Spermidine N-acetyltransferase Research Articles

Functional polyamine metabolic enzymes and pathways encoded by the virosphere

Viruses produce more viruses by manipulating the metabolic and replication systems of their host cells. Many have acquired metabolic genes from ancestral hosts and use the encoded enzymes to subvert host metabolism. The polyamine spermidine is required for bacteriophage and eukaryotic virus replication, and herein, we have identified and functionally characterized diverse phage- and virus-encoded polyamine metabolic enzymes and pathways. These include pyridoxal 5'-phosphate (PLP)-dependent ornithine decarboxylase (ODC), pyruvoyl-dependent ODC and arginine decarboxylase (ADC), arginase, S-adenosylmethionine decarboxylase (AdoMetDC/speD), spermidine synthase, homospermidine synthase, spermidine N-acetyltransferase, and N-acetylspermidine amidohydrolase. We identified homologs of the spermidine-modified translation factor eIF5a encoded by giant viruses of the Imitervirales. Although AdoMetDC/speD is prevalent among marine phages, some homologs have lost AdoMetDC activity and have evolved into pyruvoyl-dependent ADC or ODC. The pelagiphages that encode the pyruvoyl-dependent ADCs infect the abundant ocean bacterium Candidatus Pelagibacter ubique, which we have found encodes a PLP-dependent ODC homolog that has evolved into an ADC, indicating that infected cells would contain both PLP- and pyruvoyl-dependent ADCs. Complete or partial spermidine or homospermidine biosynthetic pathways are found encoded in the giant viruses of the Algavirales and Imitervirales, and in addition, some viruses of the Imitervirales can release spermidine from the inactive N-acetylspermidine. In contrast, diverse phages encode spermidine N-acetyltransferase that can sequester spermidine into its inactive N-acetyl form. Together, the virome-encoded enzymes and pathways for biosynthesis and release or biochemical sequestration of spermidine or its structural analog homospermidine consolidate and expand evidence supporting an important and global role of spermidine in virus biology.

Free spermidine evokes superoxide radicals that manifest toxicity.

Spermidine and other polyamines alleviate oxidative stress, yet excess spermidine seems toxic to Escherichia coli unless it is neutralized by SpeG, an enzyme for the spermidine N-acetyl transferase function. Thus, wild-type E. coli can tolerate applied exogenous spermidine stress, but ΔspeG strain of E. coli fails to do that. Here, using different reactive oxygen species (ROS) probes and performing electron paramagnetic resonance spectroscopy, we provide evidence that although spermidine mitigates oxidative stress by lowering overall ROS levels, excess of it simultaneously triggers the production of superoxide radicals, thereby causing toxicity in the ΔspeG strain. Furthermore, performing microarray experiment and other biochemical assays, we show that the spermidine-induced superoxide anions affected redox balance and iron homeostasis. Finally, we demonstrate that while RNA-bound spermidine inhibits iron oxidation, free spermidine interacts and oxidizes the iron to evoke superoxide radicals directly. Therefore, we propose that the spermidine-induced superoxide generation is one of the major causes of spermidine toxicity in E. coli.

Clonal diversity of methicillin-resistant Staphylococcus aureus (MRSA) from bloodstream infections in northern Japan: Identification of spermidine N-acetyltransferase gene (speG) in staphylococcal cassette chromosomes (SCCs) associated with type II and IV SCCmec

ObjectivesMethicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of bloodstream infections (BSIs). We aimed to study molecular epidemiological characteristics of MRSA isolates from BSIs in northern Japan to elucidate the recent trend of their clonal diversity. MethodsMRSA isolates (n = 277) were collected from blood samples of patients who attended healthcare facilities in Hokkaido, the northern main island of Japan, for a two-year period from August 2017. Genotypes, virulence factors/drug-resistance determinants, and structure of SCCmec complex were analysed by PCR and sequencing analysis. ResultsSCCmec-IIa (n = 171, 61.7%) with coagulase genotype (coa-) II, ST5/ST764/ST2389 was the most common genetic trait, followed by SCCmec-IVa (n = 78, 28.2%), and IVl (n = 10, 3.6%). Among the MRSA-IVa, 14 isolates (5.1% of all the isolates) had genetic features identical to USA300 clone (ST8/coa-IIIa/spa-t008 having ΦSa2USA and ACME-I), while PVL/ACME-negative MRSA-IVa isolates (n = 64) were classified into coa-IIa/IIIa/VIIa/VIIb, with coa-VIIa/spa-t1784/ST1 being dominant. Other minor clones included ST8-SCCmec-I, and ST30/ST45/ST81/ST121/ST1232-SCCmec-V, among which the ST1232 isolate harboured PVL genes. Spermidine N-acetyltransferase gene (speG), which is typically present in ACME-I of USA300 clone, was also identified in two isolates, ACME-II’-positive ST764-MRSA-IIa and ACME-negative ST1-MRSA-IVa, showing resistance to spermine. speG of these isolates was located in additional SCCs adjacent to SCCmec. ConclusionsOur present study revealed clonal diversity of MRSA from BSIs in Japan, with increased prevalence of ST8-USA300. Distinct types of speG-carrying SCCs associated with SCCmec-II or IV were identified.

