The Agonistic Activity of the Human Epidermal Growth Factor is Reduced by the D46G Substitution.

BACKGROUND: Resistance to anti-tumor agents targeting the epidermal growth factor receptor (EGFR) reduces treatment response and requires the development of novel EGFR antagonists. Mutant epidermal growth factor (EGF) forms with reduced agonistic activity could be promising agents in cancer treatment. METHODS: EGF D46G affinity to EGFR domain III was assessed with affinity chromatography. EGF D46G acute toxicity in Af albino mice at 320 and 3200 µg/kg subcutaneous doses was evaluated. EGF D46G activity in human epidermoid carcinoma cells at 10 ng/mL concentration in serum-free medium and in subcutaneous Ehrlich ascites carcinoma mice model at 320 µg/kg dose was studied. RESULTS: The D46G substitution decreases the thermal stability of EGF complexes with EGFR domain III by decreasing the ability of the C-terminus to be released from the intermolecular ß- sheet. However, with remaining binding sites for EGFR domain I, EGF D46G effectively competes with other EGF-like growth factors for binding to EGFR and does not demonstrate toxic effects in mice. EGF D46G inhibits the proliferation of human epidermoid carcinoma cells compared to native EGF. A single subcutaneous administration of EGF D46G along with Ehrlich carcinoma cells injection inhibits the proliferation of these cells and delays tumor formation for up to seven days. CONCLUSION: EGF D46G can be defined as a partial EGFR agonist as this mutant form demonstrates reduced agonistic activity compared to native EGF. The study emphasizes the role of the EGF C-terminus in establishing interactions with EGFR domain III, which are necessary for EGFR activation and subsequent proliferation of cells.

Recognition of the HLA-DRB3*03:65 allele in a Chinese individual.

We report a novel HLA-DRB3*03 allele, now named DRB3*03:65, identified by next-generation sequencing.

QMix: An Efficient Program to Automatically Estimate Multi-Matrix Mixture Models for Amino Acid Substitution Process.

The single-matrix amino acid (AA) substitution models are widely used in phylogenetic analyses; however, they are unable to properly model the heterogeneity of AA substitution rates among sites. The multi-matrix mixture models can handle the site rate heterogeneity and outperform the single-matrix models. Estimating multi-matrix mixture models is a complex process and no computer program is available for this task. In this study, we implemented a computer program of the so-called QMix based on the algorithm of LG4X and LG4M with several enhancements to automatically estimate multi-matrix mixture models from large datasets. QMix employs QMaker algorithm instead of XRATE algorithm to accurately and rapidly estimate the parameters of models. It is able to estimate mixture models with different number of matrices and supports multi-threading computing to efficiently estimate models from thousands of genes. We re-estimate mixture models LG4X and LG4M from 1471 HSSP alignments. The re-estimated models (HP4X and HP4M) are slightly better than LG4X and LG4M in building maximum likelihood trees from HSSP and TreeBASE datasets. QMix program required about 10 hours on a computer with 18 cores to estimate a mixture model with four matrices from 200 HSSP alignments. It is easy to use and freely available for researchers.

PHACTboost: A Phylogeny-Aware Pathogenicity Predictor for Missense Mutations via Boosting.

Most algorithms that are used to predict the effects of variants rely on evolutionary conservation. However, a majority of such techniques compute evolutionary conservation by solely using the alignment of multiple sequences while overlooking the evolutionary context of substitution events. We had introduced PHACT, a scoring-based pathogenicity predictor for missense mutations that can leverage phylogenetic trees, in our previous study. By building on this foundation, we now propose PHACTboost, a gradient boosting tree-based classifier that combines PHACT scores with information from multiple sequence alignments, phylogenetic trees, and ancestral reconstruction. By learning from data, PHACTboost outperforms PHACT. Furthermore, the results of comprehensive experiments on carefully constructed sets of variants demonstrated that PHACTboost can outperform 40 prevalent pathogenicity predictors reported in the dbNSFP, including conventional tools, metapredictors, and deep learning-based approaches as well as more recent tools such as AlphaMissense, EVE, and CPT-1. The superiority of PHACTboost over these methods was particularly evident in case of hard variants for which different pathogenicity predictors offered conflicting results. We provide predictions of 215 million amino acid alterations over 20,191 proteins. PHACTboost is available at https://github.com/CompGenomeLab/PHACTboost. PHACTboost can improve our understanding of genetic diseases and facilitate more accurate diagnoses.

