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Native amine dehydrogenases offer sustainable access to chiral amines, so the search for scaffolds capable of converting more diverse carbonyl compounds is required to reach the full potential of this alternative to conventional synthetic reductive aminations. Here we report a multidisciplinary strategy combining bioinformatics, chemoinformatics and biocatalysis to extensively screen billions of sequences in silico and to efficiently find native amine dehydrogenases features using computational approaches. In this way, we achieve a comprehensive overview of the initial native amine dehydrogenase family, extending it from 2,011 to 17,959 sequences, and identify native amine dehydrogenases with non-reported substrate spectra, including hindered carbonyls and ethyl ketones, and accepting methylamine and cyclopropylamine as amine donor. We also present preliminary model-based structural information to inform the design of potential ( R )-selective amine dehydrogenases, as native amine dehydrogenases are mostly ( S )-selective. This integrated strategy paves the way for expanding the resource of other enzyme families and in highlighting enzymes with original features. Sustainable chemistry can benefit from biocatalysis, but a high diversity of enzymes is needed. Here, the authors screen billions of protein sequences to provide an overview of the native amine dehydrogenase family for amine synthesis.

Gamma-Aminobutyric acid (GABA) accumulates in plants following exposure to heavy metals. To investigate the role of GABA in cadmium (Cd) tolerance and elucidate the underlying mechanisms, GABA (0, 25 and 50 µM) was applied to Cd-treated maize plants. Vegetative growth parameters were improved in both Cd-treated and control plants due to GABA application. Cd uptake and translocation were considerably inhibited by GABA. Antioxidant enzyme activity was enhanced in plants subjected to Cd. Concurrently GABA caused further increases in catalase and superoxide dismutase activities, which led to a significant reduction in hydrogen peroxide, superoxide anion and malondealdehyde contents under stress conditions. Polyamine biosynthesis-responsive genes, namely ornithine decarboxylase and spermidine synthase, were induced by GABA in plants grown under Cd shock. GABA suppressed polyamine oxidase, a gene related to polyamine catabolism, when plants were exposed to Cd. Consequently, different forms of polyamines were elevated in Cd-exposed plants following GABA application. The maximum quantum efficiency of photosystem II (F v /F m ) was decreased by Cd-exposed plants, but was completely restored by GABA to the same value in the control. These results suggest a multifaceted contribution of GABA, through regulation of Cd uptake, production of reactive oxygen species and polyamine metabolism, in response to Cd stress.

It is estimated that the etiology of 20–30% of epithelial cancers is directly associated with inflammation, although the direct molecular events linking inflammation and carcinogenesis are poorly defined. In the context of gastrointestinal disease, the bacterium enterotoxigenic Bacteroides fragilis (ETBF) is a significant source of chronic inflammation and has been implicated as a risk factor for colorectal cancer. Spermine oxidase (SMO) is a polyamine catabolic enzyme that is highly inducible by inflammatory stimuli resulting in increased reactive oxygen species (ROS) and DNA damage. We now demonstrate that purified B. fragilis toxin (BFT) up-regulates SMO in HT29/c1 and T84 colonic epithelial cells, resulting in SMO-dependent generation of ROS and induction of γ-H2A.x, a marker of DNA damage. Further, ETBF-induced colitis in C57BL/6 mice is associated with increased SMO expression and treatment of mice with an inhibitor of polyamine catabolism, N1,N4-bis(2,3-butandienyl)-1,4-butanediamine (MDL 72527), significantly reduces ETBF-induced chronic inflammation and proliferation. Most importantly, in the multiple intestinal neoplasia (Min) mouse model, treatment with MDL 72527 reduces ETBF-induced colon tumorigenesis by 69% (P < 0.001). The results of these studies indicate that SMO is a source of bacteria-induced ROS directly associated with tumorigenesis and could serve as a unique target for chemoprevention.

