[Genetic analysis and prenatal diagnosis for a Chinese pedigree affected with co-morbid Ornithine carbamoyl transferase deficiency and MECP2 duplication syndrome].

OBJECTIVE: To explore the genetic basis for a Chinese pedigree affected with co-morbid Ornithine carbamoyl transferase deficiency (OTCD) and MECP2 duplication syndrome. METHODS: A proband who was admitted to the Neonatal Intensive Care Unit of Gansu Provincial Maternal and Child Health Care Hospital on December 19, 2017 was selected as the study subject. High-throughput sequencing and multiplex ligation-dependent probe amplification (MLPA) were carried out for her pedigree, and short tandem repeat-based linkage analysis and chromosome copy number variation sequencing (CNV-seq) were used for the prenatal diagnosis. RESULTS: The proband, a 3-day-old female, was found to harbor heterozygous deletion of exons 7-9 of the OTC gene. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as likely pathogenic (PVS1+PM2_Supporting+PP4). The proband was diagnosed with OTCD , which was in keeping with her acute encephalopathy and metabolic abnormalities (manifesting as hyperammonemia, decreased blood citrulline, and increased urine orotic acid). Prenatal diagnosis was carried out for the subsequent pregnancy. The fetus did not harbor the exons 7-9 deletion of the OTC gene, but was found to carry a duplication in Xq28 region (which encompassed the whole region of MECP2 duplication syndrome) and was positive for the SRY sequence. The same duplication was also found in the proband and her mother. Considering the possible existence of X-chromosome inactivation, the proband was diagnosed with two X-linked recessive disorders including OTCD and MECP2 duplication syndrome, and the fetus was determined as a male affected with the MECP2 duplication syndrome. CONCLUSION: Discoveries of the pathogenic variants underlying the OTCD and MECP2 duplication syndrome have enabled clinical intervention, treatment, genetic counseling and prenatal diagnosis for this pedigree.

Genetic analysis of acyl-CoA carboxylases involved in lipid accumulation in Rhodococcus jostii RHA1.

In actinomycetes, the acyl-CoA carboxylases, including the so-called acetyl-CoA carboxylases (ACCs), are biotin-dependent enzymes that exhibit broad substrate specificity and diverse domain and subunit arrangements. Bioinformatic analyses of the Rhodococcus jostii RHA1 genome found that this microorganism contains a vast arrange of putative acyl-CoA carboxylases domains and subunits. From the thirteen putative carboxyltransferase domains, only the carboxyltransferase subunit RO01202 and the carboxyltransferase domain present in the multidomain protein RO04222 are highly similar to well-known essential ACC subunits from other actinobacteria. Mutant strains in each of these genes showed that none of these enzymes is essential for R. jostii growth in rich or in minimal media with high nitrogen concentration, presumably because of their partial overlapping activities. A mutant strain in the ro04222 gene showed a decrease in triacylglycerol and mycolic acids accumulation in rich and minimal medium, highlighting the relevance of this multidomain ACC in the biosynthesis of these lipids. On the other hand, RO01202, a carboxyltransferase domain of a putative ACC complex, whose biotin carboxylase and biotin carboxyl carrier protein domain were not yet identified, was found to be essential for R. jostii growth only in minimal medium with low nitrogen concentration. The results of this study have identified a new component of the TAG-accumulating machinery in the oleaginous R. jostii RHA1. While non-essential for growth and TAG biosynthesis in RHA1, the activity of RO04222 significantly contributes to lipogenesis during single-cell oil production. Furthermore, this study highlights the high functional diversity of ACCs in actinobacteria, particularly regarding their essentiality under different environmental conditions. KEY POINTS: • R. jostii possess a remarkable heterogeneity in their acyl-carboxylase complexes. • RO04222 is a multidomain acetyl-CoA carboxylase involved in lipid accumulation. • RO01202 is an essential carboxyltransferase only at low nitrogen conditions.

Endowing homodimeric carbamoyltransferase GdmN with iterative functions through structural characterization and mechanistic studies.

Iterative enzymes, which catalyze sequential reactions, have the potential to improve the atom economy and diversity of industrial enzymatic processes. Redesigning one-step enzymes to be iterative biocatalysts could further enhance these processes. Carbamoyltransferases (CTases) catalyze carbamoylation, an important modification for the bioactivity of many secondary metabolites with pharmaceutical applications. To generate an iterative CTase, we determine the X-ray structure of GdmN, a one-step CTase involved in ansamycin biosynthesis. GdmN forms a face-to-face homodimer through unusual C-terminal domains, a previously unknown functional form for CTases. Structural determination of GdmN complexed with multiple intermediates elucidates the carbamoylation process and identifies key binding residues within a spacious substrate-binding pocket. Further structural and computational analyses enable multi-site enzyme engineering, resulting in an iterative CTase with the capacity for successive 7-O and 3-O carbamoylations. Our findings reveal a subclade of the CTase family and exemplify the potential of protein engineering for generating iterative enzymes.