Genomic signatures associated with recurrent scale loss in cyprinid fish.

Scale morphology represents a fundamental feature of fish and a key evolutionary trait underlying fish diversification. Despite frequent and recurrent scale loss throughout fish diversification, comprehensive genome-wide analyses of the genomic signatures associated with scale loss in divergent fish lineages remain scarce. In the current study, we investigated genome-wide signatures, specifically convergent protein-coding gene loss, amino acid substitutions, and cis-regulatory sequence changes, associated with recurrent scale loss in two divergent Cypriniformes lineages based on large-scale genomic, transcriptomic, and epigenetic data. Results demonstrated convergent changes in many genes related to scale formation in divergent scaleless fish lineages, including loss of P/Q-rich scpp genes (e.g. scpp6 and scpp7), accelerated evolution of non-coding elements adjacent to the fgf and fgfr genes, and convergent amino acid changes in genes (e.g. snap29) under relaxed selection. Collectively, these findings highlight the existence of a shared genetic architecture underlying recurrent scale loss in divergent fish lineages, suggesting that evolutionary outcomes may be genetically repeatable and predictable in the convergence of scale loss in fish.

Single Amino Acid Substitution in the Matrix Protein of Rabies Virus Is Associated with Neurovirulence in Mice.

Rabies is a fatal encephalitic infectious disease caused by the rabies virus (RABV). RABV is highly neurotropic and replicates in neuronal cell lines in vitro. The RABV fixed strain, HEP-Flury, was produced via passaging in primary chicken embryonic fibroblast cells. HEP-Flury showed rapid adaptation when propagated in mouse neuroblastoma (MNA) cells. In this study, we compared the growth of our previously constructed recombinant HEP (rHEP) strain-based on the sequence of the HEP (HEP-Flury) strain-with that of the original HEP strain. The original HEP strain exhibited higher titer than rHEP and a single substitution at position 80 in the matrix (M) protein M(D80N) after incubation in MNA cells, which was absent in rHEP. In vivo, intracerebral inoculation of the rHEP-M(D80N) strain with this substitution resulted in enhanced viral growth in the mouse brain and a significant loss of body weight in the adult mice. The number of viral antigen-positive cells in the brains of adult mice inoculated with the rHEP-M(D80N) strain was significantly higher than that with the rHEP strain at 5 days post-inoculation. Our findings demonstrate that a single amino acid substitution in the M protein M(D80N) is associated with neurovirulence in mice owing to adaptation to mouse neuronal cells.

Relative frequency of genomic mutations in SARS-CoV-2 recovered from southern Brazilian cases of COVID-19 through the Gamma, Delta and Omicron waves.

