Emphasis on the importance of comprehensive clinical and genetic analysis - spinal muscular atrophy combined with phenylketonuria: A case report.

RATIONALE: Both spinal muscular atrophy (SMA) and Phenylketonuria (PKU) are caused by biallelic pathogenic mutations. However, there has been no report on case who suffering from both diseases simultaneously. SMA mainly affects the motor function while PKU may have an impact on both the intelligence and motor function. But if only 1 disease is treated while neglecting the other, the treatment effect will be compromised. Here, for the first time, we report a case from China diagnosed with both these diseases and treated properly. PATIENT CONCERNS: A boy was admitted to the Children's Hospital Affiliated to Shandong University (Jinan, China) due to "limb weakness for 19 months" when he was 22 months old. Considering that the child's motor function development is delayed, we made a comprehensive examinations including inherited metabolic diseases and found a significantly increase of phenylalanine concentration in the blood which indicating PKU. Combined with his typical clinical manifestations of SMA, target capture sequencing followed by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) technologies were used for genetic confirmation. DIAGNOSES: SMA and PKU was confirmed. INTERVENTIONS: The child was treated with risdiplam and low phenylalanine formula immediately when he was diagnosed with both SMA and PKU. OUTCOMES: The child showed remarkable improvement in motor function and significant decrease of blood phenylalanine concentration after treatment. LESSONS: To our knowledge, this is the first reported case of SMA combined with PKU. This case expands our understanding of diagnosis for synchronous SMA and PKU and highlights the importance of comprehensive examinations and the utilizing of various genetic testing methods to make an accurate diagnosis of genetic diseases, which may help avoiding the progressive damage caused by certain genetic disease with insidious clinical symptoms.

Propofol rescues voltage-dependent gating of HCN1 channel epilepsy mutants.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels1 are essential for pacemaking activity and neural signalling2,3. Drugs inhibiting HCN1 are promising candidates for management of neuropathic pain4 and epileptic seizures5. The general anaesthetic propofol (2,6-di-iso-propylphenol) is a known HCN1 allosteric inhibitor6 with unknown structural basis. Here, using single-particle cryo-electron microscopy and electrophysiology, we show that propofol inhibits HCN1 by binding to a mechanistic hotspot in a groove between the S5 and S6 transmembrane helices. We found that propofol restored voltage-dependent closing in two HCN1 epilepsy-associated polymorphisms that act by destabilizing the channel closed state: M305L, located in the propofol-binding site in S5, and D401H in S6 (refs. 7,8). To understand the mechanism of propofol inhibition and restoration of voltage-gating, we tracked voltage-sensor movement in spHCN channels and found that propofol inhibition is independent of voltage-sensor conformational changes. Mutations at the homologous methionine in spHCN and an adjacent conserved phenylalanine in S6 similarly destabilize closing without disrupting voltage-sensor movements, indicating that voltage-dependent closure requires this interface intact. We propose a model for voltage-dependent gating in which propofol stabilizes coupling between the voltage sensor and pore at this conserved methionine-phenylalanine interface in HCN channels. These findings unlock potential exploitation of this site to design specific drugs targeting HCN channelopathies.

Contribution of Phe112, Ser114, and Tyr115 to Drug-Binding Selectivity in the A Variant of α1-Acid Glycoprotein.

The plasma protein α1-acid glycoprotein (AGP) primarily affects the pharmacokinetics of basic drugs. There are two AGP variants in humans, A and F1*S, exhibiting distinct drug-binding selectivity. Elucidation of the drug-binding selectivity of human AGP variants is essential for drug development and personalized drug therapy. Herein, we aimed to establish the contribution of amino acids 112 and 114 of human AGP to drug-binding selectively. Both amino acids are located in the drug-binding region and differ between the variants. Phe112/Ser114 of the A variant and its equivalent residues in the F1*S variant (Leu112/Phe114) were swapped with each other. Binding experiments were then conducted using the antiarrhythmic drug disopyramide, which selectively binds to the A variant. A significant decrease in the bound fraction was observed in each singly mutated A protein (Phe112Leu or Ser114Phe). Moreover, the bound fraction of the double A mutant (Phe112Leu/Ser114Phe) was decreased to that of wild-type F1*S. Intriguingly, the double F1*S mutant (Leu112Phe/Phe114Ser), in which residues were swapped with those of the A variant, showed only partial restoration in binding. The triple F1*S mutant (Leu112Phe/Phe114Ser/Asp115Tyr), where position 115 is thought to contribute to the difference in pocket size between variants, showed a further recovery in binding to 70% of that of wild-type A. These results were supported by thermodynamic analysis and acridine orange binding, which selectively binds the A variant. Together, these data indicate that, in addition to direct interaction with Phe112 and Ser114, the binding pocket size contributed by Tyr115 is important for the drug-binding selectivity of the A variant.

