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The generation of an active [FeFe]-hydrogenase requires the synthesis of a complex metal center, the H-cluster, by three dedicated maturases: the radical S-adenosyl-l-methionine (SAM) enzymes HydE and HydG, and the GTPase HydF. A key step of [FeFe]-hydrogenase maturation is the synthesis of the dithiomethylamine (DTMA) bridging ligand, a process recently shown to involve the aminomethyl-lipoyl-H-protein from the glycine cleavage system, whose methylamine group originates from serine and ammonium. Here we use functional assays together with electron paramagnetic resonance and electron-nuclear double resonance spectroscopies to show that serine or aspartate together with their respective ammonia-lyase enzymes can provide the nitrogen for DTMA biosynthesis during in vitro [FeFe]-hydrogenase maturation. We also report bioinformatic analysis of the hyd operon, revealing a strong association with genes encoding ammonia-lyases, suggesting important biochemical and metabolic connections. Together, our results provide evidence that ammonia-lyases play an important role in [FeFe]-hydrogenase maturation by delivering the ammonium required for dithiomethylamine ligand synthesis., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In cyanobacteria, metabolic pathways that use the nitrogen-rich amino acid arginine play a pivotal role in nitrogen storage and mobilization. The N-terminal domains of two recently identified bacterial enzymes: ArgZ from Synechocystis and AgrE from Anabaena , have been found to contain an arginine dihydrolase. This enzyme provides catabolic activity that converts arginine to ornithine, resulting in concomitant release of CO 2 and ammonia. In Synechocystis , the ArgZ-mediated ornithine-ammonia cycle plays a central role in nitrogen storage and remobilization. The C-terminal domain of AgrE contains an ornithine cyclodeaminase responsible for the formation of proline from ornithine and ammonia production, indicating that AgrE is a bifunctional enzyme catalyzing two sequential reactions in arginine catabolism. Here, the crystal structures of AgrE in three different ligation states revealed that it has a tetrameric conformation, possesses a binding site for the arginine dihydrolase substrate l-arginine and product l-ornithine, and contains a binding site for the coenzyme NAD(H) required for ornithine cyclodeaminase activity. Structure-function analyses indicated that the structure and catalytic mechanism of arginine dihydrolase in AgrE are highly homologous with those of a known bacterial arginine hydrolase. We found that in addition to other active-site residues, Asn-71 is essential for AgrE's dihydrolase activity. Further analysis suggested the presence of a passage for substrate channeling between the two distinct AgrE active sites, which are situated ∼45 Å apart. These results provide structural and functional insights into the bifunctional arginine dihydrolase-ornithine cyclodeaminase enzyme AgrE required for arginine catabolism in Anabaena ., (© 2020 Lee and Rhee.)

The data-independent acquisition (DIA) approach has recently been introduced as a novel mass spectrometric method that promises to combine the high content aspect of shotgun proteomics with the reproducibility and precision of selected reaction monitoring. Here, we evaluate, whether SWATH-MS type DIA effectively translates into a better protein profiling as compared with the established shotgun proteomics. We implemented a novel DIA method on the widely used Orbitrap platform and used retention-time-normalized (iRT) spectral libraries for targeted data extraction using Spectronaut. We call this combination hyper reaction monitoring (HRM). Using a controlled sample set, we show that HRM outperformed shotgun proteomics both in the number of consistently identified peptides across multiple measurements and quantification of differentially abundant proteins. The reproducibility of HRM in peptide detection was above 98%, resulting in quasi complete data sets compared with 49% of shotgun proteomics. Utilizing HRM, we profiled acetaminophen (APAP)(1)-treated three-dimensional human liver microtissues. An early onset of relevant proteome changes was revealed at subtoxic doses of APAP. Further, we detected and quantified for the first time human NAPQI-protein adducts that might be relevant for the toxicity of APAP. The adducts were identified on four mitochondrial oxidative stress related proteins (GATM, PARK7, PRDX6, and VDAC2) and two other proteins (ANXA2 and FTCD). Our findings imply that DIA should be the preferred method for quantitative protein profiling., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Although the proteins of the lysine fermentation pathway were biochemically characterized more than thirty years ago, the genes encoding the proteins that catalyze three steps of this pathway are still unknown. We combined gene context, similarity of enzymatic mechanisms, and molecular weight comparisons with known proteins to select candidate genes for these three orphan proteins. We used a wastewater metagenomic collection of sequences to find and characterize the missing genes of the lysine fermentation pathway. After recombinant protein production and purification following cloning in Escherichia coli, we demonstrated that these genes (named kdd, kce, and kal) encode a l-erythro-3,5-diaminohexanoate dehydrogenase, a 3-keto-5-aminohexanoate cleavage enzyme, and a 3-aminobutyryl-CoA ammonia lyase, respectively. Because all of the genes of the pathway are now identified, we used this breakthrough to detect lysine-fermenting bacteria in sequenced genomes. We identified twelve bacteria that possess these genes and thus are expected to ferment lysine, and their gene organization is discussed.