The presence of different mutations in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome can be related to changes in coronavirus disease (COVID-19) infection. Besides, these viral alterations associated with factors such as massive number of positive cases, vaccination and reinfections can be important in the viral evolution process. As well as, mutations found at low frequencies may have a more neutral action and consequently be less inclined to negative selection, facilitating their spread through the population. Related to that, we aimed to present mutations that are possibly relevant in the process of viral evolution found in 115 SARS-CoV-2 sequences from samples of individuals residing in the metropolitan region of Porto Alegre in the state of Rio Grande do Sul, Brazil. The genome from clinical samples was sequenced using High-Throughput Sequencing (HTS) and analyzed using a workflow to map reads and find variations/SNPs. The samples were separated into 3 groups considering the sample lineage. Of the total number of analyzed sequences, 35 were from the Gamma lineage, 35 from Delta and 45 from Omicron. Amino acid changes present in frequencies lower than 80% of the reads in the sequences were evaluated. 11 common mutations among the samples were found in the Gamma lineage, 1 in the ORF1ab gene, 7 in the S gene, 2 in the ORF6 gene and 1 in the ORF7a gene. While in the Delta lineage, a total of 11 mutations distributed in the ORF1ab, S, ORF7a and N genes, 2, 7, 1 and 1 mutation were found in each gene, respectively. And finally, in the Omicron, 16 mutations were identified, 2 in the ORF1ab gene, 12 in the S gene and 2 in the M gene. In conclusion, we emphasize that genomic surveillance can be a useful tool to assess how mutations play a key role in virus adaptation, and its process of susceptibility to new hosts showing the possible signs of viral evolution.

Evolutionary genetics of pulmonary anatomical adaptations in deep-diving cetaceans.

BACKGROUND: Cetaceans, having experienced prolonged adaptation to aquatic environments, have undergone evolutionary changes in their respiratory systems. This process of evolution has resulted in the emergence of distinctive phenotypic traits, notably the abundance of elastic fibers and thickened alveolar walls in their lungs, which may facilitate alveolar collapse during diving. This structure helps selective exchange of oxygen and carbon dioxide, while minimizing nitrogen exchange, thereby reducing the risk of DCS. Nevertheless, the scientific inquiry into the mechanisms through which these unique phenotypic characteristics govern the diving behavior of marine mammals, including cetaceans, remains unresolved. RESULTS: This study entails an evolutionary analysis of 42 genes associated with pulmonary fibrosis across 45 mammalian species. Twenty-one genes in cetaceans exhibited accelerated evolution, featuring specific amino acid substitutions in 14 of them. Primarily linked to the development of the respiratory system and lung morphological construction, these genes play a crucial role. Moreover, among marine mammals, we identified eight genes undergoing positive selection, and the evolutionary rates of three genes significantly correlated with diving depth. Specifically, the SFTPC gene exhibited convergent amino acid substitutions. Through in vitro cellular experiments, we illustrated that convergent amino acid site mutations in SFTPC contribute positively to pulmonary fibrosis in marine mammals, and the presence of this phenotype can induce deep alveolar collapse during diving, thereby reducing the risk of DCS during diving. CONCLUSIONS: The study unveils pivotal genetic signals in cetaceans and other marine mammals, arising through evolution. These genetic signals may influence lung characteristics in marine mammals and have been linked to a reduced risk of developing DCS. Moreover, the research serves as a valuable reference for delving deeper into human diving physiology.

Case report: Novel NUS1 variant in a Chinese patient with tremors and intellectual disability.

Introduction: Nuclear undecaprenyl pyrophosphate synthase 1 (NUS1) gene variants are associated with a range of phenotypes, including epilepsy, intellectual disability, cerebellar ataxia, Parkinson's disease, dystonia, and congenital disorders of glycosylation. Additionally, cases describing genotypes and clinical features are rare. Case Presentation: Herein, we report the case of a 23-year-old Chinese female patient who presented with tremors, intellectual disability, and epilepsy. A history of carbon monoxide exposure, brain trauma, or encephalitis was not present in this case. Trio whole-exome sequencing analysis revealed a de novo pathogenic variant of c.750del in exon 4, leading to p.Leu251* amino acid substitution. Genetic analysis failed to identify the identical mutations in the remaining family members who underwent screening. The patient was diagnosed with a rare congenital disease, "congenital glycosylation disorder, type 1aa, autosomal dominant, type 55, with seizures (MRD-55)." Conclusion: We provide further evidence for the role of variants in NUS1 in the development of tremors, epilepsy, and intellectual disabilities. These findings expand our understanding of the clinical phenotypes of NUS1 variants.

