthipeptides is much more than anticipated, while lanthionine synthetases must have been co-evolved among various species of the genus Paenibacillus. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report showing diversity and distribution of different classes of lanthipeptides among various species of the genus Paenibacillus. This study also reveals the novel lanthipeptide sequences which may be further developed as potential antimicrobials for therapeutic applications.

Structural and Kinetic Insight into the Biosynthesis of H2S and l-Lanthionine from l-Cysteine by a Pyridoxal l-Phosphate-Dependent Enzyme from Fusobacterium nucleatum.

Fusobacterium nucleatum is a common oral bacterium and a major producer of H2S, a toxic gas linked to the pathogenesis of periodontal disease. The bacterium encodes a fold type II pyridoxal l-phosphate (PLP)-dependent enzyme, Fn1220 or lanthionine synthase (LS), that generates H2S and l-lanthionine (a component of the peptidoglycan layer) through ß-replacement of l-cysteine by a second molecule of l-cysteine. Herein, we show through detailed kinetic analysis that LS elicits catalytic promiscuity as demonstrated for other fold type II PLP-dependent homologues, namely, O-acetylserine sulfhydrylase (OASS) and cystathionine ß-synthase (CBS). Like OASS, LS can assimilate H2S by catalyzing the ß-replacement of O-acetyl-l-serine by sulfide to form l-cysteine. However, the turnover for this reaction in LS is slower than that of other studied OASS enzymes due to slower conversion to the α-aminoacrylate intermediate. Similar to yeast and human CBS, LS can generate H2S and l-cystathionine through ß-replacement of l-cysteine by a second molecule of l-homocysteine; however, whereas this is the main H2S-forming reaction in CBS, it is not for LS. LS shows a marked preference for forming H2S and l-lanthionine through the condensation of 2 equiv of l-cysteine. Sequence alignment of LS with other CBS and OASS enzymes and inspection of the LS crystal structure in the external aldimine state with l-lanthionine reveal that LS possesses a unique loop that engages in hydrogen-bond contact with the product, providing a structural rationale for the enzyme's catalytic preference for H2S and l-lanthionine biosynthesis.

Nitrile-synthesizing enzyme: Screening, purification and characterization.

Cyanide is known as a toxic compound for almost all living organisms. We have searched for cyanide-resistant bacteria from the soil and stock culture collection of our laboratory, and have found the existence of a lot of microorganisms grown on culture media containing 10 mM potassium cyanide. Almost all of these cyanide-resistant bacteria were found to show ß-cyano-L-alanine (ß-CNAla) synthetic activity. ß-CNAla synthase is known to catalyze nitrile synthesis: the formation of ß-CNAla from potassium cyanide and O-acetyl-L-serine or L-cysteine. We found that some microorganisms were able to detoxify cyanide using O-methyl-DL-serine, O-phospho-L-serine and ß-chloro-DL-alanine. In addition, we purified ß-CNAla synthase from Pseudomonas ovalis No. 111 in nine steps, and characterized the purified enzyme. This enzyme has a molecular mass of 60,000 and appears to consist of two identical subunits. The purified enzyme exhibits a maximum activity at pH 8.5-9.0 at an optimal temperature of 40-50°C. The enzyme is specific for O-acetyl-L-serine and ß-chloro-DL-alanine. The Km value for O-acetyl-L-serine is 10.0 mM and Vmax value is 3.57 µmol/min/mg.

Insights into the Biosynthesis of Dehydroalanines in Goadsporin.

Dehydroalanines in goadsporin are proposed to be formed by GodF and GodG, which show slight homology to the N-terminal glutamylation and C-terminal elimination domains, respectively, of LanB, a class I lanthipeptide dehydratase. Although similar, separated-type LanBs are conserved among thiopeptides and indispensable for their biosynthesis and biological activities, these enzymes had not yet been characterized. Here, we identified goadsporin B, which has unmodified Ser4 and Ser14, from both godF and godG disruptants. The godG disruptant also produced goadsporin C, a glutamylated-Ser4 variant of goadsporin B. These results suggested that dehydroalanines are formed by glutamylation and glutamate elimination. NMR analysis revealed for the first time that the glutamyl group was attached to a serine via an ester bond, by the catalysis of LanB-type enzymes. Our findings provide insights into the function of separated-type LanBs involved in the biosynthesis of goadsporin and thiopeptides.