Polyamines (PAs) are essential organic polycations for cell viability along the whole phylogenetic scale. In mammals, they are involved in the most important physiological processes: cell proliferation and viability, nutrition, fertility, as well as nervous and immune systems. Consequently, altered polyamine metabolism is involved in a series of pathologies. Due to their pathophysiological importance, PA metabolism has evolved to be a very robust metabolic module, interconnected with the other essential metabolic modules for gene expression and cell proliferation/differentiation. Two different PA sources exist for animals: PA coming from diet and endogenous synthesis. In the first section of this work, the molecular characteristics of PAs are presented as determinant of their roles in living organisms. In a second section, the metabolic specificities of mammalian PA metabolism are reviewed, as well as some obscure aspects on it. This second section includes information on mammalian cell/tissue-dependent PA-related gene expression and information on crosstalk with the other mammalian metabolic modules. The third section presents a synthesis of the physiological processes described as modulated by PAs in humans and/or experimental animal models, the molecular bases of these regulatory mechanisms known so far, as well as the most important gaps of information, which explain why knowledge around the specific roles of PAs in human physiology is still considered a “mysterious” subject. In spite of its robustness, PA metabolism can be altered under different exogenous and/or endogenous circumstances so leading to the loss of homeostasis and, therefore, to the promotion of a pathology. The available information will be summarized in the fourth section of this review. The different sections of this review also point out the lesser-known aspects of the topic. Finally, future prospects to advance on these still obscure gaps of knowledge on the roles on PAs on human physiopathology are discussed.

Nicotinamide N -methyltransferase (NNMT) expression is increased in white adipose tissue and liver of obese and diabetic mice, Nnmt knockdown protects against diet-induced obesity by altering the availability of adipose S -adenosylmethionine and NAD + , rendering Nnmt a novel target for treating obesity and type 2 diabetes. NNMT a possible drug target in diabetes Nicotinamide N -methyltransferase (NNMT), an enzyme that methylates nicotinamide (vitamin B3) using S -adenosylmethionine (SAM) as a methyl donor, is present at high levels in adipose tissue and is increased in some cancers, neurodegenerative diseases, obesity and diabetes. Barbara Kahn and colleagues report that NNMT is elevated in adipose tissue and liver in obese and diabetic mice. Knockdown of NNMT in adipose tissue protects against diet-induced obesity and its metabolic consequences such as glucose intolerance and fatty liver. NNMT inhibition in adipocytes leads to consumption of metabolic substrates coupled with increased energy expenditure, resulting in increased leanness. These findings identify NNMT as a potential target for treating obesity and type 2 diabetes. In obesity and type 2 diabetes, Glut4 glucose transporter expression is decreased selectively in adipocytes 1 . Adipose-specific knockout or overexpression of Glut4 alters systemic insulin sensitivity 2 . Here we show, using DNA array analyses, that nicotinamide N -methyltransferase ( Nnmt ) is the most strongly reciprocally regulated gene when comparing gene expression in white adipose tissue (WAT) from adipose-specific Glut4 -knockout or adipose-specific Glut4 -overexpressing mice with their respective controls. NNMT methylates nicotinamide (vitamin B3) using S -adenosylmethionine (SAM) as a methyl donor 3 , 4 . Nicotinamide is a precursor of NAD + , an important cofactor linking cellular redox states with energy metabolism 5 . SAM provides propylamine for polyamine biosynthesis and donates a methyl group for histone methylation 6 . Polyamine flux including synthesis, catabolism and excretion, is controlled by the rate-limiting enzymes ornithine decarboxylase (ODC) and spermidine–spermine N 1 -acetyltransferase (SSAT; encoded by Sat1 ) and by polyamine oxidase (PAO), and has a major role in energy metabolism 7 , 8 . We report that NNMT expression is increased in WAT and liver of obese and diabetic mice. Nnmt knockdown in WAT and liver protects against diet-induced obesity by augmenting cellular energy expenditure. NNMT inhibition increases adipose SAM and NAD + levels and upregulates ODC and SSAT activity as well as expression, owing to the effects of NNMT on histone H3 lysine 4 methylation in adipose tissue. Direct evidence for increased polyamine flux resulting from NNMT inhibition includes elevated urinary excretion and adipocyte secretion of diacetylspermine, a product of polyamine metabolism. NNMT inhibition in adipocytes increases oxygen consumption in an ODC-, SSAT- and PAO-dependent manner. Thus, NNMT is a novel regulator of histone methylation, polyamine flux and NAD + -dependent SIRT1 signalling, and is a unique and attractive target for treating obesity and type 2 diabetes.