A conserved phenylalanine motif among teleost fish provides insight for improving electromagnetic perception.

Magnetoreceptive biology as a field remains relatively obscure; compared with the breadth of species believed to sense magnetic fields, it remains under-studied. Here, we present grounds for the expansion of magnetoreception studies among teleosts. We begin with the electromagnetic perceptive gene (EPG) from Kryptopterus vitreolus and expand to identify 72 teleosts with homologous proteins containing a conserved three-phenylalanine (3F) motif. Phylogenetic analysis provides insight as to how EPG may have evolved over time and indicates that certain clades may have experienced a loss of function driven by different fitness pressures. One potential factor is water type with freshwater fish significantly more likely to possess the functional motif version (FFF), and saltwater fish to have the non-functional variant (FXF). It was also revealed that when the 3F motif from the homologue of Brachyhypopomus gauderio (B.g.) is inserted into EPG-EPG(B.g.)-the response (as indicated by increased intracellular calcium) is faster. This indicates that EPG has the potential to be engineered to improve upon its response and increase its utility to be used as a controller for specific outcomes.

Transcriptomics and metabolomics reveal the mechanism of metabolites changes in Cymbidium tortisepalum var. longibracteatum colour mutation cultivars.

BACKGROUND: Foliage color is considered an important ornamental character of Cymbidium tortisepalum (C. tortisepalum), which significantly improves its horticultural and economic value. However, little is understood on the formation mechanism underlying foliage-color variations. METHODS: In this study, we applied a multi-omics approach based on transcriptomics and metabolomics, to investigate the biomolecule mechanisms of metabolites changes in C. tortisepalum colour mutation cultivars. RESULTS: A total of 508 genes were identified as differentially expressed genes (DEGs) between wild and foliage colour mutation C. tortisepalum cultivars based on transcriptomic data. KEGG enrichment of DEGs showed that genes involved in phenylalanine metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis and brassinosteroid biosynthesis were most significantly enriched. A total of 420 metabolites were identified in C. tortisepalum using UPLC-MS/MS-based approach and 115 metabolites differentially produced by the mutation cultivars were identified. KEGG enrichment indicated that the most metabolites differentially produced by the mutation cultivars were involved in glycerophospholipid metabolism, tryptophan metabolism, isoflavonoid biosynthesis, flavone and flavonol biosynthesis. Integrated analysis of the metabolomic and transcriptomic data showed that there were four significant enrichment pathways between the two cultivars, including phenylalanine metabolism, phenylpropanoid biosynthesis, flavone and flavonol biosynthesis and flavonoid biosynthesis. CONCLUSION: The results of this study revealed the mechanism of metabolites changes in C. tortisepalum foliage colour mutation cultivars, which provides a new reference for breeders to improve the foliage color of C. tortisepalum.

Comprehensive analyses of phenylalanine hydroxylase variants and phenotypic characteristics of patients in the eastern region of Türkiye.