Conformational Selection Governs Carrier Domain Positioning in Staphylococcus aureus Pyruvate Carboxylase.

Biotin-dependent enzymes employ a carrier domain to efficiently transport reaction intermediates between distant active sites. The translocation of this carrier domain is critical to the interpretation of kinetic and structural studies, but there have been few direct attempts to investigate the dynamic interplay between ligand binding and carrier domain positioning in biotin-dependent enzymes. Pyruvate carboxylase (PC) catalyzes the MgATP-dependent carboxylation of pyruvate where the biotinylated carrier domain must translocate ∼70 Šfrom the biotin carboxylase domain to the carboxyltransferase domain. Many prior studies have assumed that carrier domain movement is governed by ligand-induced conformational changes, but the mechanism underlying this movement has not been confirmed. Here, we have developed a system to directly observe PC carrier domain positioning in both the presence and absence of ligands, independent of catalytic turnover. We have incorporated a cross-linking trap that reports on the interdomain conformation of the carrier domain when it is positioned in proximity to a neighboring carboxyltransferase domain. Cross-linking was monitored by gel electrophoresis, inactivation kinetics, and intrinsic tryptophan fluorescence. We demonstrate that the carrier domain positioning equilibrium is sensitive to substrate analogues and the allosteric activator acetyl-CoA. Notably, saturating concentrations of biotin carboxylase ligands do not prevent carrier domain trapping proximal to the neighboring carboxyltransferase domain, demonstrating that carrier domain positioning is governed by conformational selection. This model of carrier domain translocation in PC can be applied to other multi-domain enzymes that employ large-scale domain motions to transfer intermediates during catalysis.

Progress and Challenges in Quantifying Carbonyl-Metabolomic Phenomes with LC-MS/MS.

Carbonyl-containing metabolites widely exist in biological samples and have important physiological functions. Thus, accurate and sensitive quantitative analysis of carbonyl-containing metabolites is crucial to provide insight into metabolic pathways as well as disease mechanisms. Although reversed phase liquid chromatography electrospray ionization mass spectrometry (RPLC-ESI-MS) is widely used due to the powerful separation capability of RPLC and high specificity and sensitivity of MS, but it is often challenging to directly analyze carbonyl-containing metabolites using RPLC-ESI-MS due to the poor ionization efficiency of neutral carbonyl groups in ESI. Modification of carbonyl-containing metabolites by a chemical derivatization strategy can overcome the obstacle of sensitivity; however, it is insufficient to achieve accurate quantification due to instrument drift and matrix effects. The emergence of stable isotope-coded derivatization (ICD) provides a good solution to the problems encountered above. Thus, LC-MS methods that utilize ICD have been applied in metabolomics including quantitative targeted analysis and untargeted profiling analysis. In addition, ICD makes multiplex or multichannel submetabolome analysis possible, which not only reduces instrument running time but also avoids the variation of MS response. In this review, representative derivatization reagents and typical applications in absolute quantification and submetabolome profiling are discussed to highlight the superiority of the ICD strategy for detection of carbonyl-containing metabolites.

Toxic oligomers of the amyloidogenic HypF-N protein form pores in mitochondrial membranes.

Studies on the amyloidogenic N-terminal domain of the E. coli HypF protein (HypF-N) have contributed significantly to a detailed understanding of the pathogenic mechanisms in neurodegenerative diseases characterised by the formation of misfolded oligomers, by proteins such as amyloid-ß, α-synuclein and tau. Given that both cell membranes and mitochondria are increasingly recognised as key targets of oligomer toxicity, we investigated the damaging effects of aggregates of HypF-N on mitochondrial membranes. Essentially, we found that HypF-N oligomers characterised by high surface hydrophobicity (type A) were able to trigger a robust permeabilisation of mito-mimetic liposomes possessing cardiolipin-rich membranes and dysfunction of isolated mitochondria, as demonstrated by a combination of mitochondrial shrinking, lowering of mitochondrial membrane potential and cytochrome c release. Furthermore, using single-channel electrophysiology recordings we obtained evidence that the type A aggregates induced currents reflecting formation of ion-conducting pores in mito-mimetic planar phospholipid bilayers, with multi-level conductances ranging in the hundreds of pS at negative membrane voltages. Conversely, HypF-N oligomers with low surface hydrophobicity (type B) could not permeabilise or porate mitochondrial membranes. These results suggest an inherent toxicity of membrane-active aggregates of amyloid-forming proteins to mitochondria, and that targeting of oligomer-mitochondrial membrane interactions might therefore afford protection against such damage.