Phosphoserine Lyase Deoxyribozymes: DNA-Catalyzed Formation of Dehydroalanine Residues in Peptides.

Dehydroalanine (Dha) is a nonproteinogenic electrophilic amino acid that is a synthetic intermediate or product in the biosynthesis of several bioactive cyclic peptides such as lantibiotics, thiopeptides, and microcystins. Dha also enables labeling of proteins and synthesis of post-translationally modified proteins and their analogues. However, current chemical approaches to introducing Dha into peptides have substantial limitations. Using in vitro selection, here we show that DNA can catalyze Zn(2+) or Zn(2+)/Mn(2+)-dependent formation of Dha from phosphoserine (pSer), i.e., exhibit pSer lyase activity, a fundamentally new DNA-catalyzed reaction. Two new pSer lyase deoxyribozymes, named Dha-forming deoxyribozymes 1 and 2 (DhaDz1 and DhaDz2), each function with multiple turnover on the model hexapeptide substrate that was used during selection. Using DhaDz1, we generated Dha from pSer within an unrelated linear 13-mer peptide. Subsequent base-promoted intramolecular cyclization of homocysteine into Dha formed a stable cystathionine (thioether) analogue of the complement inhibitor compstatin. These findings establish the fundamental catalytic ability of DNA to eliminate phosphate from pSer to form Dha and suggest that with further development, pSer lyase deoxyribozymes will have broad practical utility for site-specific enzymatic synthesis of Dha from pSer in peptide substrates.

Formylglycine, a post-translationally generated residue with unique catalytic capabilities and biotechnology applications.

Formylglycine (fGly) is a catalytically essential residue found almost exclusively in the active sites of type I sulfatases. Formed by post-translational oxidation of cysteine or serine side chains, this aldehyde-functionalized residue participates in a unique and highly efficient catalytic mechanism for sulfate ester hydrolysis. The enzymes that produce fGly, formylglycine-generating enzyme (FGE) and anaerobic sulfatase-maturating enzyme (anSME), are as unique and specialized as fGly itself. FGE especially is structurally and mechanistically distinct, and serves the sole function of activating type I sulfatase targets. This review summarizes the current state of knowledge regarding the mechanism by which fGly contributes to sulfate ester hydrolysis, the molecular details of fGly biogenesis by FGE and anSME, and finally, recent biotechnology applications of fGly beyond its natural catalytic function.

Substrate control in stereoselective lanthionine biosynthesis.

Enzymes are typically highly stereoselective catalysts that enforce a reactive conformation on their native substrates. We report here a rare example in which the substrate controls the stereoselectivity of an enzyme-catalysed Michael-type addition during the biosynthesis of lanthipeptides. These natural products contain thioether crosslinks formed by a cysteine attack on dehydrated Ser and Thr residues. We demonstrate that several lanthionine synthetases catalyse highly selective anti-additions in which the substrate (and not the enzyme) determines whether the addition occurs from the re or si face. A single point mutation in the peptide substrate completely inverted the stereochemical outcome of the enzymatic modification. Quantum mechanical calculations reproduced the experimentally observed selectivity and suggest that conformational restraints imposed by the amino-acid sequence on the transition states determine the face selectivity of the Michael-type cyclization.

Vitamin B-6 restriction reduces the production of hydrogen sulfide and its biomarkers by the transsulfuration pathway in cultured human hepatoma cells.