Staphylococcus petrasii diagnostics and its pathogenic potential enhanced by mobile genetic elements

Staphylococcus petrasii is recently described coagulase negative staphylococcal species and an opportunistic human pathogen, still often misidentified in clinical specimens. Four subspecies are distinguished in S. petrasii by polyphasic taxonomical analyses, however a comparative study has still not been done on the majority of isolates and their genome properties have not yet been thoroughly analysed. Here, we describe the phenotypic and genotypic characteristics of 65 isolates and the results of de novo sequencing, whole genome assembly and annotation of draft genomes of five strains. The strains were identified by MALDI-TOF mass spectrometry to the species level and the majority of the strains were identified to the subspecies level by fingerprinting methods, (GTG)5 repetitive PCR and ribotyping. Macrorestriction profiling by pulsed-field gel electrophoresis was confirmed to be a suitable strain typing method. Comparative genomics revealed the presence of new mobile genetic elements carrying antimicrobial resistance factors such as staphylococcal cassette chromosome (SCC) mec, transposones, phage-inducible genomic islands, and plasmids. Their mosaic structure and similarity across coagulase-negative staphylococci and Staphylococcus aureus suggest the possible exchange of these elements. Numerous putative virulence factors such as adhesins, autolysins, exoenzymes, capsule formation genes, immunomodulators, the phage-associated sasX gene, and SCC-associated spermidine N-acetyltransferase gene, pseudouridine and sorbitol utilization operons might explain clinical manifestations of S. petrasii isolates. The increasing recovery of S. petrasii isolates from human clinical material, the multi-drug resistance including methicillin resistance of S. petrasii subsp. jettensis strains, and virulence factors homologous to other pathogenic staphylococci demonstrate the importance of the species in human disease.

The spermidine acetyltransferase SpeG regulates transcription of the small RNA rprA.

Spermidine N-acetyltransferase (SpeG) acetylates and thus neutralizes toxic polyamines. Studies indicate that SpeG plays an important role in virulence and pathogenicity of many bacteria, which have evolved SpeG-dependent strategies to control polyamine concentrations and survive in their hosts. In Escherichia coli, the two-component response regulator RcsB is reported to be subject to Nε-acetylation on several lysine residues, resulting in reduced DNA binding affinity and reduced transcription of the small RNA rprA; however, the physiological acetylation mechanism responsible for this behavior has not been fully determined. Here, we performed an acetyltransferase screen and found that SpeG inhibits rprA promoter activity in an acetylation-independent manner. Surface plasmon resonance analysis revealed that SpeG can physically interact with the DNA-binding carboxyl domain of RcsB. We hypothesize that SpeG interacts with the DNA-binding domain of RcsB and that this interaction might be responsible for SpeG-dependent inhibition of RcsB-dependent rprA transcription. This work provides a model for SpeG as a modulator of E. coli transcription through its ability to interact with the transcription factor RcsB. This is the first study to provide evidence that an enzyme involved in polyamine metabolism can influence the function of the global regulator RcsB, which integrates information concerning envelope stresses and central metabolic status to regulate diverse behaviors.

Small angle X-ray scattering data and structure factor fitting for the study of the quaternary structure of the spermidine N-acetyltransferase SpeG.

Here we describe the treatment of the small-angle X-ray Scattering (SAXS) data used during SpeG quaternary structure study as part of the research article “Substrate induced allosteric change in the quaternary structure of the spermidine N-acetyltransferase SpeG” published in Journal of Molecular Biology [1]. These data were collected on two separate area detectors as separate dilution series of the SpeG and the SpeG with spermine samples along with data from their companion buffers. The data were radially integrated, corrected for incident beam variation, and scaled to absolute units. After subtraction of volume-fraction scaled buffer scattering and division by the SpeG concentration, multiple scattering curves free of an inter-molecular structure factor were derived from the dilution series. Rather than extrapolating to infinite dilution, the structure factor contribution was estimated by fitting to the full set of data provided by dividing the scattering curves of a dilution series by the curve from the most dilute sample in that series.

Substrate-Induced Allosteric Change in the Quaternary Structure of the Spermidine N-Acetyltransferase SpeG

The spermidine N-acetyltransferase SpeG is a dodecameric enzyme that catalyzes the transfer of an acetyl group from acetyl coenzyme A to polyamines such as spermidine and spermine. SpeG has an allosteric polyamine-binding site and acetylating polyamines regulate their intracellular concentrations. The structures of SpeG from Vibrio cholerae in complexes with polyamines and cofactor have been characterized earlier. Here, we present the dodecameric structure of SpeG from V. cholerae in a ligand-free form in three different conformational states: open, intermediate and closed. All structures were crystallized in C2 space group symmetry and contain six monomers in the asymmetric unit cell. Two hexamers related by crystallographic 2-fold symmetry form the SpeG dodecamer. The open and intermediate states have a unique open dodecameric ring. This SpeG dodecamer is asymmetric except for the one 2-fold axis and is unlike any known dodecameric structure. Using a fluorescence thermal shift assay, size-exclusion chromatography with multi-angle light scattering, small-angle X-ray scattering analysis, negative-stain electron microscopy and structural analysis, we demonstrate that this unique open dodecameric state exists in solution. Our combined results indicate that polyamines trigger conformational changes and induce the symmetric closed dodecameric state of the protein when they bind to their allosteric sites.