Identification of Critical Amino Acid Residues of a Two-Component Sensor Protein for Signal Sensing in Porphyromonas gingivalis Fimbriation via Random Mutant Library Construction.

Porphyromonas gingivalis (Pg) utilizes FimA fimbriae to colonize the gingival sulcus and evade the host immune system. The biogenesis of all FimA-related components is positively regulated by the FimS-FimR two-component system, making the FimS sensory protein an attractive target for preventing Pg infection. However, the specific environmental signal received by FimS remains unknown. We constructed random Pg mutant libraries to identify critical amino acid residues for signal sensing by FimS. Optimized error-prone polymerase chain reaction (PCR) was used to introduce a limited number of random mutations in the periplasmic-domain-coding sequence of fimS, and expression vectors carrying various mutants were generated by inverse PCR. More than 500 transformants were obtained from the fimS-knockout Pg strain using the Escherichia coli-Pg conjugal transfer system, whereas only ~100 transformants were obtained using electroporation. Four and six transformant strains showed increased and decreased fimA expression, respectively. Six strains had single amino acid substitutions in the periplasmic domain, indicating critical residues for signal sensing by FimS. This newly developed strategy should be generally applicable and contribute to molecular genetics studies of Pg, including the elucidation of structure-function relationships of proteins of interest.

Confirmation and characterization of the first case of acetolactate synthase (ALS)-inhibitor resistance in Japanese brome (Bromus japonicus) in the US.

BACKGROUND: Japanese brome (Bromus japonicus Thumb.) is one of the problematic annual weeds in winter wheat (Triticum aestivum L.) and is generally controlled by acetolactate synthase (ALS) inhibitors. Repeated use of the ALS inhibitor propoxycarbazone-Na resulted in the evolution of resistance to this herbicide in three B. japonicus populations, i.e., R1, R2, and R3 in Kansas (KS). However, the level of resistance and mechanism conferring resistance in these populations is unknown. The objectives of this research were to (i) evaluate the level of resistance to propoxycarbazone-Na in R1, R2, and R3 in comparison with a known susceptible population (S1), (ii) investigate the mechanism of resistance involved in conferring ALS-inhibitor resistance, and (iii) investigate the cross-resistance to other ALS inhibitors. RESULTS: Dose-response (0 to 16x; x = 44 g ai ha-1 of propoxycarbazone-Na) assay indicated 167, 125, and 667-fold resistance in R1, R2 and R3 populations, respectively, compared to S1 population. ALS gene sequencing confirmed the mutations resulting in amino acid substitutions, i.e., Pro-197-Thr (R3, R1)/Ser (R2, R1) bestowing resistance to these ALS inhibitors. Such amino acid substitutions also showed differential cross-resistance to sulfosulfuron, mesosulfuron-methyl, pyroxsulam, and imazamox among resistant populations. Pretreatment with malathion (a cytochrome P450 enzyme-inhibitor) followed by imazamox treatment suggested cross-resistance to this herbicide possibly via metabolism only in R3 population. CONCLUSION: Overall, these results confirm the first case of target-site based resistance to ALS inhibitors in B. japonicus in the US, highlighting the need for exploring herbicides with alternative modes of action to enhance weed control in winter wheat. © 2024 Society of Chemical Industry.

Fit for Purpose Approach To Evaluate Detection of Amino Acid Substitutions in Shotgun Proteomics.