A peripherally associated 58-kDa Golgi protein (58K) of unknown function has been previously described (Bloom, G. S., and Brashear, T. A. (1989) J. Biol. Chem. 264, 16083-16092). To molecularly characterize 58K, we used a monoclonal anti-58K antibody (monoclonal antibody 58K-9) to screen a rat liver cDNA expression library. Positive clones were isolated, characterized, and partially sequenced. The obtained sequences show a high level of identity with sequences of porcine formiminotransferase cyclodeaminase (FTCD), suggesting that 58K is rat FTCD. Rat FTCD is structurally similar to porcine FTCD, a metabolic enzyme involved in conversion of histidine to glutamic acid, and exists in dimeric, tetrameric, and octameric complexes resistant to proteolysis. To define parameters of FTCD association with the Golgi, comparison of its behavior with various Golgi and ER-to-Golgi intermediate compartment marker proteins was examined under specific conditions. The results show that extraction parameters of FTCD are similar to those of GM130, a tightly associated Golgi matrix protein. FTCD appears to be a dynamic component of the Golgi, and a proportion of FTCD molecules cycle between the Golgi and earlier compartments of the secretory pathway. FTCD remains associated with Golgi fragments during microtubule disruption and is not released into cytosol during brefeldin A treatment. Instead, FTCD relocates from the Golgi, but the time course of its redistribution is distinct from that of mannosidase II relocation. FTCD is already dispersed into small punctate structures at a time when mannosidase II is still largely localized to Golgi structures. FTCD is not observed in tubules originating from the Golgi and containing mannosidase II. Instead, it appears to redistribute in small vesicles arranged in a linear "pearls on a string" pattern. These results suggest that FTCD relocation is temporally and spatially distinct from mannosidase II relocation and that FTCD provides a novel marker to study Golgi dynamics.

We have isolated and characterized cDNA clones encoding the porcine liver octameric enzyme, 5-formiminotetrahydrofolate:L-glutamate N-formiminotransferase (EC 2.1.2.5)-formiminotetrahydrofolate cyclodeaminase (EC 4.3.1.4). The cDNA encodes a novel amino acid sequence of 541 residues which contains exact matches to two sequences derived by automated sequence analysis of CNBr cleavage fragments isolated from the porcine enzyme. The recombinant enzyme has been expressed as a soluble protein in Escherichia coli at levels 4-fold higher than those observed in liver, and is bifunctional, displaying both transferase and deaminase activities. With a calculated subunit molecular mass of 58,926 Da, it is similar in size to the enzyme isolated from porcine liver. Purification of the enzyme from E. coli involves chromatography on a novel polyglutamate column which might interact with the folylpolyglutamate binding site of the protein. The purified recombinant enzyme has a transferase specific activity of 39-41 units/mg/min.

