HMGCL-induced ß-hydroxybutyrate production attenuates hepatocellular carcinoma via DPP4-mediated ferroptosis susceptibility.

BACKGROUND: Metabolic disorder is an essential characteristic of tumor development. Ketogenesis is a heterogeneous factor in multiple cancers, but the effect of ketogenesis on hepatocellular carcinoma (HCC) is elusive. METHODS: We aimed to explain the role of ketogenesis-related hydroxy-methyl-glutaryl-CoA lyase (HMGCL) on HCC suppression. Expression pattern of HMGCL in HCC specimens was evaluated by immunohistochemistry (IHC). HMGCL was depleted or overexpressed in HCC cells to investigate the functions of HMGCL in vitro and in vivo. The anti-tumor function of HMGCL was studied in subcutaneous xenograft and Trp53Δhep/Δhep; c-Myc-driven HCC mouse models. The mechanism of HMGCL-mediated tumor suppression was studied by IHC, western blot (WB) and Cut & Tag. RESULTS: HMGCL depletion promoted HCC proliferation and metastasis, whereas its overexpression reversed this trend. As HMGCL catalyzes ß-hydroxy-butyric acid (ß-OHB) production, we discovered that HMGCL increased acetylation at histone H3K9, which further promoted the transcription of dipeptidyl peptidase 4 (DPP4), a key protein maintains intracellular lipid peroxidation and iron accumulation, leading to HCC cells vulnerability to erastin- and sorafenib-induced ferroptosis. CONCLUSION: Our study identified a critical role of HMGCL on HCC suppression, of which HMGCL regulated H3K9 acetylation through ß-OHB and modulating the expression of DPP4 in a dose-dependent manner, which led to ferroptosis in HCC cells.

HOGA1 gene pathogenic variants in primary hyperoxaluria type III: Spectrum of pathogenic sequence variants, and phenotypic association.

Primary hyperoxalurias (PH) are a group of rare heterogeneous disorders characterized by deficiencies in glyoxylate metabolism. To date, three genes have been identified to cause three types of PH (I, II, and III). The HOGA1 gene caused type III in around 10% of the PH cases. Disease-associated pathogenic variants have been reported from several populations and a comprehensive spectrum of these mutations and genotype-phenotype correlation has never been presented. In this study, we describe new cases of the HOGA1 gene pathogenic variants identified in our population. We report the first case of ESKD with successful kidney transplantation with 5 years of follow-up. Furthermore, a comprehensive overview of PH type III associated HOGA1 gene variants was carried out. Compiling the data from the literature, we reviewed 57 distinct HOGA1 gene pathogenic variants in 175 patients worldwide. The majority of reported variants are missense variants that predicted a loss of function mechanism as the underlying pathology. There has been evidence of the presence of founder mutations in several populations like Europeans, Ashkenazi Jews, Arab, and Chinese populations. No significant genotype-phenotype correlation was identified concerning the ages of onset of the disease and biochemical and metabolic parameters. Nephrocalcinosis was rare in patients with disease-associated variants. Most of the patients were presented with urolithiasis early in life; only five cases reported disease progression after the second decade of life. The establishment of impairment of renal function in 8% of all the reported cases makes this type a relatively severe form of primary hyperoxaluria, not a benign etiology as suggested previously.

Recombinant human N-acetylneuraminate lyase as a tool to study clinically relevant mutant variants.

N-acetylneuraminic acid (sialic acid) is an abundantly found carbohydrate moiety covering the surface of all vertebrate cells and secreted glycoproteins. The human N-acetylneuraminate pyruvate lyase (NPL) interconverts sialic acid to N-acetylmannosamine and pyruvate, and mutations of the NPL gene were found to cause sialuria and impair the functionality of muscles. Here we report the soluble and functional expression of human NPL in Escherichia coli, which allowed us to study the biochemical properties of two clinically relevant NLP mutations (Asn45Asp and Arg63Cys). The Asn45Asp mutant variant was enzymatically active, but had lower expression levels and showed reduced stability when compared to the wild-type NPL variant. Expression trials of the Arg63Cys mutant did not yield any recombinant protein and consequently, no enzymatic activity was detected. The locations of these clinically relevant amino acid substitutions are also discussed by using a human NPL homology model.