Polyamine oxidases (PAOs) are enzymes degrading the polyamine molecules and have important roles in plant growth, development and in stress tolerance. Despite their significance, their genomic organization and functional roles in Sorghum bicolor , a drought-tolerant staple crop, remain largely unexplored. In this study, a comprehensive comparative genomics analysis was conducted and identified six PAO genes in sorghum phylogenetically clustered into four clades, with sorghum exhibiting lineage-specific expansion via segmental and tandem duplications. Structural modelling identified conserved FAD-dependent oxidase cores across all SbPAO proteins and identified a novel motif (GLRLYRTSGDNSVLYDHDLEDYALYDYEGAQVPRETVLK) unique to sorghum PAOs, potentially linked to flavin-dependent oxidoreductase activity. SbPAO5 and SbPAO6 exhibited the most elaborated fold in three-dimentional modeling. SbPAO4 and SbPAO5 possess peroxisomal targeting signals (PTS1). These structural and targeting divergences collectively suggest subfunctionalization within the SbPAO family. Collinearity analysis highlighted strong syntenic conservation with rice and maize suggesting evolutionary and functional conservation of PAOs in grasses. Promoter sequence analysis revealed presence of several stress and hormones responsive elements, aligning with tissue- and genotype-specific expression patterns under drought. In the tolerant genotype Dorado, SbPAO4–6 were dynamically upregulated in leaves and grains, correlating with spermidine accumulation and enhanced stress resilience. Elevated spermidine level in sensitive genotype Giza 15 suggested back conversion of spermine to spermidine by upregulation of SbPAO5 . Co-expression analysis linked SbPAO5 and SbPAO6 to stress signalling and metabolic hubs implicating their roles in integrated stress adaptation. These findings establish a foundation for genomic organization and evolutionary relationships of PAO genes in sorghum and prioritizes SbPAO5 and SbPAO6 as candidates for stress-resistant breeding and enhanced production in sorghum.

Helicobacter pylori is the primary cause of gastric cancer, and there is a need to discover new molecular targets for therapeutic intervention in H. pylori disease progression. We have previously shown that spermine oxidase (SMOX), the enzyme that catabolizes the back-conversion of the polyamine spermine to spermidine, is upregulated during infection and is associated with increased cancer risk in humans. We sought to determine the direct role of SMOX in gastric carcinogenesis during H. pylori infection. In this study, we demonstrate that transgenic FVB/N insulin-gastrin (INS-GAS) mice that develop gastric carcinoma with H. pylori infection were protected from cancer development with Smox deletion. RNA sequencing revealed that genes associated with the immune system and cancer were downregulated in the infected Smox –/– mice. Furthermore, there was a decrease in cell proliferation and DNA damage in infected Smox –/– animals. There was significant generation of adducts of the highly reactive electrophile acrolein, a byproduct of SMOX activity, in gastric tissues from H. pylori -infected humans and wild-type, but not Smox –/– mice. Genetic deletion of Smox in murine organoids or chemical inhibition of SMOX in human gastric epithelial cells significantly reduced generation of acrolein induced by H. pylori . Additionally, acrolein-induced DNA damage in gastric epithelial cells was ablated with the electrophile scavenger 2-hydroxybenzylamine (2-HOBA). Gastric acrolein adduct levels were attenuated in infected INS-GAS mice treated with 2-HOBA, which exhibit reduced gastric carcinoma. These findings implicate SMOX and acrolein in H. pylori -induced carcinogenesis, thus indicating their potential as therapeutic targets.