OBJECTIVES: Phenylalanine hydroxylase (PAH) is predominantly a hepatic enzyme that catalyzes phenylalanine (Phe) into tyrosine, which is the rate-limiting step in Phe catabolism. Biallelic variants in the PAH gene cause PAH enzyme deficiency. Phenylketonuria (PKU) is an autosomal recessive disorder that causes neurologic, behavioral, and dermatological findings. PKU could be divided clinically into three types based on the blood Phe levels: classic phenylketonuria (cPKU), mild-moderate phenylketonuria (mPKU), and mild hyperphenylalaninemia (MHP). This study aimed to determine the phenotypic and genotypic characteristics of Turkish PKU patients in the eastern region of Türkiye. METHODS: Demographic characteristics, serum Phe levels, treatments, and PAH variants of 163 patients with PKU and hyperphenylalaninemia (HPA) were retrospectively evaluated. Blood Phe levels of the patients were analyzed with the high-performance liquid chromatography method. For PAH gene analysis, next-generation sequencing was performed. RESULTS: Of the 163 patients included in the study, 38 (23.3 %) had cPKU, 16 (9.8 %) had mPKU, and 109 (66.9 %) had MHP. Homozygous variants in the PAH gene were detected in 66 (40.5 %) of the patients, while compound heterozygous variants were detected in 97 (59.5 %) patients. Two novel and 35 recurrent variants in the PAH gene were detected. Of the two novel variants, one was missense (p.Phe351Leu) and the other was frameshift (p.Met276Cysfs*65). The most frequently detected variants were p.Thr380Met (18 %), p.Arg261Gln (16.8 %), and p.Ala300Ser (12.8 %). All patients with the homozygous c.1066-11G>A variant exhibited cPKU phenotype. The c.898G>T (p.Ala300Ser), c.1139C>T (p.Thr380Met), and c.1208C>T (p.Ala403Val) variants were statistically related to mild phenotype. On the other hand, c.592_613del (p.Tyr198Serfs*136), c.1028A>G (p.Tyr343Cys), and c.782G>A (p.Arg261Gln) variants were more frequently detected in the cPKU group. CONCLUSIONS: Our study, conducted with patients from the eastern region of Türkiye, demonstrates the genetic heterogeneity in the Turkish population. Simultaneously, our research contributes to genotype-phenotype correlation and expands the genotypic spectrum by identifying novel variants.

The critical role of residues Phe120 and Val161 of (2 R,3 R)­2,3­butanediol dehydrogenase from Neisseria gonorrhoeae as probed by molecular docking and site-directed mutagenesis.

NAD+-dependent (2 R,3 R)­2,3­butanediol dehydrogenase (BDH) from Neisseria gonorrhoeae (NgBDH) is a representative member of the medium-chain dehydrogenase/reductase (MDR) superfamily. To date, little information is available on the substrate binding sites and catalytic residues of BDHs from this superfamily. In this work, according to molecular docking studies, we found that conserved residues Phe120 and Val161 form strong hydrophobic interactions with both (2 R,3 R)­2,3­butanediol (RR-BD) and meso-2,3­butanediol (meso-BD) and that mutations of these residues to alanine or threonine impair substrate binding. To further evaluate the roles of these two residues, Phe120 and Val161 were mutated to alanine or threonine. Kinetic analysis revealed that, relative to those of wild type, the apparent KM values of the Phe120Ala mutant for RR-BD and meso-BD increased 36- and 369-fold, respectively; the catalytic efficiencies of this mutant with RR-BD and meso-BD decreased approximately 586- and 3528-fold, respectively; and the apparent KM values of the Val161Ala mutant for RR-BD and meso-BD increased 4- and 37-fold, respectively, the catalytic efficiencies of this mutant with RR-BD and meso-BD decreased approximately 3- and 28-fold, respectively. Additionally, the Val161Thr mutant slightly decreased catalytic efficiencies (twofold with RR-BD; 7.3-fold with meso-BD) due to an increase in KM (sixfold for RR-BD; 24-fold for meso-BD) and a slight increase (2.8-fold with RR-BD; 3.3-fold with meso-BD) in kcat. These findings validate the critical roles of Phe120 and Val161 of NgBDH in substrate binding and catalysis. Overall, the current study provides a better understanding of the substrate binding and catalysis of BDHs within the MDR superfamily.

Genetically encoded site-specific 19F unnatural amino acid incorporation in V. natriegens for in-cell NMR analysis.

Nuclear magnetic resonance (NMR) spectroscopy NMR is a well-established technique for probing protein structure, dynamics and conformational changes. Taking advantage of the high signal sensitivity and broad chemical shift range of 19F nuclei, 19F NMR has been applied to investigate protein function at atomic resolution. In this report, we extend the unnatural amino acid site-specific incorporation into V. natriegens, an alternate protein expression system. The unnatural amino acid L-4-trifluoromethylphenylalanine (tfmF) was site-specifically introduced into the mitogen-activated protein kinase MEKK3 in V. natriegens using genetically encoded technology, which will be an extensive method for in-cell protein structure and dynamic investigation.

Cyclo-diphenylalanine production in Aspergillus nidulans through stepwise metabolic engineering.