Amino acid substitutions (AASs) alter proteins from their genome-expected sequences. Accumulation of substitutions in proteins underlies numerous diseases and antibiotic mechanisms. Accurate global detection of AASs and their frequencies is crucial for understanding these mechanisms. Shotgun proteomics provides an untargeted method for measuring AASs but introduces biases when extrapolating from the genome to identify AASs. To characterize these biases, we created a "ground-truth" approach using the similarities betweenEscherichia coli and Salmonella typhimurium to model the complexity of AAS detection. Shotgun proteomics on mixed lysates generated libraries representing ∼100,000 peptide-spectra and 4161 peptide sequences with a single AAS and defined stoichiometry. Identifying S. typhimurium peptide-spectra with only the E. coli genome resulted in 64.1% correctly identified library peptides. Specific AASs exhibit variable identification efficiencies. There was no inherent bias from the stoichiometry of the substitutions. Short peptides and AASs localized near peptide termini had poor identification efficiency. We identify a new class of "scissor substitutions" that gain or lose protease cleavage sites. Scissor substitutions also had poor identification efficiency. This ground-truth AAS library reveals various sources of bias, which will guide the application of shotgun proteomics to validate AAS hypotheses.

Estimating amino acid substitution models from genome datasets: a simulation study on the performance of estimated models.

Estimating parameters of amino acid substitution models is a crucial task in bioinformatics. The maximum likelihood (ML) approach has been proposed to estimate amino acid substitution models from large datasets. The quality of newly estimated models is normally assessed by comparing with the existing models in building ML trees. Two important questions remained are the correlation of the estimated models with the true models and the required size of the training datasets to estimate reliable models. In this article, we performed a simulation study to answer these two questions based on simulated data. We simulated genome datasets with different numbers of genes/alignments based on predefined models (called true models) and predefined trees (called true trees). The simulated datasets were used to estimate amino acid substitution model using the ML estimation methods. Our experiments showed that models estimated by the ML methods from simulated datasets with more than 100 genes have high correlations with the true models. The estimated models performed well in building ML trees in comparison with the true models. The results suggest that amino acid substitution models estimated by the ML methods from large genome datasets are a reliable tool for analyzing amino acid sequences.

The phosphorylation of the movement protein TGBp1 regulates the accumulation of the Bamboo mosaic virus.

Phosphorylation and dephosphorylation of viral movement proteins plays a crucial role in regulating virus movement. Our study focused on investigating the movement protein TGBp1 of Bamboo mosaic virus (BaMV), which is a single-stranded positive-sense RNA virus. Specifically, we examined four potential phosphorylation sites (S15, S18, T58, and S247) within the TGBp1 protein. To study the impact of phosphorylation, we introduced amino acid substitutions at the selected sites. Alanine substitutions were used to prevent phosphorylation, while aspartate substitutions were employed to mimic phosphorylation. Our findings suggest that mimicking phosphorylation at S15, S18 and T58 of TGBp1 might be linked to silencing suppressor activities. The phosphorylated form at these sites exhibits a loss of silencing suppressor activity, leading to reduced viral accumulation in the inoculated leaves. Furthermore, mimicking phosphorylation at residues S15 and S18 could diminish viral accumulation at the single-cell level, while doing so at residue T58 could influence virus movement. However, mimicking phosphorylation at residue S247 does not appear to be relevant to both functions of TGBp1. Overall, our study provides insights into the functional significance of specific phosphorylation sites in BaMV TGBp1, illuminating the regulatory mechanisms involved in virus movement and silencing suppression.

Bioinformatics insights into CENP-T and CENP-W protein-protein interaction disruptive amino acid substitution in the CENP-T-W complex.