Trodusquemine displaces protein misfolded oligomers from cell membranes and abrogates their cytotoxicity through a generic mechanism.

The onset and progression of numerous protein misfolding diseases are associated with the presence of oligomers formed during the aberrant aggregation of several different proteins, including amyloid-ß (Aß) in Alzheimer's disease and α-synuclein (αS) in Parkinson's disease. These small, soluble aggregates are currently major targets for drug discovery. In this study, we show that trodusquemine, a naturally-occurring aminosterol, markedly reduces the cytotoxicity of αS, Aß and HypF-N oligomers to human neuroblastoma cells by displacing the oligomers from cell membranes in the absence of any substantial morphological and structural changes to the oligomers. These results indicate that the reduced toxicity results from a mechanism that is common to oligomers from different proteins, shed light on the origin of the toxicity of the most deleterious species associated with protein aggregation and suggest that aminosterols have the therapeutically-relevant potential to protect cells from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases.

Translational Detection of Nonproteinogenic Amino Acids Using an Engineered Complementary Cell-Free Protein Synthesis Assay.

We developed a simple and rapid method for analyzing nonproteinogenic amino acids that does not require conventional chromatographic equipment. In this technique, nonproteinogenic amino acids were first converted to a proteinogenic amino acid through in vitro metabolism in a cell extract. The proteinogenic amino acid generated from the nonproteinogenic precursors were then incorporated into a reporter protein using a cell-free protein synthesis system. The titers of the nonproteinogenic amino acids could be readily quantified by measuring the activity of reporter proteins. This method, which combines the enzymatic conversion of target amino acids with translational analysis, makes amino acid analysis more accessible while minimizing the cost and time requirements. We anticipate that the same strategy could be extended to the detection of diverse biochemical molecules with clinical and industrial implications.

Studies of an insecticidal inhibitor of acetyl-CoA carboxylase in the nematode C. elegans.

We have studied the mode of action of the insecticide spirotetramat in the nematode Caenorhabditis elegans. A combination of symptomology, forward genetics and genome editing show that spirotetramat acts on acetyl-CoA carboxylase (ACC) in C. elegans, as it does in insects. We found C. elegans embryos exposed to spirotetramat show a cell division defect which closely resembles the phenotype of loss-of-function mutations in the gene pod-2, which encodes ACC. We then identified two mutations in the carboxyl transferase domain of pod-2 (ACC) which confer resistance and were confirmed using CRISPR/Cas9. One of these mutations substitutes an invertebrate-specific amino acid with one ubiquitous in other taxa; this residue may, therefore, be a determinant of the selectivity of spirotetramat for invertebrates. Such a mutation may also be the target of selection for resistance in the field. Our study is a further demonstration of the utility of C. elegans in studying bioactive chemicals.

Differential Interactome and Innate Immune Response Activation of Two Structurally Distinct Misfolded Protein Oligomers.

The formation of misfolded protein oligomers during early stages of amyloid aggregation and the activation of neuroinflammatory responses are two key events associated with neurodegenerative diseases. Although it has been established that misfolded oligomers are involved in the neuroinflammatory process, the links between their structural features and their functional effects on the immune response remain unknown. To explore such links, we took advantage of two structurally distinct soluble oligomers (type A and B) of protein HypF-N and compared the elicited microglial inflammatory responses. By using confocal microscopy, protein pull-down, and high-throughput mass spectrometry, we found that, even though both types bound to a common pool of microglial proteins, type B oligomers-with a lower solvent-exposed hydrophobicity-showed enhanced protein binding, correlating with the observed inflammatory response. Furthermore, the interactome associated with inflammatory-mediated neurodegeneration revealed previously unidentified receptors and signaling molecules likely to be involved in the oligomer-elicited innate immune response.

The Toxicity of Misfolded Protein Oligomers Is Independent of Their Secondary Structure.