High-Level Production of Lysine in the Yeast Saccharomyces cerevisiae by Rational Design of Homocitrate Synthase.

Homocitrate synthase (HCS) catalyzes the aldol condensation of 2-oxoglutarate (2-OG) and acetyl coenzyme A (AcCoA) to form homocitrate, which is the first enzyme of the lysine biosynthetic pathway in the yeast Saccharomyces cerevisiae. The HCS activity is tightly regulated via feedback inhibition by the end product lysine. Here, we designed a feedback inhibition-insensitive HCS of S. cerevisiae (ScLys20) for high-level production of lysine in yeast cells. In silico docking of the substrate 2-OG and the inhibitor lysine to ScLys20 predicted that the substitution of serine with glutamate at position 385 would be more suitable for desensitization of the lysine feedback inhibition than the substitution from serine to phenylalanine in the already known Ser385Phe variant. Enzymatic analysis revealed that the Ser385Glu variant is far more insensitive to feedback inhibition than the Ser385Phe variant. We also found that the lysine contents in yeast cells expressing the Ser385Glu variant were 4.62- and 1.47-fold higher than those of cells expressing the wild-type HCS and Ser385Phe variant, respectively, due to the extreme desensitization to feedback inhibition. In this study, we obtained highly feedback inhibition-insensitive HCS using in silico docking and enzymatic analysis. Our results indicate that the rational engineering of HCS for feedback inhibition desensitization by lysine could be useful for constructing new yeast strains with higher lysine productivity. IMPORTANCE A traditional method for screening toxic analogue-resistant mutants has been established for the breeding of microbes that produce high levels of amino acids, including lysine. However, another efficient strategy is required to further improve their productivity. Homocitrate synthase (HCS) catalyzes the first step of lysine biosynthesis in the yeast Saccharomyces cerevisiae, and its activity is subject to feedback inhibition by lysine. Here, in silico design of a key enzyme that regulates the biosynthesis of lysine was utilized to increase the productivity of lysine. We designed HCS for the high-level production of lysine in yeast cells by in silico docking simulation. The engineered HCS exhibited much less sensitivity to lysine and conferred higher production of lysine than the already known variant obtained by traditional breeding. The combination of in silico design and experimental analysis of a key enzyme will contribute to advances in metabolic engineering for the construction of industrial microorganisms.

Origin and evolution of nonulosonic acid synthases and their relationship with bacterial pathogenicity revealed by a large-scale phylogenetic analysis.

Nonulosonic acids (NulOs) are a group of nine-carbon monosaccharides with different functions in nature. N-acetylneuraminic acid (Neu5Ac) is the most common NulO. It covers the membrane surface of all human cells and is a central molecule in the process of self-recognition via SIGLECS receptors. Some pathogenic bacteria escape the immune system by copying the sialylation of the host cell membrane. Neu5Ac production in these bacteria is catalysed by the enzyme NeuB. Some bacteria can also produce other NulOs named pseudaminic and legionaminic acids, through the NeuB homologues PseI and LegI, respectively. In Opisthokonta eukaryotes, the biosynthesis of Neu5Ac is catalysed by the enzyme NanS. In this study, we used publicly available data of sequences of NulOs synthases to investigate its distribution within the three domains of life and its relationship with pathogenic bacteria. We mined the KEGG database and found 425 NeuB sequences. Most NeuB sequences (58.74 %) from the KEGG orthology database were classified as from environmental bacteria; however, sequences from pathogenic bacteria showed higher conservation and prevalence of a specific domain named SAF. Using the HMM profile we identified 13 941 NulO synthase sequences in UniProt. Phylogenetic analysis of these sequences showed that the synthases were divided into three main groups that can be related to the lifestyle of these bacteria: (I) predominantly environmental, (II) intermediate and (III) predominantly pathogenic. NeuB was widely distributed in the groups. However, LegI and PseI were more concentrated in groups II and III, respectively. We also found that PseI appeared later in the evolutionary process, derived from NeuB. We use this same methodology to retrieve sialic acid synthase sequences from Archaea and Eukarya. A large-scale phylogenetic analysis showed that while the Archaea sequences are spread across the tree, the eukaryotic NanS sequences were grouped in a specific branch in group II. None of the bacterial NanS sequences grouped with the eukaryotic branch. The analysis of conserved residues showed that the synthases of Archaea and Eukarya present a mutation in one of the three catalytic residues, an E134D change, related to a Neisseria meningitidis reference sequence. We also found that the conservation profile is higher between NeuB of pathogenic bacteria and NanS of eukaryotes than between NeuB of environmental bacteria and NanS of eukaryotes. Our large-scale analysis brings new perspectives on the evolution of NulOs synthases, suggesting their presence in the last common universal ancestor.