BACKGROUND: Pyridoxal 5'-phosphate (PLP) functions as a coenzyme in many cellular processes including one-carbon metabolism and the interconversion and catabolism of amino acids. PLP-dependent enzymes, cystathionine ß-synthase and cystathionine γ-lyase, function in transsulfuration but also have been implicated in the production of the endogenous gaseous signaling molecule hydrogen sulfide (H2S) concurrent with the formation of the biomarkers lanthionine and homolanthionine. OBJECTIVE: Our objective was to determine if H2S production and concurrent biomarker production is affected by vitamin B-6 restriction in a cell culture model. METHODS: We used cultured human hepatoma cells and evaluated static intracellular profiles of amino acids and in vivo kinetics of H2S biomarker formation. Cells were cultured for 6 wk in media containing concentrations of pyridoxal that represented severe vitamin B-6 deficiency (15 nmol/L pyridoxal), marginal deficiency (56 nmol/L pyridoxal), adequacy (210 nmol/L pyridoxal), and standard medium formulation providing a supraphysiologic pyridoxal concentration (1800 nmol/L pyridoxal). RESULTS: Intracellular concentrations of lanthionine and homolanthionine in cells cultured at 15 nmol/L pyridoxal were 50% lower (P < 0.002) and 47% lower (P < 0.0255), respectively, than observed in cells cultured at 1800 nmol/L pyridoxal. Extracellular homocysteine and cysteine were 58% and 46% higher, respectively, in severely deficient cells than in adequate cells (P < 0.002). Fractional synthesis rates of lanthionine (P < 0.01) and homolanthionine (P < 0.006) were lower at 15 and 56 nmol/L pyridoxal than at both higher pyridoxal concentrations. The rate of homocysteine remethylation and the fractional rate of homocysteine production from methionine were not affected by vitamin B-6 restriction. In vitro studies of cell lysates using direct measurement of H2S also had a reduced extent of H2S production in the 2 lower vitamin B-6 conditions. CONCLUSION: In view of the physiologic roles of H2S, these results suggest a mechanism that may be involved in the association between human vitamin B-6 inadequacy and its effects on human health.

Insights into the evolution of lanthipeptide biosynthesis.

Lanthipeptides are a group of posttranslationally modified peptide natural products that contain multiple thioether crosslinks. These crosslinks are formed by dehydration of Ser/Thr residues followed by addition of the thiols of Cys residues to the resulting dehydroamino acids. At least four different pathways to these polycyclic natural products have evolved, reflecting the high efficiency and evolvability of a posttranslational modification route to generate conformationally constrained peptides. The wealth of genomic information that has been made available in recent years has started to provide insights into how these remarkable pathways and their posttranslational modification machineries may have evolved. In this review, we discuss a model for the evolution of the lanthipeptide biosynthetic enzymes that has recently been developed based on the currently available data.

Catabolism of exogenous lactate reveals it as a legitimate metabolic substrate in breast cancer.

Lactate accumulation in tumors has been associated with metastases and poor overall survival in cancer patients. Lactate promotes angiogenesis and metastasis, providing rationale for understanding how it is processed by cells. The concentration of lactate in tumors is a balance between the amount produced, amount carried away by vasculature and if/how it is catabolized by aerobic tumor or stromal cells. We examined lactate metabolism in human normal and breast tumor cell lines and rat breast cancer: 1. at relevant concentrations, 2. under aerobic vs. hypoxic conditions, 3. under conditions of normo vs. hypoglucosis. We also compared the avidity of tumors for lactate vs. glucose and identified key lactate catabolites to reveal how breast cancer cells process it. Lactate was non-toxic at clinically relevant concentrations. It was taken up and catabolized to alanine and glutamate by all cell lines. Kinetic uptake rates of lactate in vivo surpassed that of glucose in R3230Ac mammary carcinomas. The uptake appeared specific to aerobic tumor regions, consistent with the proposed "metabolic symbiont" model; here lactate produced by hypoxic cells is used by aerobic cells. We investigated whether treatment with alpha-cyano-4-hydroxycinnamate (CHC), a MCT1 inhibitor, would kill cells in the presence of high lactate. Both 0.1 mM and 5 mM CHC prevented lactate uptake in R3230Ac cells at lactate concentrations at ≤ 20 mM but not at 40 mM. 0.1 mM CHC was well-tolerated by R3230Ac and MCF7 cells, but 5 mM CHC killed both cell lines ± lactate, indicating off-target effects. This study showed that breast cancer cells tolerate and use lactate at clinically relevant concentrations in vitro (± glucose) and in vivo. We provided additional support for the metabolic symbiont model and discovered that breast cells prevailingly take up and catabolize lactate, providing rationale for future studies on manipulation of lactate catabolism pathways for therapy.