The self-assembly of proteins into structured fibrillar aggregates is associated with a range of neurodegenerative diseases, including Alzheimer's and Parkinson's diseases, in which an important cytotoxic role is thought to be played by small soluble oligomers accumulating during the aggregation process or released by mature fibrils. As the structural characteristics of such species and their links with toxicity are still not fully defined, we have compared six examples of preformed misfolded protein oligomers with different ß-sheet content, as determined using Fourier transform infrared spectroscopy, and with different toxicity, as determined by three cellular readouts of cell viability. The results show the absence of any measurable correlation between the nature of their secondary structure and their cellular toxicity, both when comparing the six types of oligomers as a group and when comparing species in subgroups characterized by either the same size or the same exposure of hydrophobic moieties.

Discovery and characterization of a novel C-terminal peptide carboxyl methyltransferase in a lassomycin-like lasso peptide biosynthetic pathway.

Lasso peptides belong to a peculiar family of ribosomally synthesized and post-translationally modified peptides (RiPPs)-natural products with an unusual isopeptide-bonded slipknot structure. Except for assembling of this unusual lasso fold, several further post-translational modifications of lasso peptides, including C-terminal methylation, phosphorylation/poly-phosphorylation, citrullination, and acetylation, have been reported recently. However, most of their biosynthetic logic have not been elucidated except the phosphorylated paeninodin lasso peptide. Herein, we identified two novel lassomycin-like lasso peptide biosynthetic pathways and, for the first time, characterized a novel C-terminal peptide carboxyl methyltransferase involved in these pathways. Our investigations revealed that this new family of methyltransferase could specifically methylate the C terminus of precursor peptide substrates, eventually leading to lassomycin-like C-terminal methylated lasso peptides. Our studies offer another rare insight into the extraordinary strategies of chemical diversification adopted by lasso peptide biosynthetic machinery and predicated two valuable sources for methylated lasso peptide discovery.

Nanoscale Discrimination between Toxic and Nontoxic Protein Misfolded Oligomers with Tip-Enhanced Raman Spectroscopy.

Highly toxic protein misfolded oligomers associated with neurological disorders such as Alzheimer's and Parkinson's diseases are nowadays considered primarily responsible for promoting synaptic failure and neuronal death. Unraveling the relationship between structure and neurotoxicity of protein oligomers appears pivotal in understanding the causes of the pathological process, as well as in designing novel diagnostic and therapeutic strategies tuned toward the earliest and presymptomatic stages of the disease. Here, it is benefited from tip-enhanced Raman spectroscopy (TERS) as a surface-sensitive tool with spatial resolution on the nanoscale, to inspect the spatial organization and surface character of individual protein oligomers from two samples formed by the same polypeptide sequence and different toxicity levels. TERS provides direct assignment of specific amino acid residues that are exposed to a large extent on the surface of toxic species and buried in nontoxic oligomers. These residues, thanks to their outward disposition, might represent structural factors driving the pathogenic behavior exhibited by protein misfolded oligomers, including affecting cell membrane integrity and specific signaling pathways in neurodegenerative conditions.

New secondary metabolites from an engineering mutant of endophytic Streptomyces sp. CS.

In previous work, a series of bioactive natural products had been isolated from the plant endophytic Streptomyces sp. CS, which was isolated from Maytenus hookeri. To mine new active metabolites, we describe introducing an alien carbamoyltransferase (asm21) gene into the strain CS by conjugal transfer. As a result, three recombinatorial mutants named CS/asm21-1, CS/asm21-2 and CS/asm21-4 were successfully constructed. Three mutants and wild type CS were cultured on solid medium, and the extracts were detected and analyzed by liquid chromatography-mass spectrometry (LC-MS). The LC-MS profiles showed several unknown peaks that were present in the spectra of extracts of the CS/asm21-4 cultured on oatmeal solid medium. Then, three new naphthomycins O-Q (1-3), a new macrolide hookerolide (4) as well as nine known compounds were obtained from the solid cultured medium. Their structures were identified by spectra data. These new compounds showed moderate antimicrobial activities.

Structural differences between toxic and nontoxic HypF-N oligomers.

We have studied two misfolded oligomeric forms of the protein HypF-N, which show similar morphologies but very different toxicities. We measured over 80 intermolecular distance-dependent parameters for each oligomer type using FRET, in conjunction with solution- and solid-state NMR and other biophysical techniques. The results indicate that the formation of a highly organised hydrogen bonded core in the toxic oligomers results in the exposure of a larger number of hydrophobic residues than in the nontoxic species, causing the former to form aberrant interactions with cellular components.

Preliminary studies on the antibacterial mechanism of Yanglingmycin.