Paraburkholderia phymatum Homocitrate Synthase NifV Plays a Key Role for Nitrogenase Activity during Symbiosis with Papilionoids and in Free-Living Growth Conditions.

Homocitrate is an essential component of the iron-molybdenum cofactor of nitrogenase, the bacterial enzyme that catalyzes the reduction of dinitrogen (N2) to ammonia. In nitrogen-fixing and nodulating alpha-rhizobia, homocitrate is usually provided to bacteroids in root nodules by their plant host. In contrast, non-nodulating free-living diazotrophs encode the homocitrate synthase (NifV) and reduce N2 in nitrogen-limiting free-living conditions. Paraburkholderia phymatum STM815 is a beta-rhizobial strain, which can enter symbiosis with a broad range of legumes, including papilionoids and mimosoids. In contrast to most alpha-rhizobia, which lack nifV, P. phymatum harbors a copy of nifV on its symbiotic plasmid. We show here that P. phymatum nifV is essential for nitrogenase activity both in root nodules of papilionoid plants and in free-living growth conditions. Notably, nifV was dispensable in nodules of Mimosa pudica despite the fact that the gene was highly expressed during symbiosis with all tested papilionoid and mimosoid plants. A metabolome analysis of papilionoid and mimosoid root nodules infected with the P. phymatum wild-type strain revealed that among the approximately 400 measured metabolites, homocitrate and other metabolites involved in lysine biosynthesis and degradation have accumulated in all plant nodules compared to uninfected roots, suggesting an important role of these metabolites during symbiosis.

Listeria monocytogenes MenI Encodes a DHNA-CoA Thioesterase Necessary for Menaquinone Biosynthesis, Cytosolic Survival, and Virulence.

Listeria monocytogenes is a Gram-positive, intracellular pathogen that is highly adapted to invade and replicate in the cytosol of eukaryotic cells. Intermediate metabolites in the menaquinone biosynthesis pathway are essential for the cytosolic survival and virulence of L. monocytogenes, independent of the production of menaquinone (MK) and aerobic respiration. Determining which specific intermediate metabolite(s) are essential for cytosolic survival and virulence has been hindered by the lack of an identified 1,4-dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) thioesterase essential for converting DHNA-CoA to DHNA in the MK synthesis pathway. Using the recently identified Escherichia coli DHNA-CoA thioesterase as a query, homology sequence analysis revealed a single homolog in L. monocytogenes, LMRG_02730 Genetic deletion of LMRG_02730 resulted in an ablated membrane potential, indicative of a nonfunctional electron transport chain (ETC) and an inability to aerobically respire. Biochemical kinetic analysis of LMRG_02730 revealed strong activity toward DHNA-CoA, similar to its E. coli homolog, further demonstrating that LMRG_02730 is a DHNA-CoA thioesterase. Functional analyses in vitro, ex vivo, and in vivo using mutants directly downstream and upstream of LMRG_02730 revealed that DHNA-CoA is sufficient to facilitate in vitro growth in minimal medium, intracellular replication, and plaque formation in fibroblasts. In contrast, protection against bacteriolysis in the cytosol of macrophages and tissue-specific virulence in vivo requires the production of 1,4-dihydroxy-2-naphthoate (DHNA). Taken together, these data implicate LMRG_02730 (renamed MenI) as a DHNA-CoA thioesterase and suggest that while DHNA, or an unknown downstream product of DHNA, protects the bacteria from killing in the macrophage cytosol, DHNA-CoA is necessary for intracellular bacterial replication.

In vitro characterization of 3-chloro-4-hydroxybenzoic acid building block formation in ambigol biosynthesis.