Ethanolamine ammonia-lyase is a bacterial enzyme that catalyzes the adenosylcobalamin-dependent conversion of certain vicinal amino alcohols to oxo compounds and ammonia. Studies of ethanolamine ammonia-lyase from Clostridium sp. and Escherichia coli have suggested that the enzyme is a heterodimer composed of subunits of Mr approximately 55,000 and 35,000. Using a partial Sau3A Salmonella typhimurium library ligated into pBR328 and selecting by complementation of a mutant lacking ethanolamine ammonia-lyase activity, we have cloned the genes for the 2 subunits of the S. typhimurium enzyme. The genes were localized to a 6.5-kilobase fragment of S. typhimurium DNA, from which they could be expressed in E. coli under noninducing conditions. Sequencing of a 2526-base pair portion of this 6.5-kilobase DNA fragment revealed two open reading frames separated by 21 base pairs. The open reading frames encoded proteins of 452 and 286 residues whose derived N-terminal sequences were identical to the N-terminal sequences of the 2 subunits of the E. coli ethanolamine ammonia-lyase, except that residue 16 of the large subunit was asparagine in the E. coli sequence and aspartic acid in the S. typhimurium sequence.

The porphobilinogen deaminase gene encodes the third enzyme of the heme biosynthetic pathway. This gene is expressed in a tissue-specific manner and gives rise to two isoenzymatic forms encoded by mRNA species differing in their 5' extremity. Recent studies in human demonstrated that the tissue-specific expression of the porphobilinogen deaminase gene is determined in erythropoietic cells, by the utilization of a specific promoter situated 3' to the housekeeping promoter used in other cell types. This results, through differential splicing, in the mutually exclusive presence of either exon 1 or exon 2 in mature mRNAs. Here, we report the cloning and sequencing of the porphobilinogen deaminase gene from mouse. The overall organization of the mouse gene is similar to that of the human one. In the housekeeping promoter, only a short stretch of homology is found including two potential Sp1 binding sites; in contrast, more extensive similarity appears in the erythroid-specific promoter including two motifs also found in globin gene, a CACCC box, and a recently described Ery F1 consensus binding sequence. We derived a set of single-stranded probes corresponding to different parts of the mouse gene to carry out a detailed analysis of the transcriptional unit in various cell types, using a run-on transcription assay on isolated nuclei. In liver cells, the first (non-erythropoietic) exon is more actively transcribed than parts of the gene situated downstream, suggesting that the elongation of transcripts is blocked within the 5' part of the first intron. In erythropoietic cells, the downstream promoter becomes activated; surprisingly, the initiation of transcription is also enhanced from the upstream (housekeeping) promoter and most of the transcripts initiated at the housekeeping promoter stop downstream of the first exon, between the two promoters.

4-Coumarate:CoA ligase (EC 6.2.1.12) from cell suspension cultures of parsley (Petroselinum hortense) was extensively purified. The enzyme behaved as a monomer with a molecular weight of approximately 60,000. A rabbit antiserum to the purified enzyme was obtained and used to determine the rates of 4-coumarate:CoA ligase synthesis under various conditions of induction, such as short term or continuous irradiation and treatment of the cells with an "elicitor" preparation from a fungal pathogen. In all cases, the time course of changes in the rate of synthesis, measured both in vivo and in vitro, was very similar for 4-coumarate:CoA ligase and a closely related enzyme, phenylalanine ammonia-lyase (EC 4.3.1.5). The results suggest that the mRNA activities encoding the two enzymes are regulated in a coordinated manner.

Phenylalanine ammonia-lyase (PAL) catalyzes the first reaction in the biosynthesis from phenylalanine of a wide variety of phenylpropanoid natural products including lignin, flavonoid pigments, and phytoalexins. In bean (Phaseolus vulgaris L.), PAL is encoded by a family of three genes. We show here by RNase protection with gene-specific probes that these genes are expressed differentially during development and in response to different environmental cues. While all three genes are expressed at high levels in roots, only PAL1 and PAL2 are expressed in shoots and only PAL1 is expressed in leaves. Strikingly, PAL2 is expressed at very high levels in petals, where PAL1 is only very weakly expressed and PAL3 is not expressed. All three genes are induced by mechanical wounding of hypocotyls, but fungal infection only activates PAL1 and PAL3. Illumination of etiolated hypocotyls activates PAL1 and PAL2 but not PAL3. Corresponding differential patterns of synthesis of specific PAL polypeptide isoforms were observed by two-dimensional gel electrophoretic analysis of in vitro translation products encoded by RNA isolated from hypocotyls stimulated by light, wounding, or infection. The specific isoforms encoded by transcripts of the three PAL genes were identified by inhibition of synthesis in vitro with gene-specific anti-sense transcripts followed by comparative two-dimensional gel electrophoretic analysis of the pattern of translation products. These data indicate that selective expression of PAL genes encoding functional variants is governed by a complex set of regulatory networks for developmental and environmental control of phenylpropanoid biosynthesis.