A Novel Polyamine Allosteric Site of SpeG from Vibrio cholerae Is Revealed by Its Dodecameric Structure

Spermidine N-acetyltransferase, encoded by the gene speG, catalyzes the initial step in the degradation of polyamines and is a critical enzyme for determining the polyamine concentrations in bacteria. In Escherichia coli, studies have shown that SpeG is the enzyme responsible for acetylating spermidine under stress conditions and for preventing spermidine toxicity. Not all bacteria contain speG, and many bacterial pathogens have developed strategies to either acquire or silence it for pathogenesis. Here, we present thorough kinetic analyses combined with structural characterization of the VCA0947 SpeG enzyme from the important human pathogen Vibrio cholerae. Our studies revealed the unexpected presence of a previously unknown allosteric site and an unusual dodecameric structure for a member of the Gcn5-related N-acetyltransferase superfamily. We show that SpeG forms dodecamers in solution and in crystals and describe its three-dimensional structure in several ligand-free and liganded structures. Importantly, these structural data define the first view of a polyamine bound in an allosteric site of an N-acetyltransferase. Kinetic characterization of SpeG from V. cholerae showed that it acetylates spermidine and spermine. The behavior of this enzyme is complex and exhibits sigmoidal curves and substrate inhibition. We performed a detailed non-linear regression kinetic analysis to simultaneously fit families of substrate saturation curves to uncover a simple kinetic mechanism that explains the apparent complexity of this enzyme. Our results provide a fundamental understanding of the bacterial SpeG enzyme, which will be key toward understanding the regulation of polyamine levels in bacteria during pathogenesis.

Mathematical Modeling of Polyamine Metabolism in Mammals

Polyamines are considered as essential compounds in living cells, since they are involved in cell proliferation, transcription, and translation processes. Furthermore, polyamine homeostasis is necessary to cell survival, and its deregulation is involved in relevant processes, such as cancer and neurodegenerative disorders. Great efforts have been made to elucidate the nature of polyamine homeostasis, giving rise to relevant information concerning the behavior of the different components of polyamine metabolism, and a great amount of information has been generated. However, a complex regulation at transcriptional, translational, and metabolic levels as well as the strong relationship between polyamines and essential cell processes make it difficult to discriminate the role of polyamine regulation itself from the whole cell response when an experimental approach is given in vivo. To overcome this limitation, a bottom-up approach to model mathematically metabolic pathways could allow us to elucidate the systemic behavior from individual kinetic and molecular properties. In this paper, we propose a mathematical model of polyamine metabolism from kinetic constants and both metabolite and enzyme levels extracted from bibliographic sources. This model captures the tendencies observed in transgenic mice for the so-called key enzymes of polyamine metabolism, ornithine decarboxylase, S-adenosylmethionine decarboxylase and spermine spermidine N-acetyl transferase. Furthermore, the model shows a relevant role of S-adenosylmethionine and acetyl-CoA availability in polyamine homeostasis, which are not usually considered in systemic experimental studies.

Spermidine acetylation in N1 and N8 position in rat brain and in N-ethyl- N-nitrosourea-induced gliomas

N-ethyl- N-nitrosourea-induced rat gliomas showed a stimulation of cytosolic spermidine N-acetyltransferase activity compared with normal brain, with an increased formation of N 1 and N 8-acetylspermidine, suggesting the activation of two enzymes acetylating the polyamine in N 1 and N 8 position, respectively. The enhancement in cytosolic spermidine N 1-acetyltransferase was probably responsible for an activation of the polyamine interconversion pathway in gliomas, as indicated by an accumulation of N 1-acetylspermidine and a marked increase in putrescine. Spermidine N 8-acetyltransferase, although acetylating histones at a high rate, seemed distinct from the nuclear enzyme, as, unlike from the latter, it showed very low affinity for putrescine and was not inhibited by methylglyoxal bis(guanylhydrazone).

Purification of methylglyoxal bis(guanylhydrazone)-induced spermidine N-acetyltransferase from baby hamster kidney cells (BHK-21/C13)

Purification of methylglyoxal bis(guanylhydrazone)-induced spermidine N-acetyltransferase from baby hamster kidney cells (BHK-21/C13)

Properties of spermidine N-acetyltransferase from livers of rats treated with carbon tetrachloride and its role in the conversion of spermidine into putrescine.

Properties of spermidine N-acetyltransferase from livers of rats treated with carbon tetrachloride and its role in the conversion of spermidine into putrescine.