The cyanobacterium Fischerella ambigua is a natural producer of polychlorinated aromatic compounds, the ambigols A-E. The biosynthetic gene cluster (BGC) of these highly halogenated triphenyls has been recently identified by heterologous expression. It consists of 10 genes named ab1-10. Two of the encoded enzymes, i.e. Ab2 and Ab3, were identified by in vitro and in vivo assays as cytochrome P450 enzymes responsible for biaryl and biaryl ether formation. The key substrate for these P450 enzymes is 2,4-dichlorophenol, which in turn is derived from the precursor 3-chloro-4-hydroxybenzoic acid. Here, the biosynthetic steps leading towards 3-chloro-4-hydroxybenzoic acid were investigated by in vitro assays. Ab7, an isoenzyme of a 3-deoxy-7-phosphoheptulonate (DAHP) synthase, is involved in chorismate biosynthesis by the shikimate pathway. Chorismate in turn is further converted by a dedicated chorismate lyase (Ab5) yielding 4-hydroxybenzoic acid (4-HBA). The stand alone adenylation domain Ab6 is necessary to activate 4-HBA, which is subsequently tethered to the acyl carrier protein (ACP) Ab8. The Ab8 bound substrate is chlorinated by Ab10 in meta position yielding 3-Cl-4-HBA, which is then transfered by the condensation (C) domain to the peptidyl carrier protein and released by the thioesterase (TE) domain of Ab9. The released product is then expected to be the dedicated substrate of the halogenase Ab1 producing the monomeric ambigol building block 2,4-dichlorophenol.

Chorismate- and isochorismate converting enzymes: versatile catalysts acting on an important metabolic node.

Chorismate and isochorismate represent an important branching point connecting primary and secondary metabolism in bacteria, fungi, archaea and plants. Chorismate- and isochorismate-converting enzymes are potential targets for new bioactive compounds, as well as valuable biocatalysts for the in vivo and in vitro synthesis of fine chemicals. The diversity of the products of chorismate- and isochorismate-converting enzymes is reflected in the enzymatic three-dimensional structures and molecular mechanisms. Due to the high reactivity of chorismate and its derivatives, these enzymes have evolved to be accurately tailored to their respective reaction; at the same time, many of them exhibit a fascinating flexibility regarding side reactions and acceptance of alternative substrates. Here, we give an overview of the different (sub)families of chorismate- and isochorismate-converting enzymes, their molecular mechanisms, and three-dimensional structures. In addition, we highlight important results of mutagenetic approaches that generate a broader understanding of the influence of distinct active site residues for product formation and the conversion of one subfamily into another. Based on this, we discuss to what extent the recent advances in the field might influence the general mechanistic understanding of chorismate- and isochorismate-converting enzymes. Recent discoveries of new chorismate-derived products and pathways, as well as biocatalytic conversions of non-physiological substrates, highlight how this vast field is expected to continue developing in the future.

Identification of a bacteria-produced benzisoxazole with antibiotic activity against multi-drug resistant Acinetobacter baumannii.

The emergence of multi-drug resistant pathogenic bacteria represents a serious and growing threat to national healthcare systems. Most pressing is an immediate need for the development of novel antibacterial agents to treat Gram-negative multi-drug resistant infections, including the opportunistic, hospital-derived pathogen, Acinetobacter baumannii. Herein we report a naturally occurring 1,2-benzisoxazole with minimum inhibitory concentrations as low as 6.25 µg ml-1 against clinical strains of multi-drug resistant A. baumannii and investigate its possible mechanisms of action. This molecule represents a new chemotype for antibacterial agents against A. baumannii and is easily accessed in two steps via de novo synthesis. In vitro testing of structural analogs suggest that the natural compound may already be optimized for activity against this pathogen. Our results demonstrate that supplementation of 4-hydroxybenzoate in minimal media was able to reverse 1,2-benzisoxazole's antibacterial effects in A. baumannii. A search of metabolic pathways involving 4-hydroxybenzoate coupled with molecular modeling studies implicates two enzymes, chorismate pyruvate-lyase and 4-hydroxybenzoate octaprenyltransferase, as promising leads for the target of 3,6-dihydroxy-1,2-benzisoxazole.

Involvement of subdomain II in the recognition of acetyl-CoA revealed by the crystal structure of homocitrate synthase from Sulfolobus acidocaldarius.