Cyclo-diphenylalanine (cFF) is a symmetrical aromatic diketopiperazine (DKP) found wide-spread in microbes, plants, and resulting food products. As different bioactivities continue being discovered and relevant food and pharmaceutical applications gradually emerge for cFF, there is a growing need for establishing convenient and efficient methods to access this type of compound. Here, we present a robust cFF production system which entailed stepwise engineering of the filamentous fungal strain Aspergillus nidulans A1145 as a heterologous expression host. We first established a preliminary cFF producing strain by introducing the heterologous nonribosomal peptide synthetase (NRPS) gene penP1 to A. nidulans A1145. Key metabolic pathways involving shikimate and aromatic amino acid biosynthetic support were then engineered through a combination of gene deletions of competitive pathway steps, over-expressing feedback-insensitive enzymes in phenylalanine biosynthesis, and introducing a phosphoketolase-based pathway, which diverted glycolytic flux toward the formation of erythrose 4-phosphate (E4P). Through the stepwise engineering of A. nidulans A1145 outlined above, involving both heterologous pathway addition and native pathway metabolic engineering, we were able to produce cFF with titers reaching 611 mg/L in shake flask culture and 2.5 g/L in bench-scale fed-batch bioreactor culture. Our study establishes a production platform for cFF biosynthesis and successfully demonstrates engineering of phenylalanine derived diketopiperazines in a filamentous fungal host.

Crucial Residue for Tuning Thermal Relaxation Kinetics in the Biliverdin-binding Cyanobacteriochrome Photoreceptor Revealed by Site-saturation Mutagenesis.

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors distantly related to the phytochromes sensing red and far-red light reversibly. Only the cGMP phosphodiesterase/Adenylate cyclase/FhlA (GAF) domain is needed for chromophore incorporation and proper photoconversion. The CBCR GAF domains covalently ligate linear tetrapyrrole chromophores and show reversible photoconversion between two light-absorbing states. In most cases, the two light-absorbing states are stable under dark conditions, but in some cases, the photoproduct state undergoes thermal relaxation back to the dark-adapted state during thermal relaxation. In this study, we examined the engineered CBCR GAF domain, AnPixJg2_BV4. AnPixJg2_BV4 covalently binds biliverdin IX-alpha (BV) and shows reversible photoconversion between a far-red-absorbing Pfr dark-adapted state and an orange-absorbing Po photoproduct state. Because the BV is an intrinsic chromophore of mammalian cells and absorbs far-red light penetrating into deep tissues, BV-binding CBCR molecules are useful for the development of optogenetic and bioimaging tools used in mammals. To obtain a better developmental platform molecule, we performed site-saturation random mutagenesis on the Phe319 position. We succeeded in obtaining variant molecules with higher chromophore-binding efficiency and higher molar extinction coefficient. Furthermore, we observed a wide variation in thermal relaxation kinetics, with an 81-fold difference between the slowest and fastest rates. Both molecules with relatively slow and fast thermal relaxation would be advantageous for optogenetic control.

Single amino acid mutation of nectin-1 provides remarkable resistance against lethal pseudorabies virus infection in mice.

An approach to genetically engineered resistance to pseudorabies virus (PRV) infection was examined by using a mouse model with defined point mutation in primary receptor for alphaherpesviruses, nectin-1, by the CRISPR/Cas9 system. It has become clear that phenylalanine at position 129 of nectin-1 is important for binding to viral glycoprotein D (gD), and mutation of phenylalanine 129 to alanine (F129A) prevents nectin-1 binding to gD and virus entry in vitro. Here, to assess the antiviral potential of the single amino acid mutation of nectin-1, F129A, in vivo, we generated genome-edited mutant mouse lines; F129A and 135 knockout (KO). The latter, 135 KO used as a nectin-1 knockout line for comparison, expresses a carboxy-terminal deleted polypeptide consisting of 135 amino acids without phenylalanine 129. In the challenge with 10 LD50 PRV via intranasal route, perfect protection of disease onset was induced by expression of the mutation of nectin-1, F129A (survival rate: 100% in F129A and 135 KO versus 0% in wild type mice). Neither viral DNA/antigens nor pathological changes were detected in F129A, suggesting that viral entry was prevented at the primary site in natural infection. In the challenge with 50 LD50 PRV, lower but still strong protective effect against disease onset was observed (survival rate: 57% in F129A and 75% in 135 KO versus 0% in wild type mice). The present results indicate that single amino acid mutation of nectin-1 F129A provides significant resistance against lethal pseudorabies.

