Guanidine production by plant homoarginine-6-hydroxylases.

Metabolism and biological functions of the nitrogen-rich compound guanidine have long been neglected. The discovery of four classes of guanidine-sensing riboswitches and two pathways for guanidine degradation in bacteria hint at widespread sources of unconjugated guanidine in nature. So far, only three enzymes from a narrow range of bacteria and fungi have been shown to produce guanidine, with the ethylene-forming enzyme (EFE) as the most prominent example. Here, we show that a related class of Fe2+- and 2-oxoglutarate-dependent dioxygenases (2-ODD-C23) highly conserved among plants and algae catalyze the hydroxylation of homoarginine at the C6-position. Spontaneous decay of 6-hydroxyhomoarginine yields guanidine and 2-aminoadipate-6-semialdehyde. The latter can be reduced to pipecolate by pyrroline-5-carboxylate reductase but more likely is oxidized to aminoadipate by aldehyde dehydrogenase ALDH7B in vivo. Arabidopsis has three 2-ODD-C23 isoforms, among which Din11 is unusual because it also accepted arginine as substrate, which was not the case for the other 2-ODD-C23 isoforms from Arabidopsis or other plants. In contrast to EFE, none of the three Arabidopsis enzymes produced ethylene. Guanidine contents were typically between 10 and 20 nmol*(g fresh weight)-1 in Arabidopsis but increased to 100 or 300 nmol*(g fresh weight)-1 after homoarginine feeding or treatment with Din11-inducing methyljasmonate, respectively. In 2-ODD-C23 triple mutants, the guanidine content was strongly reduced, whereas it increased in overexpression plants. We discuss the implications of the finding of widespread guanidine-producing enzymes in photosynthetic eukaryotes as a so far underestimated branch of the bio-geochemical nitrogen cycle and propose possible functions of natural guanidine production.

Identification of a homoarginine biosynthetic gene from a microcystin biosynthetic pathway in Fischerella sp. PCC 9339.

Microcystins (MCs) are a family of chemically diverse toxins produced by numerous distantly related cyanobacteria. They are potent inhibitors of eukaryotic protein phosphatases 1 and 2A and are responsible for the toxicosis and death of wild and domestic animals around the world. Microcystins are synthesized on large enzyme complexes comprised of peptide synthetases, polyketide synthases, and additional modifying enzymes. Bioinformatic analysis identified the presence of an additional uncharacterized enzyme in the microcystin (mcy) biosynthetic gene cluster in Fischerella sp. PCC 9339, which we named McyK, that lacked a clearly defined role in the biosynthesis of microcystin. Further bioinformatic analysis suggested that McyK belongs to the inosamine-phosphate amidinotransferase family and could be involved in synthesizing homo amino acids. Quadrupole time-of-flight tandem mass spectrometry (Q-TOFMS/MS) analysis confirmed that Fischerella sp. PCC 9339 produces MC-Leucine2-Homoarginine4(MC-LHar) and [Aspartic acid3]MC-Leucine2-Homoarginine4 ([Asp3]MC-LHar) as the dominant chemical variants. We hypothesized that the McyK enzyme might be involved in the production of microcystin variants containing homoarginine (Har) in the strain. Heterologous expression of a codon-optimized mcyK gene in Escherichia coli confirmed that McyK is responsible for the synthesis of L-Har. These results confirm the production of MC-LHar, a novel microcystin chemical variant [Asp3]MC-LHar, and a new microcystin biosynthetic enzyme involved in supply of the rare homo-amino acid Har to the microcystin biosynthetic pathway in Fischerella sp. PCC 9339. This study provides new insights into the logic underpinning the biosynthesis of microcystin chemical variants and broadens our knowledge of structural diversity of the microcystin family of toxins.

Insufficiency of plasmatic arginine/homoarginine during the initial postoperative phase among patients with tumors affecting the medulla oblongata heightens the likelihood of neurogenic pulmonary oedema following surgery.