Dipeptidyl peptidase IV activity in commercial solutions of human serum albumin.

Due to the heterogeneous nature of commercial human serum albumin (cHSA), other components, such as the protease dipeptidyl peptidase IV (DPP-IV), possibly contribute to the therapeutic effect of cHSA. Here, we provide evidence for the first time that DPP-IV activity contributes to the formation of aspartate-alanine diketopiperazine (DA-DKP), a known immunomodulatory molecule from the N terminus of human albumin. cHSA was assayed for DPP-IV activity using a specific DPP-IV substrate and inhibitor. DPP-IV activity was assayed at 37 and 60°C because cHSA solutions are pasteurized at 60°C. DPP-IV activity in cHSA was compared with other sources of albumin such as a recombinant albumin (rHSA). In addition, the production of DA-DKP was measured by negative electrospray ionization/liquid chromatography mass spectrometry (ESI(-)/LCMS). Significant levels of DPP-IV activity were present in cHSA. This activity was abolished using a specific DPP-IV inhibitor. Fully 70 to 80% DPP-IV activity remained at 60°C compared with the 37°C incubate. No DPP-IV activity was present in rHSA, suggesting that DPP-IV activity is present only in HSA produced using the Cohn fractionation process. The formation of DA-DKP at 60°C was observed with the DPP-IV inhibitor significantly decreasing this formation. DPP-IV activity in cHSA results in the production of DA-DKP, which could account for some of the clinical effects of cHSA.

Androgen-responsive and nonresponsive prostate cancer cells present a distinct glycolytic metabolism profile.

Prostate cancer (PCa) progresses from an early stage, confined to prostate, to a more aggressive metastasized cancer related with loss of androgen responsiveness. Although, it has been recognized that PCa cells have unique metabolic features, their glycolytic profile in androgen-dependent and androgen-independent stages of disease is much less known. Hence, the main purpose of this study was to compare glucose metabolism in androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) PCa cells. Cell culture medium was collected and differences in glucose consumption and, lactate and alanine production were measured using Proton Nuclear Magnetic Resonance ((1)H NMR) spectra analysis. The mRNA and protein expression of glucose transporters (GLUT1 and GLUT3), phosphofructokinase 1 (PFK1), lactate dehydrogenase (LDH) and monocarboxylate transporter (MCT4) were determined by real-time PCR and Western Blot, respectively. The obtained results demonstrate that androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) cells consumed similar amounts of glucose, whereas PC3 cells present higher lactate production. This increase in lactate production was concomitant with higher levels of MCT4 protein, increased LDH activity and higher lactate/alanine ratio, also suggesting increased levels of oxidative stress in PC3 cells. However, protein levels of LDH, associated with lactate metabolism, and GLUT3, involved in glucose uptake, were decreased in PC3 comparatively with LNCaP. Androgen-responsive and nonresponsive PCa cells present distinct glycolytic metabolism profiles, which suggest that targeting LDH and MCT4 metabolic pathways may be an important step for the development of new diagnostic and therapeutic strategies in the different stages of PCa.

Metabolic modulation induced by oestradiol and DHT in immature rat Sertoli cells cultured in vitro.