The success of protein evolution campaigns is strongly dependent on the sequence context in which mutations are introduced, stemming from pervasive non-additive interactions between a protein’s amino acids (‘intra-gene epistasis’). Our limited understanding of such epistasis hinders the correct prediction of the functional contributions and adaptive potential of mutations. Here we present a straightforward unique molecular identifier (UMI)-linked consensus sequencing workflow (UMIC-seq) that simplifies mapping of evolutionary trajectories based on full-length sequences. Attaching UMIs to gene variants allows accurate consensus generation for closely related genes with nanopore sequencing. We exemplify the utility of this approach by reconstructing the artificial phylogeny emerging in three rounds of directed evolution of an amine dehydrogenase biocatalyst via ultrahigh throughput droplet screening. Uniquely, we are able to identify lineages and their founding variant, as well as non-additive interactions between mutations within a full gene showing sign epistasis. Access to deep and accurate long reads will facilitate prediction of key beneficial mutations and adaptive potential based on in silico analysis of large sequence datasets. The success of protein evolution is dependent on the sequence context mutations are introduced into. Here the authors present UMIC-seq that allows consensus generation for closely related genes by using unique molecular identifiers linked to gene variants.

ABSTRACT Eukaryotic Translation Initiation Factor 5A (EIF5A) is a translation factor regulated by hypusination, a unique posttranslational modification catalyzed by deoxyhypusine synthetase (DHPS) and deoxyhypusine hydroxylase (DOHH) starting from the polyamine spermidine. Emerging data are showing that hypusinated EIF5A regulates key cellular processes such as autophagy, senescence, polyamine homeostasis, energy metabolism, and plays a role in cancer. However, the effects of EIF5A inhibition in preclinical cancer models, the mechanism of action, and specific translational targets are still poorly understood. We show here that hypusinated EIF5A promotes growth of colorectal cancer (CRC) cells by directly regulating MYC biosynthesis at specific pausing motifs. Inhibition of EIF5A hypusination with the DHPS inhibitor GC7 or through lentiviral-mediated knockdown of DHPS or EIF5A reduces the growth of various CRC cells. Multiplex gene expression analysis reveals that inhibition of hypusination impairs the expression of transcripts regulated by MYC, suggesting the involvement of this oncogene in the observed effect. Indeed, we demonstrate that EIF5A regulates MYC elongation without affecting its mRNA content or protein stability, by alleviating ribosome stalling at five distinct pausing motifs in MYC CDS. Of note, we show that blockade of the hypusination axis elicits a remarkable growth inhibitory effect in preclinical models of CRC and significantly reduces the size of polyps in APC Min/+ mice, a model of human familial adenomatous polyposis (FAP). Together, these data illustrate an unprecedented mechanism, whereby the tumor-promoting properties of hypusinated EIF5A are linked to its ability to regulate MYC elongation and provide a rationale for the use of DHPS/EIF5A inhibitors in CRC therapy.

Deoxyhypusine synthase (DHPS) is an enzyme encoded by the DHPS gene, with high expression in various cancers, including ovarian cancer (OC). DHPS regulates the translation initiation factor EIF5A, and EIF5A2 knockout inhibits OC tumor growth and metastasis by blocking the epithelial-to-mesenchymal transition (EMT) and the TGFβ pathway. In this study, we show that DHPS is amplified in OC patients, and its elevated expression correlates with poor survival. Using lentiviral CRISPR/Cas9 vectors for DHPS knockout, we observed EMT inhibition in SKOV3 and OVCAR8 cells through suppressed hypusination and reduced EIF5A2 expression. Inhibition of DHPS activity with GC7 similarly blocked hypusination and EMT. Disrupting DHPS expression, either genetically or pharmacologically, inhibited primary tumor growth and metastasis in OC mouse models. These findings suggest that targeting DHPS and inhibiting hypusination could be promising strategies for OC treatment.