BACKGROUND: This prospective clinical study aims to investigate the fluctuations of neurotransmitters in peripheral venous blood during the perioperative period and to identify independent predictors for postoperative neurogenic pulmonary oedema (NPE) in patients with medulla oblongata-involved tumours. MATERIALS AND METHODS: Peripheral venous blood samples of the enroled patients at seven perioperative time points, as well as their medical records and radiologic data were collected. High-performance liquid chromatography-tandem mass spectrometry was utilized to detect the concentrations of 39 neurotransmitters in these samples. The study applied univariate and multivariate generalized estimating equation (GEE) logistic regression analyses to explore independent predictors of postoperative NPE, and one-way repeated-measures ANOVA to compare the concentrations of the same neurotransmitter at different perioperative time points. RESULTS: The study included 36 patients with medulla oblongata-involved tumours from January to December 2019, and found that 13.9% of them experienced postoperative NPE. The absence of intraoperative use of sevoflurane ( P =0.008), decreased concentrations of arginine ( P =0.026) and homoarginine ( P =0.030), and prolonged postoperative tracheal extubation ( P <0.001) were identified as independent risk factors for postoperative NPE in medulla oblongata-involved tumour patients. Pairwise comparison analysis revealed that the perioperative decreases in arginine and homoarginine concentrations mainly occurred within the postoperative 8 h. CONCLUSION: This study demonstrates that NPE is not uncommon in patients with medulla oblongata-involved tumours. The absence of intraoperative use of sevoflurane, decreased concentrations of plasmatic arginine and homoarginine, and prolonged postoperative tracheal extubation are independent predictors of postoperative NPE. These two neurotransmitters' concentrations dropped mainly within the early postoperative hours and could serve as potential early warning indicators of postoperative NPE in clinical practice.

Systemic Effects of Homoarginine Supplementation on Arginine Metabolizing Enzymes in Rats with Heart Failure with Preserved Ejection Fraction.

A restoration of low homoarginine (hArg) levels in obese ZSF1 rats (O-ZSF1) before (S1-ZSF1) and after (S2-ZSF1) the manifestation of heart failure with preserved ejection fraction (HFpEF) did not affect the worsening of cardiac HFpEF characteristics. Here, potential regulation of key enzymes of arginine metabolism in other organs was analyzed. Arginase 2 (ARG2) was reduced >35% in the kidney and small intestine of hArg-supplemented rats compared to O-ZSF1. Glycine amidinotransferase (GATM) was 29% upregulated in the kidneys of S1-ZSF1. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) levels were reduced >50% in the livers of O-ZSF1 but restored in S2-ZSF1 compared to healthy rats (L-ZSF1). In the skeletal muscle, iNOS was lower in O-ZSF1 and further decreased in S1-ZSF1 and S2-ZSF1 compared to L-ZSF1. iNOS levels were lower in the liver of the S2-ZSF1 group but higher in the kidneys of S1-ZSF1 compared to L-ZSF1. Supplementation with hArg in an in vivo HFpEF model resulted in the inhibition of renal ARG2 and an increase in GATM expression. This supplementation might contribute to the stabilization of intestinal iNOS and ARG2 imbalances, thereby enhancing barrier function. Additionally, it may offer protective effects in skeletal muscle by downregulating iNOS. In the conceptualization of hArg supplementation studies, the current disease progression stage as well as organ-specific enzyme regulation should be considered.

Structure-activity study of oncocin: On-resin guanidinylation and incorporation of homoarginine, 4-hydroxyproline or 4,4-difluoroproline residues.

Antimicrobial peptides (AMPs) often display guanidinium functionalities, and hence robust synthetic procedures are needed to facilitate access to analogues with unnatural homologues of arginine (Arg = R). Initially, a resin-bound Arg/Pro-rich fluoren-9-yl-methyloxycarbonyl-protected fragment (Fmoc-RPRPPR) of the AMP oncocin (i.e., VDKPPYLPRPRPPRRIYNR-NH2) was employed in a comparative on-resin assessment of commercial guanidinylation reagents head-to-head with the recently studied bis-Boc-protected triazole-based reagent, 1H-triazole-1-[N,N'-bis(tert-butoxycarbonyl)]-carboxamidine, which was synthesized by a chromatography-free procedure. This reagent was found to enable quantitative conversion in solid-phase peptide synthesis (SPPS) of peptides displaying homoarginine (Har) residues and/or an N-terminal guanidinium group. SPPS was used to obtain analogues of the 18-mer oncocin with single as well as multiple Arg → Har modifications. In addition, the effect of replacement of proline (Pro) residues in oncocin was explored by incorporating single or multiple trans-4-hydroxy-l-proline (Hyp) or 4,4-difluoro-l-proline (Dfp) residues, which both affected hydrophobicity. The resulting peptide library was tested against both Gram-negative and Gram-positive bacteria. Analysis of the minimal inhibitory concentrations (MICs) showed that analogues, displaying modifications at positions 4, 5 and 12 (originally Pro residues), had retained or slightly improved antimicrobial activity. Next, an oncocin analogue with two stabilizing l-Arg → d-Arg replacements in the C-terminal part was further modified by triple-replacement of Pro by either Dfp or Hyp in positions 4, 5, and 12. The resulting analogue displaying three Pro → Dfp modifications proved to possess the best activity profile: MICs of 1-2 µg/mL against E. coli and Klebsiella pneumoniae, less than 1% hemolysis at 800 µg/mL, and an IC50 above 1280 µg/mL in HepG2 cells. Thus, incorporation of bis-fluorinated Pro residues appears to constitute a novel tool in structure-activity studies aimed at optimization of Pro-rich AMPs.