Systems Biology and Inborn Error of Metabolism: Analytical Strategy in Investigating Different Biochemical/Genetic Parameters.

Inborn errors of metabolism (IEM) are a group of about 500 rare genetic diseases with large diversity and complexity due to number of metabolic pathways involved in. Establishing a correct diagnosis and identifying the specific clinical phenotype is consequently a difficult task. However, an inclusive diagnosis able in capturing the different clinical phenotypes is mandatory for successful treatment. However, in contrast with Garrod's basic assumption "one-gene one-disease," no "simple" correlation between genotype-phenotype can be vindicated in IEMs. An illustrative example of IEM is Phenylketonuria (PKU), an autosomal recessive inborn error of L-phenylalanine (Phe) metabolism, ascribed to variants of the phenylalanine hydroxylase (PAH) gene encoding for the enzyme complex phenylalanine-hydroxylase. Blood values of Phe allow classifying PKU into different clinical phenotypes, albeit the participation of other genetic/biochemical pathways in the pathogenetic mechanisms remains elusive. Indeed, it has been shown that the most serious complications, such as cognitive impairment, are not only related to the gene dysfunction but also to the patient's background and the participation of several nongenetic factors.Therefore, a Systems Biology-based strategy is required in addressing IEM complexity, and in identifying the interplay between different pathways in shaping the clinical phenotype. Such an approach should entail the concerted investigation of genomic, transcriptomics, proteomics, metabolomics profiles altogether with phenylalanine and amino acids metabolism. Noticeably, this "omic" perspective could be instrumental in planning personalized treatment, tailored accordingly to the disease profile and prognosis.

Impact of N221S missense mutation in human ribonucleotide reductase small subunit b on mitochondrial DNA depletion syndrome.

The impact of N221S mutation in hRRM2B gene, which encodes the small subunit of human ribonucleotide reductase (RNR), on RNR activity and the pathogenesis of mitochondrial DNA depletion syndrome (MDDS) was investigated. Our results demonstrate that N221 mutations significantly reduce RNR activity, suggesting its role in the development of MDDS. We proposed an allosteric regulation pathway involving a chain of three phenylalanine residues on the αE helix of RNR small subunit ß. This pathway connects the C-terminal loop of ß2, transfers the activation signal from the large catalytic subunit α to ß active site, and controls access of oxygen for radical generation. N221 is near this pathway and likely plays a role in regulating RNR activity. Mutagenesis studies on residues involved in the phenylalanine chain and the regulation pathway were conducted to confirm our proposed mechanism. We also performed molecular dynamic simulation and protein contact network analysis to support our findings. This study sheds new light on RNR small subunit regulation and provides insight on the pathogenesis of MDDS.

Efficient in vivo prime editing corrects the most frequent phenylketonuria variant, associated with high unmet medical need.

The c.1222C>T (p.Arg408Trp) variant in the phenylalanine hydroxylase gene (PAH) is the most frequent cause of phenylketonuria (PKU), the most common inborn error of metabolism. This autosomal-recessive disorder is characterized by accumulation of blood phenylalanine (Phe) to neurotoxic levels. Using real-world data, we observed that despite dietary and medical interventions, most PKU individuals harboring at least one c.1222C>T variant experience chronic, severe Phe elevations and do not comply with Phe monitoring guidelines. Motivated by these findings, we generated an edited c.1222C>T hepatocyte cell line and humanized c.1222C>T mouse models, with which we demonstrated efficient in vitro and in vivo correction of the variant with prime editing. Delivery via adeno-associated viral (AAV) vectors reproducibly achieved complete normalization of blood Phe levels in PKU mice, with up to 52% whole-liver corrective PAH editing. These studies validate a strategy involving prime editing as a potential treatment for a large proportion of individuals with PKU.

Use of non-canonical amino acids in genetic code expansion-based therapeutics: Effects on mouse gut microbiota.