The antibacterial mechanism of Yanglingmycin, a new dihydrooxazole antibiotic, was preliminarily investigated by symptomatology observation and physical and biochemical analysis. The electron microscopy observation exhibited that the bacterial cell became elongated, appeared breakage or even cavities on the cell surface after treated with Yanglingmycin. The content of reducing sugar and the activity levels of alanine transaminase and aspartate transaminase in treated group had a significant increase compared to control group. These results indicated that the integrity of bacteria cell membrane was damaged by the antibiotic. Furthermore, the activity of Accase and carboxyltransferase could be effectively inhibited by Yanglingmycin. Meanwhile, the addition of exogenous fatty acid resulted in the decrease or even loss of the antibacterial activity of Yanglingmycin. These findings implied that Yanglingmycin might take effect by inhibiting the activity of Accase, which resulted in the blockade of fatty acids and lipids biosynthesis.

A revised model for the control of fatty acid synthesis by master regulator Spo0A in Bacillus subtilis.

In starving Bacillus subtilis cells, the accDA operon encoding two subunits of the essential acetyl-CoA carboxylase (ACC) has been proposed to be tightly regulated by direct binding of the master regulator Spo0A to a cis element (0A box) in the promoter region. When the 0A box is mutated, biofilm formation and sporulation have been reported to be impaired. Here, we present evidence that two 0A boxes, one previously known (0A-1) and another newly discovered (0A-2) in the accDA promoter region are positively and negatively regulated by Spo0A∼P respectively. Cells with mutated 0A boxes experience slight delays in sporulation, but eventually sporulate with high efficiency. In contrast, cells harboring a single mutated 0A-2 box are deficient for biofilm formation, while cells harboring either a mutated 0A-1 box or both mutated 0A boxes form biofilms. We further show that the essential ACC enzyme localizes on or near the cell membrane by directly observing a functional GFP fusion to one of the enzyme's subunits. Collectively, we propose a revised model in which accDA is primarily transcribed by a major σA -RNA polymerase, while Spo0A∼P plays an additional role in the fine-tuning of accDA expression upon starvation to support proper biofilm formation and sporulation.

Introduction of an acetyl-CoA carboxylation bypass into Escherichia coli for enhanced free fatty acid production.

This study investigated the effect of the methylmalonyl-CoA carboxyltransferase (MMC) of Propionibacterium freudenreichii on production of free fatty acid (FFA) in Escherichia coli. Overexpression of the MMC exhibited a 44% increase in FFA titer. Co-overexpression of MMC and phosphoenolpyruvate carboxylase (PPC), which supplies the MMC precursor, further improved the titer by 40%. Expression of malic enzyme (MaeB) led to a 23% increase in FFA titer in the acetyl-CoA carboxylase (ACC)-overexpressing cells, but no increase in the MMC-overexpressing cells. The highest FFA production in the MMC-overexpressing strain was achieved through the addition of aspartic acid, which can be converted into oxaloacetate (OAA), resulting in a 120% increased titer compared with that in the ACC-overexpressing strain. These findings demonstrate that MMC provides an alternative pathway for malonyl-CoA synthesis and increases fatty acid production.

Proteolytic cleavage orchestrates cofactor insertion and protein assembly in [NiFe]-hydrogenase biosynthesis.

Metalloenzymes catalyze complex and essential processes, such as photosynthesis, respiration, and nitrogen fixation. For example, bacteria and archaea use [NiFe]-hydrogenases to catalyze the uptake and release of molecular hydrogen (H2). [NiFe]-hydrogenases are redox enzymes composed of a large subunit that harbors a NiFe(CN)2CO metallo-center and a small subunit with three iron-sulfur clusters. The large subunit is synthesized with a C-terminal extension, cleaved off by a specific endopeptidase during maturation. The exact role of the C-terminal extension has remained elusive; however, cleavage takes place exclusively after assembly of the [NiFe]-cofactor and before large and small subunits form the catalytically active heterodimer. To unravel the functional role of the C-terminal extension, we used an enzymatic in vitro maturation assay that allows synthesizing functional [NiFe]-hydrogenase-2 of Escherichia coli from purified components. The maturation process included formation and insertion of the NiFe(CN)2CO cofactor into the large subunit, endoproteolytic cleavage of the C-terminal extension, and dimerization with the small subunit. Biochemical and spectroscopic analysis indicated that the C-terminal extension of the large subunit is essential for recognition by the maturation machinery. Only upon completion of cofactor insertion was removal of the C-terminal extension observed. Our results indicate that endoproteolytic cleavage is a central checkpoint in the maturation process. Here, cleavage temporally orchestrates cofactor insertion and protein assembly and ensures that only cofactor-containing protein can continue along the assembly line toward functional [NiFe]-hydrogenase.