Effect of Spacer Length Modification of the Cationic Side Chain on the Energetics of Antimicrobial Peptide Binding to Membrane-Mimetic Bilayers.

Understanding the mechanism of action of the antimicrobial peptide (AMP) in terms of its structure and energetics is the key to designing new potent and selective AMPs. Recently, we reported a membranolytic 14-residue-long lysine-rich cationic antimicrobial peptide (LL-14: NH3+-LKWLKKLLKWLKKL-CONH2) against Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus, which is limited by cytotoxicity and expected to undergo facile protease degradation. Aliphatic side-chain-length modification of the cationic amino-acid residues (Lys and Arg) is a popular strategy for designing protease-resistant AMPs. However, the effect of the peptide side-chain length modifications on the membrane binding affinity and its relation to the atomic structure remain an unsolved problem. We report computer simulations that quantitatively calculated the difference in peptide binding affinity to membrane-mimetic-bilayer models (bacterial: 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)/1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) bilayer and mammalian: 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer) upon decreasing or increasing the spacer length of the cationic lysine residues of LL-14 (as well as their arginine analogues). We show that the peptide/bilayer interaction energetics varies drastically in response to spacer length modification. The strength of peptide discrimination depends strongly on the nature of the bilayer (bacterial or mammalian mimetic model). An increase in the lysine spacer length by one carbon (i.e., homolysine analogue of LL-14) is weakly/strongly disfavored by the bacterial/mammalian-membrane-mimetic bilayer. Recently, we have demonstrated an excellent correlation between the antimicrobial activity of the membranolytic cationic peptides and their binding affinity to membrane-mimetic-bilayer models. Thus, the homolysine analogue of LL-14 is a promising noncytotoxic AMP with conserved activity. On the other hand, homoarginine analogue (arginine spacer length increment by a single carbon) was preferred by both the bacteria and the mammalian mimetic bilayers and displayed the strongest affinity for the former among the peptides studied in this work. Thus, the promising most potent homoarginine analogue is likely to be cytotoxic. Shortening the Lys/Arg side chain to a three-carbon spacer (Dab/Agb) improves the binding affinity to bacterial and mammalian-membrane-mimetic bilayers. Arginine and arginine-derivative peptides exhibited stronger binding affinity to the bilayers relative to the lysine analogue. The results provide a plausible explanation to the previous experimental observations, viz., superior antimicrobial activity of the arginine peptides relative to Lys peptides and the improvement of antimicrobial activity upon substitution of Lys with Dab in the cationic peptides. The simulations revealed that the small change in the peptide hydrophobicity by Lys/Arg spacer length modification could drastically alter the energetics of peptide/bilayer binding by fine-tuning the electrostatic interactions. The energetics underlying the peptide selectivity by simple membrane-mimetic bilayer models may be beneficial for designing new selective and protease-resistant AMPs.

Diurnal Variation of L-Arginine and the Cardiovascular Risk Markers Asymmetric and Symmetric Dimethylarginine and Homoarginine in Rotating Night Shift Workers and Controls.

Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) interfere with nitric oxide (NO) formation from L-arginine via different mechanisms. ADMA is a biomarker of cardiovascular disease and mortality, whilst SDMA is a biomarker of mortality after ischemic stroke. Homoarginine, another L-arginine-derived amino acid, is associated with stroke and congestive heart failure. Acute ischemic events like myocardial infarction show a time-of-day variation in the timing of their onset, as do NO-mediated vascular function and blood pressure. We studied whether the plasma concentrations of L-arginine-related amino acid metabolites show diurnal variation in a clinical study comparing 12 non-night shift workers with 60 rotating night shift workers. The plasma concentrations of L-arginine-related biomarkers, melatonin, and cortisol were measured every 3 h during a 24-h period. In addition, 24-h blood pressure recordings were performed. In non-night shift workers, L-arginine and homoarginine plasma concentrations showed diurnal variation with a 12-h period, which were both attenuated in night shift workers. ADMA and SDMA showed a 24-h rhythmicity with no significant differences in phase between night shift and non-night shift workers. The plasma profiles of melatonin and cortisol were not significantly different between both groups, suggesting that the rotating night shift work does not have a major influence on central suprachiasmatic nuclei clock timing. In addition, systolic and diastolic blood pressure patterns were similar between both groups. Our data show diurnal variation of dimethylarginines with the timing of their acrophases corresponding to the published timing of the peak incidence of cardiac ischemic events.

Developmental dynamics of homoarginine, ADMA and SDMA plasma levels from birth to adolescence.