Vaccines and cell therapeutics based on genetic code expansion are emerging. A crucial step in these therapeutic technologies is the oral administration of non-canonical amino acids (ncAAs) to control pathogen growth and therapeutic protein levels in vivo. Investigating the toxicity effects of ncAAs can help identify more suitable candidates for developing genetic code expansion-based vaccines and cell therapeutics. In this study, we determined the effects of three ncAAs, namely, 4-acetyl-phenylalanine (pAcF), 4-iodo-phenylalanine (pIoF), and 4-methoxy-phenylalanine (pMeoF), commonly used in genetic code expansion-based vaccines and cell therapeutics, on the main organs, serum biochemical parameters, and gut microbiota in mice. We observed that pIoF and pMeoF significantly altered serum biochemical parameters to some extent. Moreover, the alterations in the mouse gut microbial composition were considerably greater after the oral administration of pIoF and pMeoF than after that of pAcF, compared with that in the control mice. These findings suggest that pAcF is more suitable than pIoF and pMeoF for application in genetic code expansion-based vaccines and cell therapeutics as it disturbs the physiological and gut microecological balance in mice to a lesser extent.

Phenylalanine-tRNA aminoacylation is compromised by ALS/FTD-associated C9orf72 C4G2 repeat RNA.

The expanded hexanucleotide GGGGCC repeat mutation in the C9orf72 gene is the main genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Under one disease mechanism, sense and antisense transcripts of the repeat are predicted to bind various RNA-binding proteins, compromise their function and cause cytotoxicity. Here we identify phenylalanine-tRNA synthetase (FARS) subunit alpha (FARSA) as the main interactor of the CCCCGG antisense repeat RNA in cytosol. The aminoacylation of tRNAPhe by FARS is inhibited by antisense RNA, leading to decreased levels of charged tRNAPhe. Remarkably, this is associated with global reduction of phenylalanine incorporation in the proteome and decrease in expression of phenylalanine-rich proteins in cellular models and patient tissues. In conclusion, this study reveals functional inhibition of FARSA in the presence of antisense RNA repeats. Compromised aminoacylation of tRNA could lead to impairments in protein synthesis and further contribute to C9orf72 mutation-associated pathology.

Point mutations including a novel Pro-197-Phe mutation confer cross-resistance to acetolactate synthase (ALS) inhibiting herbicides in Lactuca serriola in Australia.

BACKGROUND: Control of prickly lettuce has become increasingly difficult for lentil growers in southern Australia because of widespread resistance to common herbicides, a lack of alternative herbicide options and the prolific production of highly mobile seed. This study aimed to quantify acetolactate synthase (ALS)-inhibiting herbicide resistance in the Mid North (MN) and Yorke Peninsula (YP) of South Australia, characterize the resistance mutations present and investigate population structure and gene flow in this species. RESULTS: Resistance was identified in all populations tested, with average survival of 92% to chlorsulfuron and 95% to imazamox + imazapyr. Five different amino acid substitutions were identified at proline 197 of the ALS gene. There was no significant difference in the median lethal dose (LD50 ) between plants with these five different substitutions when treated with metsulfuron-methyl; however, the imidazolinone resistance level was higher in plants with a phenylalanine substitution and lower in plants with a serine. Population structure based on 701 single nucleotide polymorphisms and 271 individuals provided evidence for both independent evolution of the same mutation in different populations, as well as frequent short- to medium-distance dispersal accompanied by occasional long-distance dispersal events. The overall inbreeding coefficient (FIS ) was calculated at 0.5174, indicating an intermediate level of outcrossing despite the cross-pollination experiment showing only low outcrossing. In the structure analyses, most individuals from YP were assigned to a single cluster, whereas most individuals from MN were assigned 50% to each of two clusters, indicating some genetic differences between these two regions, but also evidence for dispersal between them. CONCLUSIONS: Use of imidazolinone herbicides has selected for mutations conferring higher levels of resistance, such as the Pro-197-Phe mutation, and resulted in further spread of resistance in this species. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Phenylketonuria (PKU) Urinary Metabolomic Phenotype Is Defined by Genotype and Metabolite Imbalance: Results in 51 Early Treated Patients Using Ex Vivo 1H-NMR Analysis.

Phenylketonuria (PKU) is a rare metabolic disorder caused by mutations in the phenylalanine hydroxylase gene. Depending on the severity of the genetic mutation, medical treatment, and patient dietary management, elevated phenylalanine (Phe) may occur in blood and brain tissues. Research has recently shown that high Phe not only impacts the central nervous system, but also other organ systems (e.g., heart and microbiome). This study used ex vivo proton nuclear magnetic resonance (1H-NMR) analysis of urine samples from PKU patients (mean 14.9 ± 9.2 years, n = 51) to identify the impact of elevated blood Phe and PKU treatment on metabolic profiles. Our results found that 24 out of 98 urinary metabolites showed a significant difference (p < 0.05) for PKU patients compared to age-matched healthy controls (n = 51) based on an analysis of urinary metabolome. These altered urinary metabolites were related to Phe metabolism, dysbiosis, creatine synthesis or intake, the tricarboxylic acid (TCA) cycle, end products of nicotinamide-adenine dinucleotide degradation, and metabolites associated with a low Phe diet. There was an excellent correlation between the metabolome and genotype of PKU patients and healthy controls of 96.7% in a confusion matrix model. Metabolomic investigations may contribute to a better understanding of PKU pathophysiology.