Kinetochores are multi-protein assemblies present at the centromere of the human chromosome and play a crucial role in cellular mitosis. The CENP-T and CENP-W chains form a heterodimer, which is an integral part of the inner kinetochore, interacting with the linker DNA on one side and the outer kinetochore on the other. Additionally, the CENP-T-W dimer interacts with other regulatory proteins involved in forming inner kinetochores. The specific roles of different amino acids in the CENP-W at the protein-protein interaction (PPI) interface during the CENP-T-W dimer formation remain incompletely understood. Since cell division goes awry in diseases like cancer, this CENP-T-W partnership is a potential target for new drugs that could restore healthy cell division. We employed molecular docking, binding free energy calculations, and molecular dynamics (MD) simulations to investigate the disruptive effects of amino acids substitutions in the CENP-W chain on CENP-T-W dimer formation. By conducting a molecular docking study and analysing hydrogen bonding interactions, we identified key residues in CENP-W (ASN-46, ARG-53, LEU-83, SER-86, ARG-87, and GLY-88) for further investigation. Through site-directed mutagenesis and subsequent binding free energy calculations, we refined the selection of mutant. We chose four mutants (N46K, R53K, L83K, and R87E) of CENP-W to assess their comparative potential in forming CENP-T-W dimer. Our analysis from 250 ns long revealed that the substitution of LEU83 and ARG53 residues in CENP-W with the LYS significantly disrupts the formation of CENP-T-W dimer. In conclusion, LEU83 and ARG53 play a critical role in CENP-T and CENP-W dimerization which is ultimately required for cellular mitosis. Our findings not only deepen our understanding of cell division but also hint at exciting drug-target possibilities.

Single Amino Acid Polymorphisms in the Fasciola hepatica Carboxylesterase Type B Gene and Their Potential Role in Anthelmintic Resistance.

The expression of the Fasciola hepatica carboxylesterase type B (CestB) gene is known to be induced upon exposure to the anthelmintic triclabendazole (TCBZ), leading to a substantial rise in enzyme-specific activity. Furthermore, the nucleotide sequence of the CestB gene displays variations that can potentially result in radical amino acid substitutions at the ligand binding site. These substitutions hold the potential to impact both the ligand-protein interaction and the catalytic properties of the enzyme. Thus, the objective of our study was to identify novel CestB polymorphisms in TCBZ-resistant parasites and field isolates obtained from a highly endemic region in Central Mexico. Additionally, we aimed to assess these amino acid polymorphisms using 3D modeling against the metabolically oxidized form of the anthelmintic TCBZSOX. Our goal was to observe the formation of TCBZSOX-specific binding pockets that might provide insights into the role of CestB in the mechanism of anthelmintic resistance. We identified polymorphisms in TCBZ-resistant parasites that exhibited three radical amino acid substitutions at positions 147, 215, and 263. These substitutions resulted in the formation of a TCBZSOX-affinity pocket with the potential to bind the anthelmintic drug. Furthermore, our 3D modeling analysis revealed that these amino acid substitutions also influenced the configuration of the CestB catalytic site, leading to alterations in the enzyme's interaction with chromogenic carboxylic ester substrates and potentially affecting its catalytic properties. However, it is important to note that the TCBZSOX-binding pocket, while significant for drug binding, was located separate from the enzyme's catalytic site, rendering enzymatic hydrolysis of TCBZSOX impossible. Nonetheless, the observed increased affinity for the anthelmintic may provide an explanation for a drug sequestration type of anthelmintic resistance. These findings lay the groundwork for the future development of a molecular diagnostic tool to identify anthelmintic resistance in F. hepatica.

Improvement of the mismatch amplification mutation assay-PCR for discrimination between Streptococcus suis serotypes 2 and 1/2.

A mismatch amplification mutation assay (MAMA)-PCR, which detects a single-nucleotide polymorphism contributed to serological difference between Streptococcus suis serotypes 2 and 1/2, is used to discriminate between these serotypes. The present study reports unusual serotype 1/2 isolates untypable by the MAMA-PCR and improvement of the MAMA-PCR for typing such isolates.

Prediction of Amino Acid Substitutions in ABL1 Protein Leading to Tumor Drug Resistance Based on "Structure-Property" Relationship Classification Models.