Guanidino compounds such as dimethylarginines (SDMA, ADMA) and L-homoarginine ((L-)hArg) can interfere with bioavailability and function of the main NO-donor L-arginine (L-Arg). High ADMA and SDMA but low L-hArg concentrations have been associated with cardio- and cerebrovascular events and mortality in adults. The role of guanidino compounds in paediatric patients remains less clear. We, therefore, compared guanidino compound levels in plasma samples of 57 individuals with chronic kidney disease (CKD) and 141 individuals without CKD from the age of 0 to 17 years, including patients with different comorbidities by correlation and regression analyses. We found highest hArg, SDMA and ADMA concentrations in neonates (Kruskal-Wallis, p < 0.001 for all). From the age of 1 year on, hArg levels increased, whereas SDMA und ADMA levels further decreased in children. SDMA and ADMA are higher in children with CKD independent of GFR (mean factor 1.92 and 1.38, respectively, p < 0.001 for both), and SDMA is strongly correlated with creatinine concentration in children with CKD (Spearman's rho 0.74, p < 0.001). We provide guanidino compound levels in a large sample covering all paediatric age groups for the first time. Our data can be used to assess the role of guanidino compounds such as hArg in disease states, i.e. cerebro- and cardiovascular disorders in childhood and adolescence.

Plasma metabolomic signatures of dual decline in memory and gait in older adults.

Older adults experiencing dual decline in memory and gait have greater dementia risk than those with memory or gait decline only, but mechanisms are unknown. Dual decline may indicate specific pathophysiological pathways to dementia which can be reflected by circulating metabolites. We compared longitudinal changes in plasma metabolite biomarkers of older adults with and without dual decline in the Baltimore Longitudinal Study of Aging (BLSA). Participants were grouped into 4 phenotypes based on annual rates of decline in verbal memory and gait speed: no decline in memory or gait, memory decline only, gait decline only, and dual decline. Repeated measures of plasma metabolomics were measured by biocrates p500 kit during the same time of memory and gait assessments. In BLSA, 18 metabolites differed across groups (q-value < 0.05). Metabolites differentially abundant were enriched for lysophosphatidylcholines (lysoPC C18:0,C16:0,C17:0,C18:1,C18:2), ceramides (d18:2/24:0,d16:1/24:0,d16:1/23:0), and amino acids (glycine) classes. Compared to no decline, the dual decline group showed greater declines in lysoPC C18:0, homoarginine synthesis, and the metabolite module containing mostly triglycerides, and showed a greater increase in indoleamine 2,3-dioxygenase (IDO) activity. Metabolites distinguishing dual decline and no decline groups were implicated in metabolic pathways of the aminoacyl-tRNA biosynthesis, valine, leucine and isoleucine biosynthesis, histidine metabolism, and sphingolipid metabolism. Older adults with dual decline exhibit the most extensive alterations in metabolic profiling of lysoPCs, ceramides, IDO activity, and homoarginine synthesis. Alterations in these metabolites may indicate mitochondrial dysfunction, compromised immunity, and elevated burden of cardiovascular and kidney pathology.

The relationship between homoarginine and liver biomarkers: a combination of epidemiological and clinical studies.

Homoarginine (hArg) is a non-essential cationic amino acid which inhibits hepatic alkaline phosphatases to exert inhibitory effects on bile secretion by targeting intrahepatic biliary epithelium. We analyzed (1) the relationship between hArg and liver biomarkers in two large population-based studies and (2) the impact of hArg supplementation on liver biomarkers. We assessed the relationship between alanine transaminase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), alkaline phosphatases (AP), albumin, total bilirubin, cholinesterase, Quick's value, liver fat, and Model for End-stage Liver Disease (MELD) and hArg in appropriately adjusted linear regression models. We analyzed the effect of L-hArg supplemention (125 mg L-hArg daily for 4 weeks) on these liver biomarkers. We included 7638 individuals (men: 3705; premenopausal women: 1866, postmenopausal women: 2067). We found positive associations for hArg and ALT (ß 0.38 µkatal/L 95% confidence interval (CI): 0.29; 0.48), AST (ß 0.29 µkatal/L 95% CI 0.17; 0.41), GGT (ß 0.033 µkatal/L 95% CI 0.014; 0.053), Fib-4 score (ß 0.08 95% CI 0.03; 0.13), liver fat content (ß 0.016% 95% CI 0.006; 0.026), albumin (ß 0.030 g/L 95% CI 0.019; 0.040), and cholinesterase (ß 0.003 µkatal/L 95% CI 0.002; 0.004) in males. In premenopausal women hArg was positively related with liver fat content (ß 0.047% 95%CI 0.013; 0.080) and inversely with albumin (ß - 0.057 g/L 95% CI - 0.073; - 0.041). In postmenopausal women hARG was positively associated with AST (ß 0.26 µkatal/L 95% CI 0.11; 0.42). hArg supplementation did not affect liver biomarkers. We summarize that hArg may be a marker of liver dysfunction and should be explored further.