Laboratory evolution of Synechocystis sp. PCC 6803 for phenylpropanoid production.

Cyanobacteria are promising as a biotechnological platform for production of various industrially relevant compounds, including aromatic amino acids and their derivatives, phenylpropanoids. In this study, we have generated phenylalanine resistant mutant strains (PRMs) of the unicellular cyanobacterium Synechocystis sp. PCC 6803, by laboratory evolution under the selective pressure of phenylalanine, which inhibits the growth of wild type Synechocystis. The new strains of Synechocystis were tested for their ability to secrete phenylalanine in the growth medium during cultivation in shake flasks as well as in a high-density cultivation (HDC) system. All PRM strains secreted phenylalanine into the culture medium, with one of the mutants, PRM8, demonstrating the highest specific production of 24.9 ± 7 mg L-1·OD750-1 or 610 ± 196 mg L-1 phenylalanine after four days of growth in HDC. We further overexpressed phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) in the mutant strains in order to determine the potential of PRMs for production of trans-cinnamic acid (tCA) and para-coumaric acid (pCou), the first intermediates of the plant phenylpropanoid pathway. Productivities of these compounds were found to be lower in the PRMs compared to respective control strains, except for PRM8 under HDC conditions. The PRM8 background strain in combination with PAL or TAL expression demonstrated a specific production of 52.7 ± 15 mg L-1·OD750-1tCA and 47.1 ± 7 mg L-1·OD750-1pCou, respectively, with a volumetric titer reaching above 1 g L-1 for both products after four days of HDC cultivation. The genomes of PRMs were sequenced in order to identify which mutations caused the phenotype. Interestingly, all of the PRMs contained at least one mutation in their ccmA gene, which encodes DAHP synthase, the first enzyme of the pathway for aromatic amino acids biosynthesis. Altogether, we demonstrate that the combination of laboratory-evolved mutants and targeted metabolic engineering can be a powerful tool in cyanobacterial strain development.

Avoidance of the use of tryptophan in buried chromosomal proteins as a mechanism for reducing photo/oxidative damage to genomes.

In cells that are exposed to terrestrial sunlight, the indole moiety in the side chain of tryptophan (Trp) can suffer photo/oxidative damage (POD) by reactive oxygen species (ROS) and/or ultraviolet light (UV-B). Trp is oxidized to produce N-formylkynurenine (NFK), a UV-A-responsive photosensitizer that further degenerates into photosensitizers capable of generating ROS through exposure to visible light. Thus, Trp-containing proteins function as both victims, and perpetrators, of POD if they are not rapidly replaced through protein turnover. The literature indicates that protein turnover and DNA repair occur poorly in chromosomal interiors. We contend, therefore, that basic chromosomal proteins (BCPs) that are enveloped by DNA should have evolved to lack Trp residues in their amino acid sequences, since these could otherwise function as 'Trojan horse-type' DNA-damaging agents. Our global analyses of protein sequences demonstrates that BCPs consistently lack Trp residues, although DNA-binding proteins in general do not display such a lack. We employ HU-B (a wild-type, Trp-lacking bacterial BCP) and HU-B F47W (a mutant, Trp-containing form of the same bacterial BCP) to demonstrate that the possession of Trp is deleterious to BCPs and associated chromosomal DNA. Basically, we show that UV-B and UV-A (a) cause no POD in HU-B, but cause extensive POD in HU-B F47W (in vitro), as well as (b) only nominal DNA damage in bacteria expressing HU-B, but extensive DNA damage in bacteria expressing F47W HU-B (in vivo). Our results suggest that Trp-lacking BCPs could have evolved to reduce scope for protein-facilitated, sunlight-mediated damage of DNA by UV-A and visible light, within chromosomal interiors that are poorly serviced by protein turnover and DNA repair machinery.