One substrate many enzymes virtual screening uncovers missing genes of carnitine biosynthesis in human and mouse.

The increasing availability of experimental and computational protein structures entices their use for function prediction. Here we develop an automated procedure to identify enzymes involved in metabolic reactions by assessing substrate conformations docked to a library of protein structures. By screening AlphaFold-modeled vitamin B6-dependent enzymes, we find that a metric based on catalytically favorable conformations at the enzyme active site performs best (AUROC Score=0.84) in identifying genes associated with known reactions. Applying this procedure, we identify the mammalian gene encoding hydroxytrimethyllysine aldolase (HTMLA), the second enzyme of carnitine biosynthesis. Upon experimental validation, we find that the top-ranked candidates, serine hydroxymethyl transferase (SHMT) 1 and 2, catalyze the HTMLA reaction. However, a mouse protein absent in humans (threonine aldolase; Tha1) catalyzes the reaction more efficiently. Tha1 did not rank highest based on the AlphaFold model, but its rank improved to second place using the experimental crystal structure we determined at 2.26 Å resolution. Our findings suggest that humans have lost a gene involved in carnitine biosynthesis, with HTMLA activity of SHMT partially compensating for its function.

LATS1 controls CTCF chromatin occupancy and hormonal response of 3D-grown breast cancer cells.

The cancer epigenome has been studied in cells cultured in two-dimensional (2D) monolayers, but recent studies highlight the impact of the extracellular matrix and the three-dimensional (3D) environment on multiple cellular functions. Here, we report the physical, biochemical, and genomic differences between T47D breast cancer cells cultured in 2D and as 3D spheroids. Cells within 3D spheroids exhibit a rounder nucleus with less accessible, more compacted chromatin, as well as altered expression of ~2000 genes, the majority of which become repressed. Hi-C analysis reveals that cells in 3D are enriched for regions belonging to the B compartment, have decreased chromatin-bound CTCF and increased fusion of topologically associating domains (TADs). Upregulation of the Hippo pathway in 3D spheroids results in the activation of the LATS1 kinase, which promotes phosphorylation and displacement of CTCF from DNA, thereby likely causing the observed TAD fusions. 3D cells show higher chromatin binding of progesterone receptor (PR), leading to an increase in the number of hormone-regulated genes. This effect is in part mediated by LATS1 activation, which favors cytoplasmic retention of YAP and CTCF removal.

Deciphering the role of recurrent FAD-dependent enzymes in bacterial phosphonate catabolism.

Phosphonates-compounds containing a direct C-P bond-represent an important source of phosphorus in some environments. The most common natural phosphonate is 2-aminoethylphosphonate (AEP). Many bacteria can break AEP down through specialized "hydrolytic" pathways, which start with the conversion of AEP into phosphonoacetaldehyde (PAA), catalyzed by the transaminase PhnW. However, the substrate scope of these pathways is very narrow, as PhnW cannot process other common AEP-related phosphonates, notably N-methyl AEP (M1AEP). Here, we describe a heterogeneous group of FAD-dependent oxidoreductases that efficiently oxidize M1AEP to directly generate PAA, thus expanding the versatility and usefulness of the hydrolytic AEP degradation pathways. Furthermore, some of these enzymes can also efficiently oxidize plain AEP. By doing so, they surrogate the role of PhnW in organisms that do not possess the transaminase and create novel versions of the AEP degradation pathways in which PAA is generated solely by oxidative deamination.

Cysteine Enrichment Mediates Co-Option of Uricase in Reptilian Skin and Transition to Uricotelism.