The human gene coding for porphobilinogen deaminase (PBG-D) is transcribed into two distinct transcription units giving two mRNAs. These units originate from two adjacent promoters distant of 3 kilobase pairs. The upstream promoter is active in all cell types, whereas the downstream promoter is active only in erythroid cells. We have studied the expression of this gene either after introduction of the corresponding human chromosome into murine erythroid cells using somatic hybrids or after transfection into both erythroid and non-erythroid cells. Using somatic hybrids, we showed that activation of the erythroid-specific promoter of the PBG-D gene did not reduce the rate of initiation of the ubiquitous promoter. Transfection experiments in erythroid cells showed that the PBG-D erythroid transcription unit, controlled by the PBG-D erythroid promoter, was correctly transcribed and regulated. Furthermore, we found that the PBG-D erythroid promoter alone was sufficient for correct expression and regulation of a reporter gene during erythroid differentiation. When the human PBG-D gene was transfected into non-erythroid cells, only the ubiquitous promoter was active. Deletion of the ubiquitous promoter did not lead to any activation of the erythroid promoter, suggesting that its inactivity in non-erythroid cells was not due to promoter occlusion but to a strict erythroid specificity.

Histidase is expressed in only two tissues of the rat, liver and epidermis. Hepatic histidase synthesis and catalytic activity undergo complex sex-specific developmental courses. To determine whether changes in functional histidase mRNA levels underlie this developmental pattern, total cellular RNA was translated in a rabbit reticulocyte cell-free lysate system. Adult liver total cellular RNA directed the synthesis of three translational products immunoreactive with anti-native histidase: a polypeptide of Mr = 75,000 (75K), which corresponds to the in vivo synthesized histidase subunit, and two higher molecular weight proteins, a major and a minor peptide of Mr = 150,000 (150K) and 140,000 (140K), respectively. These latter peptides do not seem to be aggregates or dimers of the 75K polypeptide or precursors which are post-translationally hydrolyzed to Mr = 75,000; their origin and function remain to be clarified. In contrast to in vitro translation of hepatic total cellular RNA, Western blot analysis of liver cytosol confirms the presence of only the 75K histidase subunit, with no evidence of anti-histidase immunoreactive peptides of Mr = 140,000-150,000 synthesized in vivo. Quantitation of the radioactivity in the immunocomplexed 75K histidase subunit, translated using total RNA from livers of fetuses, 19-day-old males, 35-day-old males, adult males and females, and adult kidney and brain (0, 0.007, 0.010, 0.016, 0.031, 0, and 0%, respectively, of total released proteins) indicates that, in general, levels of functional histidase subunit mRNA reflected histidase catalytic activity (0, 0.20, 0.50, 1.01, 3.00, 0 and 0 units/g of tissue) during tissue differentiation and sex specific development. The above data indicate that initial expression and subsequent increases in synthesis and activity of histidase during hepatic differentiation, postnatal development, and sex hormone regulation are due to pretranslationally controlled augmentation in the levels of functional mRNA which specifies the histidase subunit. In tissues which do not express histidase no functional histidase mRNA is evident. The levels of the RNA which translate the combined 140-150K histidase-like polypeptides (0, 0.007, 0.014, 0.035, 0.034, 0, and 0% of released proteins) also paralleled the increase in enzymatic activity during tissue differentiation and development; however, no difference between males and females was evident. The significance of these observations awaits elucidation of the nature of these RNA(s).