Sertoli cells actively metabolize glucose that is converted into lactate, which is used by developing germ cells for their energy metabolism. Androgens and oestrogens have general metabolic roles that reach far beyond reproductive processes. Hence, the main purpose of this study was to examine the effect of sex hormones on metabolite secretion/consumption in primary cultures of rat Sertoli cells. Sertoli cell-enriched cultures were maintained in a defined medium for 50 h. Glucose and pyruvate consumption, and lactate and alanine secretion were determined, by 1H-NMR (proton NMR) spectra analysis, in the presence or absence of 100 nM E2 (17ß-oestradiol) or 100 nM 5α-DHT (dihydrotestosterone). Cells cultured in the absence (control) or presence of E2 consumed the same amount of glucose (29±2 pmol/cell) at similar rates during the 50 h. After 25 h of treatment with DHT, glucose consumption and glucose consumption rate significantly increased. Control and E2-treated cells secreted similar amounts of lactate during the 50 h, while the amount of lactate secreted by DHT-treated cells was significantly lower. Such a decrease was concomitant with a significant decrease in LDH A [LDH (lactate dehydrogenase) chain A] and MCT4 [MCT (monocarboxylate transporter) isoform 4] mRNA levels after 50 h treatment in hormonally treated groups, being more pronounced in DHT-treated groups. Finally, alanine production was significantly increased in E2-treated cells after 25 h treatment, which indicated a lower redox/higher oxidative state for the cells in those conditions. Together, these results support the existence of a relation between sex hormones action and energy metabolism, providing an important assessment of androgens and oestrogens as metabolic modulators in rat Sertoli cells.

Genome-enabled determination of amino acid biosynthesis in Xanthomonas campestris pv. campestris and identification of biosynthetic pathways for alanine, glycine, and isoleucine by 13C-isotopologue profiling.

To elucidate the biosynthetic pathways for all proteinogenic amino acids in Xanthomonas campestris pv. campestris, this study combines results obtained by in silico genome analysis and by (13)C-NMR-based isotopologue profiling to provide a panoramic view on a substantial section of bacterial metabolism. Initially, biosynthesis pathways were reconstructed from an improved annotation of the complete genome of X. campestris pv. campestris B100. This metabolic reconstruction resulted in the unequivocal identification of biosynthesis routes for 17 amino acids in total: arginine, asparagine, aspartate, cysteine, glutamate, glutamine, histidine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Ambiguous pathways were reconstructed from the genome data for alanine, glycine, and isoleucine biosynthesis. (13)C-NMR analyses supported the identification of the metabolically active pathways. The biosynthetic routes for these amino acids were derived from the precursor molecules pyruvate, serine, and pyruvate, respectively. By combining genome analysis and isotopologue profiling, a comprehensive set of biosynthetic pathways covering all proteinogenic amino acids was unraveled for this plant pathogenic bacterium, which plays an important role in biotechnology as a producer of the exopolysaccharide xanthan. The data obtained lay ground for subsequent functional analyses in post-genomics and biotechnology, while the innovative combination of in silico and wet lab technology described here is promising as a general approach to elucidate metabolic pathways.

Inhibition of alanine aminotransferase in silico and in vivo promotes mitochondrial metabolism to impair malignant growth.

Cancer cells commonly exhibit increased nonoxidative D-glucose metabolism whereas induction of mitochondrial metabolism may impair malignant growth. We have first used an in silico method called elementary mode analysis to identify inhibition of ALAT (L-alanine aminotransferase) as a putative target to promote mitochondrial metabolism. We then experimentally show that two competitive inhibitors of ALAT, L-cycloserine and ß-chloro-L-alanine, inhibit L-alanine production and impair D-glucose uptake of LLC1 Lewis lung carcinoma cells. The latter inhibition is linked to an initial energy deficit, as quantified by decreased ATP content, which is then followed by an activation of AMP-activated protein kinase and subsequently increased respiration rates and mitochondrial production of reactive oxygen species, culminating in ATP replenishment in ALAT-inhibited LLC1 cells. Moreover, we observe altered phosphorylation of p38 MAPK (mitogen-activated protein kinase 14), ERK (extracellular signal-regulated kinase 1/2), and Rb1 (retinoblastoma 1) proteins, as well as decreased expression of Cdc25a (cell decision cycle 25 homolog A) and Cdk4 (cyclin-dependent kinase 4). Importantly, these sequelae of ALAT inhibition culminate in similarly reduced anchorage-dependent and anchorage-independent growth rates of LLC1 cells, together suggesting that inhibition of ALAT efficiently impairs cancer growth by counteracting the Warburg effect due to compensatory activation of mitochondrial metabolism.