Uric acid is the main means of nitrogen excretion in uricotelic vertebrates (birds and reptiles) and the end product of purine catabolism in humans and a few other mammals. While uricase is inactivated in mammals unable to degrade urate, the presence of orthologous genes without inactivating mutations in avian and reptilian genomes is unexplained. Here we show that the Gallus gallus gene we name cysteine-rich urate oxidase (CRUOX) encodes a functional protein representing a unique case of cysteine enrichment in the evolution of vertebrate orthologous genes. CRUOX retains the ability to catalyze urate oxidation to hydrogen peroxide and 5-hydroxyisourate (HIU), albeit with a 100-fold reduced efficiency. However, differently from all uricases hitherto characterized, it can also facilitate urate regeneration from HIU, a catalytic property that we propose depends on its enrichment in cysteine residues. X-ray structural analysis highlights differences in the active site compared to known orthologs and suggests a mechanism for cysteine-mediated self-aggregation under H2O2-oxidative conditions. Cysteine enrichment was concurrent with the transition to uricotelism and a shift in gene expression from the liver to the skin where CRUOX is co-expressed with ß-keratins. Therefore, the loss of urate degradation in amniotes has followed opposite evolutionary trajectories: while uricase has been eliminated by pseudogenization in some mammals, it has been repurposed as a redox-sensitive enzyme in the reptilian skin.

Identification of hidden associations among eukaryotic genes through statistical analysis of coevolutionary transitions.

Coevolution at the gene level, as reflected by correlated events of gene loss or gain, can be revealed by phylogenetic profile analysis. The optimal method and metric for comparing phylogenetic profiles, especially in eukaryotic genomes, are not yet established. Here, we describe a procedure suitable for large-scale analysis, which can reveal coevolution based on the assessment of the statistical significance of correlated presence/absence transitions between gene pairs. This metric can identify coevolution in profiles with low overall similarities and is not affected by similarities lacking coevolutionary information. We applied the procedure to a large collection of 60,912 orthologous gene groups (orthogroups) in 1,264 eukaryotic genomes extracted from OrthoDB. We found significant cotransition scores for 7,825 orthogroups associated in 2,401 coevolving modules linking known and unknown genes in protein complexes and biological pathways. To demonstrate the ability of the method to predict hidden gene associations, we validated through experiments the involvement of vertebrate malate synthase-like genes in the conversion of (S)-ureidoglycolate into glyoxylate and urea, the last step of purine catabolism. This identification explains the presence of glyoxylate cycle genes in metazoa and suggests an anaplerotic role of purine degradation in early eukaryotes.

Functional Classification of the ATM Variant c.7157C>A and In Vitro Effects of Dexamethasone.

Most of the ATM variants associated with Ataxia Telangiectasia are still classified as variants with uncertain significance. Ataxia Telangiectasia is a multisystemic disorder characterized by "typical" and "atypical" phenotypes, with early-onset and severe symptoms or with late-onset and mild symptoms, respectively. Here we classified the c.7157C > A ATM variant found in homozygosity in two brothers of Lebanese ethnicity. The brothers presented with an atypical phenotype, showing less than 50% of the positive criteria considered for classification. We performed several in silico analyses to predict the effect of c.7157C > A at the DNA, mRNA and protein levels, revealing that the alteration causes a missense substitution in a highly conserved alpha helix in the FAT domain. 3D structural analyses suggested that the variant might be pathogenic due to either loss of activity or to a structural damage affecting protein stability. Our subsequent in vitro studies showed that the second hypothesis is the most likely, as indicated by the reduced protein abundance found in the cells carrying the variant. Moreover, two different functional assays showed that the mutant protein partially retains its kinase activity. Finally, we investigated the in vitro effect of Dexamethasone showing that the drug is able to increase both protein abundance and activity. In conclusion, our results suggest that the c.7157C > A variant is pathogenic, although it causes an atypical phenotype, and that dexamethasone could be therapeutically effective on this and possibly other missense ATM variants.

Actin-Resistant DNase1L2 as a Potential Therapeutics for CF Lung Disease.