Drug resistance to anticancer drugs is a serious complication in patients with cancer. Typically, drug resistance occurs due to amino acid substitutions (AAS) in drug target proteins. The study aimed at developing and validating a new approach to the creation of structure-property relationships (SPR) classification models to predict AASs leading to drug resistance to inhibitors of tyrosine-protein kinase ABL1. The approach was based on the representation of AASs as peptides described in terms of structural formulas. The data on drug-resistant and non-resistant variants of AAS for two isoforms of ABL1 were extracted from the COSMIC database. The given training sets (approximately 700 missense variants) were used for the creation of SPR models in MultiPASS software based on substructural atom-centric multiple neighborhoods of atom (MNA) descriptors for the description of the structural formula of protein fragments and a Bayesian-like algorithm for revealing structure-property relationships. It was found that MNA descriptors of the 6th level and peptides from 11 amino acid residues were the best combination for ABL1 isoform 1 with the prediction accuracy (AUC) of resistance to imatinib (0.897) and dasatinib (0.996). For ABL1 isoform 2 (resistance to imatinib), the best combination was MNA descriptors of the 6th level, peptides form 15 amino acids (AUC value was 0.909). The prediction of possible drug-resistant AASs was made for dbSNP and gnomAD data. The six selected most probable imatinib-resistant AASs were additionally validated by molecular modeling and docking, which confirmed the possibility of resistance for the E334V and T392I variants.

Molecular Bulkiness of a Single Amino Acid in the F1 α-Subunit Determines the Robustness of Cyanobacterial ATP Synthase.

Cyanobacteria are promising photosynthetic organisms owing to their ease of genetic manipulation. Among them, Synechococcus elongatus UTEX 2973 exhibits faster growth, higher biomass production efficiency and more robust stress tolerance compared with S. elongatus PCC 7942. This is due to specific genetic differences, including four single-nucleotide polymorphisms (SNPs) in three genes. One of these SNPs alters an amino acid at position 252 of the FoF1 ATP synthase α-subunit from Tyr to Cys (αY252C) in S. elongatus 7942. This change has been shown to significantly affect growth rate and stress tolerance, specifically in S. elongatus. Furthermore, experimental substitutions with several other amino acids have been shown to alter the ATP synthesis rate in the cell. In the present study, we introduced identical amino acid substitutions into Synechocystis sp. PCC 6803 at position 252 to elucidate the amino acid's significance and generality across cyanobacteria. We investigated the resulting impact on growth, intracellular enzyme complex levels, intracellular ATP levels and enzyme activity. The results showed that the αY252C substitution decreased growth rate and high-light tolerance. This indicates that a specific bulkiness of this amino acid's side chain is important for maintaining cell growth. Additionally, a remarkable decrease in the membrane-bound enzyme complex level was observed. However, the αY252C substitution did not affect enzyme activity or intracellular ATP levels. Although the mechanism of growth suppression remains unknown, the amino acid at position 252 is expected to play an important role in enzyme complex formation.

A single amino acid substitution alters the vanillylamine synthesis activity of Capsicum pAMT.

Some Capsicum synthesize a unique pungent alkaloid called capsaicin in their fruits. In the synthetic pathway of capsaicin, vanillylamine is produced from vanillin in a reaction catalyzed by a putative aminotransferase (pAMT). Therefore, the capsaicinoids content in the fruits is thought to partially depend on the characteristics of pAMT. Comparing Yume-matsuri (yume), C. annuum variety, and red habanero (RH), C. chinense variety, the vanillylamine synthesis activity of the placental extract was higher in yume than in RH. When each recombinant pAMT (rpAMT) was generated using the Escherichia coli expression system and their activities were compared, yume rpAMT synthesized 14-fold more vanillylamine than RH rpAMT. The amino acid sequence of yume and RH pAMT deduced from the cDNAs revealed that only 7 of 459 residues differed. When a single amino acid residue-substituted rpAMT was generated in which the 56th amino acid was swapped with one other, the amount of vanillylamine synthesis of yume and RH rpAMTs was inverted. Furthermore, it was suggested that the 56th amino acid contributed to the affinity for the coenzyme pyridoxal phosphate. Differences in the vanillylamine synthesis activity of pAMT may also lead to differences in the amount of capsaicin synthesis that accumulates in the fruit. Since capsaicin is a compound with commercial value, this finding may provide new insights into the creation of a variety that can synthesize more capsaicin.