Homocitrate synthase (HCS) catalyzes the aldol condensation of α-ketoglutarate and acetyl coenzyme A to form homocitrate, which is the first committed step of lysine biosynthesis through the α-aminoadipate pathway in yeast, fungi, and some prokaryotes. We determined the crystal structure of a truncated form of HCS from a hyperthermophilic acidophilic archaeon, Sulfolobus acidocaldarius, which lacks the RAM (Regulation of amino acid metabolism) domain at the C terminus serving as the regulatory domain for the feedback inhibition by lysine, in complex with α-ketoglutarate, Mg2+ , and CoA. This structure coupled with mutational analysis revealed that a subdomain, subdomain II, connecting the N-terminal catalytic domain and C-terminal RAM domain is involved in the recognition of acetyl-CoA. This is the first structural evidence of the function of subdomain II in the related enzyme family, which will lead to a better understanding of the catalytic mechanism of HCS. DATABASES: Structural data are available in the RCSB PDB database under the accession number 6KTQ.

Malyl-CoA lyase provides glycine/glyoxylate synthesis in type I methanotrophs.

The biochemical routes for assimilation of one-carbon compounds in bacteria require many clarifications. In this study, the role of malyl-CoA lyase in the metabolism of the aerobic type I methanotroph Methylotuvimicrobium alcaliphilum 20Z has been investigated by gene inactivation and biochemical studies. The functionality of the enzyme has been confirmed by heterologous expression in Escherichia coli. The mutant strain lacking Mcl activity demonstrated the phenotype of glycine auxotrophy. The genes encoding malyl-CoA lyase are present in the genomes of all methanotrophs, except for representatives of the phylum Verrucomicrobium. We suppose that malyl-CoA lyase is the enzyme that provides glyoxylate and glycine synthesis in the type I methanotrophs supporting carbon assimilation via the serine cycle in addition to the major ribulose monophosphate cycle.

The Siderophore Synthetase IucA of the Aerobactin Biosynthetic Pathway Uses an Ordered Mechanism.

Biosynthesis of the hydroxamate siderophore aerobactin requires the activity of four proteins encoded within the iuc operon. Recently, we biochemically reconstituted the biosynthetic pathway and structurally characterized IucA and IucC, two enzymes that sequentially couple N6-acetyl-N6-hydroxylysine to the primary carboxylates of citrate. IucA and IucC are members of a family of non-ribosomal peptide synthetase-independent siderophore (NIS) synthetases that are involved in the production of other siderophores, including desferrioxamine, achromobactin, and petrobactin. While structures of several members of this family were solved previously, there is limited mechanistic insight into the reaction catalyzed by NIS synthetases. Therefore, we performed a terreactant steady-state kinetic analysis and herein provide evidence for an ordered mechanism in which the chemistry is preceded by the formation of the quaternary complex. We further probed two regions of the active site with site-directed mutagenesis and identified several residues, including a conserved motif that is present on a dynamic loop, that are important for substrate binding and catalysis.

Citrate lyase CitE in Mycobacterium tuberculosis contributes to mycobacterial survival under hypoxic conditions.

Mycobacterium tuberculosis is the causative agent of tuberculosis and has evolved an ability to survive in hostile host environments. M. tuberculosis is thought to utilize the rTCA cycle to sustain its latent growth during infection, but the enzymatic characteristics and physiological function for the key citrate lyase of the rTCA cycle, MtbCitE, in the important pathogen remain unclear. In this study, we investigated the function of MtbCitE based on its structural properties and sequence comparisons with other bacterial citrate lyase subunits. We showed that several amino acid residues were important for the citrate cleavage activity of MtbCitE. Strikingly, the citrate cleavage activity of MtbCitE was inhibited by ATP, indicating that energy metabolism might couple with the regulation of MtbCitE activity, which differed from other CitEs. More interestingly, deletion of citE from Mycobacterium bovis BCG decreased the mycobacterial survival rate under hypoxic conditions, whereas complementation with citE restored the phenotype to wild-type levels. Consistently, three key rTCA cycle enzymes were positively regulated under hypoxic conditions in mycobacteria. Therefore, we characterized a unique citrate lyase MtbCitE from M. tuberculosis and found that the CitE protein significantly contributed to mycobacterial survival under hypoxic conditions.