Production of hydrogen sulfide by two enzymes associated with biosynthesis of homocysteine and lanthionine in Fusobacterium nucleatum subsp. nucleatum ATCC 25586.

Fusobacterium nucleatum produces a large amount of the toxic metabolite hydrogen sulfide in the oral cavity. Here, we report the molecular basis of F. nucleatum H(2)S production, which is associated with two different enzymes: the previously reported Cdl (Fn1220) and the newly identified Lcd (Fn0625). SDS-PAGE analysis with activity staining revealed that crude enzyme extracts from F. nucleatum ATCC 25586 contained three major H(2)S-producing proteins. Two of the proteins with low molecular masses migrated similarly to purified Fn0625 and Fn1220. Their kinetic values suggested that Fn0625 had a lower enzymic capacity to produce H(2)S from L-cysteine (approximately 30%) than Fn1220. The Fn0625 protein degraded a variety of substrates containing betaC-S linkages to produce ammonia, pyruvate and sulfur-containing products. Unlike Fn0625, Fn1220 produced neither pyruvate nor ammonia from L-cysteine. Reversed-phase HPLC separation and mass spectrometry showed that incubation of L-cysteine with Fn1220 produced H(2)S and an uncommon amino acid, lanthionine, which is a natural constituent of the peptidoglycans of F. nucleatum ATCC 25586. In contrast, most of the sulfur-containing substrates tested, except L-cysteine, were not used by Fn1220. Real-time PCR analysis demonstrated that the fn1220 gene showed several-fold higher expression than fn0625 and housekeeping genes in exponential-phase cultures of F. nucleatum. Thus, we conclude that Fn0625 and Fn1220 produce H(2)S in distinct manners: Fn0625 carries out beta-elimination of L-cysteine to produce H(2)S, pyruvate and ammonia, whereas Fn1220 catalyses the beta-replacement of L-cysteine to produce H(2)S and lanthionine, the latter of which may be used for peptidoglycan formation in F. nucleatum.

Microbial engineering of dehydro-amino acids and lanthionines in non-lantibiotic peptides.

This minireview focuses on the use of bacteria to introduce dehydroresidues and (methyl)lanthionines in (poly)peptides. It mainly describes the broad exploitation of bacteria containing lantibiotic enzymes for the engineering of these residues in a wide variety of peptides in particular in peptides unrelated to lantibiotics. Lantibiotic dehydratases dehydrate serines and threonines present in peptides preceded by a lantibiotic leader peptide thus forming dehydroalanine and dehydrobutyrine, respectively. These dehydroresidues can be coupled to cysteines thus forming (methyl)lanthionines. This coupling is catalysed by lantibiotic cyclases. The design, synthesis, and export of microbially engineered dehydroresidue and or lanthionine-containing peptides in non-lantibiotic peptides are reviewed, illustrated by some examples which demonstrate the high relevance of these special residues. This minireview is the first with special focus on the microbial engineering of nonlantibiotic peptides by exploiting lantibiotic enzymes.

Glycolysis and the tricarboxylic acid cycle are linked by alanine aminotransferase during hypoxia induced by waterlogging of Lotus japonicus.

The role of nitrogen metabolism in the survival of prolonged periods of waterlogging was investigated in highly flood-tolerant, nodulated Lotus japonicus plants. Alanine production revealed to be a critical hypoxic pathway. Alanine is the only amino acid whose biosynthesis is not inhibited by nitrogen deficiency resulting from RNA interference silencing of nodular leghemoglobin. The metabolic changes that were induced following waterlogging can be best explained by the activation of alanine metabolism in combination with the modular operation of a split tricarboxylic acid pathway. The sum result of this metabolic scenario is the accumulation of alanine and succinate and the production of extra ATP under hypoxia. The importance of alanine metabolism is discussed with respect to its ability to regulate the level of pyruvate, and this and all other changes are discussed in the context of current models concerning the regulation of plant metabolism.