In cystic fibrosis (CF), the accumulation of viscous lung secretions rich in DNA and actin is a major cause of chronic inflammation and recurrent infections leading to airway obstruction. Mucolytic therapy based on recombinant human DNase1 reduces CF mucus viscosity and promotes airway clearance. However, the marked susceptibility to actin inhibition of this enzyme prompts the research of alternative treatments that could overcome this limitation. Within the human DNase repertoire, DNase1L2 is ideally suited for this purpose because it exhibits metal-dependent endonuclease activity on plasmid DNA in a broad range of pH with acidic optimum and is minimally inhibited by actin. When tested on CF artificial mucus enriched with actin, submicromolar concentrations of DNase1L2 reduces mucus viscosity by 50% in a few seconds. Inspection of superimposed model structures of DNase1 and DNase1L2 highlights differences at the actin-binding interface that justify the increased resistance of DNase1L2 toward actin inhibition. Furthermore, a PEGylated form of the enzyme with preserved enzymatic activity was obtained, showing interesting results in terms of activity. This work represents an effort toward the exploitation of natural DNase variants as promising alternatives to DNase1 for the treatment of CF lung disease.

Discovery of a New, Recurrent Enzyme in Bacterial Phosphonate Degradation: (R)-1-Hydroxy-2-aminoethylphosphonate Ammonia-lyase.

Phosphonates represent an important source of bioavailable phosphorus in certain environments. Accordingly, many microorganisms (particularly marine bacteria) possess catabolic pathways to degrade these molecules. One example is the widespread hydrolytic route for the breakdown of 2-aminoethylphosphonate (AEP, the most common biogenic phosphonate). In this pathway, the aminotransferase PhnW initially converts AEP into phosphonoacetaldehyde (PAA), which is then cleaved by the hydrolase PhnX to yield acetaldehyde and phosphate. This work focuses on a pyridoxal 5'-phosphate-dependent enzyme that is encoded in >13% of the bacterial gene clusters containing the phnW-phnX combination. This enzyme (which we termed PbfA) is annotated as a transaminase, but there is no obvious need for an additional transamination reaction in the established AEP degradation pathway. We report here that PbfA from the marine bacterium Vibrio splendidus catalyzes an elimination reaction on the naturally occurring compound (R)-1-hydroxy-2-aminoethylphosphonate (R-HAEP). The reaction releases ammonia and generates PAA, which can be then hydrolyzed by PhnX. In contrast, PbfA is not active toward the S enantiomer of HAEP or other HAEP-related compounds such as ethanolamine and d,l-isoserine, indicating a very high substrate specificity. We also show that R-HAEP (despite being structurally similar to AEP) is not processed efficiently by the PhnW-PhnX couple in the absence of PbfA. In summary, the reaction catalyzed by PbfA serves to funnel R-HAEP into the hydrolytic pathway for AEP degradation, expanding the scope and the usefulness of the pathway itself.

Birth of a pathway for sulfur metabolism in early amniote evolution.

Among amniotes, reptiles and mammals are differently adapted to terrestrial life. It is generally appreciated that terrestrialization required adaptive changes of vertebrate metabolism, particularly in the mode of nitrogen excretion. However, the current paradigm is that metabolic adaptation to life on land did not involve synthesis of enzymatic pathways de novo, but rather repurposing of existing ones. Here, by comparing the inventory of pyridoxal 5'-phosphate-dependent enzymes in different amniotes, we identify in silico a pathway for sulfur metabolism present in chick embryos but not in mammals. Cysteine lyase contains haem and pyridoxal 5'-phosphate co-factors and converts cysteine and sulfite into cysteic acid and hydrogen sulfide, respectively. A specific cysteic acid decarboxylase produces taurine, while hydrogen sulfide is recycled into cysteine by cystathionine beta-synthase. This reaction sequence enables the formation of sulfonated amino acids during embryo development in the egg at no cost of reduced sulfur. The pathway originated around 300 million years ago in a proto-reptile by cystathionine beta-synthase duplication, cysteine lyase neofunctionalization and cysteic acid decarboxylase co-option. Our findings indicate that adaptation to terrestrial life involved innovations in metabolic pathways, and reveal the molecular mechanisms by which such innovations arose in amniote evolution.