The Drosophila Citrate Lyase Is Required for Cell Division during Spermatogenesis.

The Drosophila melanogasterDmATPCL gene encodes for the human ATP Citrate Lyase (ACL) ortholog, a metabolic enzyme that from citrate generates glucose-derived Acetyl-CoA, which fuels central biochemical reactions such as the synthesis of fatty acids, cholesterol and acetylcholine, and the acetylation of proteins and histones. We had previously reported that, although loss of Drosophila ATPCL reduced levels of Acetyl-CoA, unlike its human counterpart, it does not affect global histone acetylation and gene expression, suggesting that its role in histone acetylation is either partially redundant in Drosophila or compensated by alternative pathways. Here, we describe that depletion of DmATPCL affects spindle organization, cytokinesis, and fusome assembly during male meiosis, revealing an unanticipated role for DmATPCL during spermatogenesis. We also show that DmATPCL mutant meiotic phenotype is in part caused by a reduction of fatty acids, but not of triglycerides or cholesterol, indicating that DmATPCL-derived Acetyl-CoA is predominantly devoted to the biosynthesis of fatty acids during spermatogenesis. Collectively, our results unveil for the first time an involvement for DmATPCL in the regulation of meiotic cell division, which is likely conserved in human cells.

Characterization of l-2-keto-3-deoxyfuconate aldolases in a nonphosphorylating l-fucose metabolism pathway in anaerobic bacteria.

The genetic context in bacterial genomes and screening for potential substrates can help identify the biochemical functions of bacterial enzymes. The Gram-negative, strictly anaerobic bacterium Veillonella ratti possesses a gene cluster that appears to be related to l-fucose metabolism and contains a putative dihydrodipicolinate synthase/N-acetylneuraminate lyase protein (FucH). Here, screening of a library of 2-keto-3-deoxysugar acids with this protein and biochemical characterization of neighboring genes revealed that this gene cluster encodes enzymes in a previously unknown "route I" nonphosphorylating l-fucose pathway. Previous studies of other aldolases in the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein superfamily used only limited numbers of compounds, and the approach reported here enabled elucidation of the substrate specificities and stereochemical selectivities of these aldolases and comparison of them with those of FucH. According to the aldol cleavage reaction, the aldolases were specific for (R)- and (S)-stereospecific groups at the C4 position of 2-keto-3-deoxysugar acid but had no structural specificity or preference of methyl groups at the C5 and C6 positions, respectively. This categorization corresponded to the (Re)- or (Si)-facial selectivity of the pyruvate enamine on the (glycer)aldehyde carbonyl in the aldol-condensation reaction. These properties are commonly determined by whether a serine or threonine residue is positioned at the equivalent position close to the active site(s), and site-directed mutagenesis markedly modified C4-OH preference and selective formation of a diastereomer. I propose that substrate specificity of 2-keto-3-deoxysugar acid aldolases was convergently acquired during evolution and report the discovery of another l-2-keto-3-deoxyfuconate aldolase involved in the same nonphosphorylating l-fucose pathway in Campylobacter jejuni.

LC-MS/MS-based metabolic profiling of Escherichia coli under heterologous gene expression stress.

Escherichia coli is frequently exploited for genetic manipulations and heterologous gene expression studies. We have evaluated the metabolic profile of E. coli strain BL21 (DE3) RIL CodonPlus after genetic modifications and subjecting to the production of recombinant protein. Three genetically variable E. coli cell types were studied, normal cells (susceptible to antibiotics) cultured in simple LB medium, cells harboring ampicillin-resistant plasmid pET21a (+), grown under antibiotic stress, and cells having recombinant plasmid pET21a (+) ligated with bacterial lactate dehydrogenase gene grown under ampicillin and standard isopropyl thiogalactoside (IPTG)-induced gene expression conditions. A total of 592 metabolites were identified through liquid chromatography-mass spectrometry/mass spectrometry analysis, feature and peak detection using XCMS and CAMERA followed by precursor identification by METLIN-based procedures. Overall, 107 metabolites were found differentially regulated among genetically modified cells. Quantitative analysis has shown a significant modulation in DHNA-CoA, p-aminobenzoic acid, and citrulline levels, indicating an alteration in vitamin K, folic acid biosynthesis, and urea cycle of E. coli cells during heterologous gene expression. Modulations in energy metabolites including NADH, AMP, ADP, ATP, carbohydrate, terpenoids, fatty acid metabolites, diadenosine tetraphosphate (Ap4A), and l-carnitine advocate major metabolic rearrangements. Our study provides a broader insight into the metabolic adaptations of bacterial cells during gene manipulation experiments that can be prolonged to improve the yield of heterologous gene products and concomitant production of valuable biomolecules.

Biochemical characterization of archaeal homocitrate synthase from Sulfolobus acidocaldarius.

The hyperthermophilic archaeon, Sulfolobus, synthesizes lysine via the α-aminoadipate pathway; however, the gene encoding homocitrate synthase, the enzyme responsible for the first and committed step of the pathway, has not yet been identified. In the present study, we identified saci_1304 as the gene encoding a novel type of homocitrate synthase fused with a Regulation of Amino acid Metabolism (RAM) domain at the C terminus in Sulfolobus acidocaldarius. Enzymatic characterization revealed that Sulfolobus homocitrate synthase was inhibited by lysine; however, the mutant enzyme lacking the RAM domain was insensitive to inhibition by lysine. The present results indicated that the RAM domain is responsible for enzyme inhibition.

Cytosolic Acetyl-CoA Generated by ATP-Citrate Lyase Is Essential for Acetylation of Cell Wall Polysaccharides.

Plant cell wall polysaccharides, including xylan, glucomannan, xyloglucan and pectin, are often acetylated. Although a number of acetyltransferases responsible for the acetylation of some of these polysaccharides have been biochemically characterized, little is known about the source of acetyl donors and how acetyl donors are translocated into the Golgi, where these polysaccharides are synthesized. In this report, we investigated roles of ATP-citrate lyase (ACL) that generates cytosolic acetyl-CoA in cell wall polysaccharide acetylation and effects of simultaneous mutations of four Reduced Wall Acetylation (RWA) genes on acetyl-CoA transport into the Golgi in Arabidopsis thaliana. Expression analyses of genes involved in the generation of acetyl-CoA in different subcellular compartments showed that the expression of several ACL genes responsible for cytosolic acetyl-CoA synthesis was elevated in interfascicular fiber cells and induced by secondary wall-associated transcriptional activators. Simultaneous downregulation of the expression of ACL genes was demonstrated to result in a substantial decrease in the degree of xylan acetylation and a severe alteration in secondary wall structure in xylem vessels. In addition, the degree of acetylation of other cell wall polysaccharides, including glucomannan, xyloglucan and pectin, was also reduced. Moreover, Golgi-enriched membrane vesicles isolated from the rwa1/2/3/4 quadruple mutant were found to exhibit a drastic reduction in acetyl-CoA transport activity compared with the wild type. These findings indicate that cytosolic acetyl-CoA generated by ACL is essential for cell wall polysaccharide acetylation and RWAs are required for its transport from the cytosol into the Golgi.

3-Methylglutaric acid in energy metabolism.

3-methylglutaric (3MG) acid is a conspicuous C6 dicarboxylic organic acid classically associated with two distinct leucine pathway enzyme deficiencies. 3MG acid is excreted in urine of individuals harboring deficiencies in 3-hydroxy-3-methylglutaryl (HMG) CoA lyase (HMGCL) or 3-methylglutaconyl CoA hydratase (AUH). Whereas 3MG CoA is not part of the leucine catabolic pathway, it is likely formed via a side reaction involving reduction of the α-ß trans double bond in the leucine pathway intermediate, 3-methylglutaconyl CoA. While the metabolic basis for the accumulation of 3MG acid in subjects with deficiencies in HMGCL or AUH is apparent, the occurrence of 3MG aciduria in a host of unrelated inborn errors of metabolism associated with compromised mitochondrial energy metabolism is less clear. Herein, a novel mitochondrial biosynthetic pathway termed "the acetyl CoA diversion pathway", provides an explanation. The pathway is initiated by defective electron transport chain function which, ultimately, inhibits acetyl CoA entry into the TCA cycle. When this occurs, 3MG acid is synthesized in five steps from acetyl CoA via a novel reaction sequence, providing a metabolic rationale for the connection between 3MG aciduria and compromised mitochondrial energy metabolism.