A [3Fe-4S] cluster and tRNA-dependent aminoacyltransferase BlsK in the biosynthesis of Blasticidin S

Blasticidin S is a peptidyl nucleoside antibiotic. Its biosynthesis involves a cryptic leucylation and two leucylated intermediates, LDBS and LBS, have been found in previous studies. Leucylation has been proposed to be a new self-resistance mechanism during blasticidin S biosynthesis, and the leucyl group was found to be important for the methylation of β-amino group of the arginine side chain. However, the responsible enzyme and its associated mechanism of the leucyl transfer process remain to be elucidated. Here, we report results investigating the leucyl transfer step forming the intermediate LDBS in blasticidin biosynthesis. A hypothetical protein, BlsK, has been characterized by genetic and in vitro biochemical experiments. This enzyme catalyzes the leucyl transfer from leucyl-transfer RNA (leucyl-tRNA) to the β-amino group on the arginine side chain of DBS. Furthermore, BlsK was found to contain an iron–sulfur cluster that is necessary for activity. These findings provide an example of an iron–sulfur protein that catalyzes an aminoacyl-tRNA (aa-tRNA)–dependent amide bond formation in a natural product biosynthetic pathway.

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In Vitro Studies of Cellular Iron–Sulfur Cluster Biosynthesis, Trafficking, and Transport

Methods in Enzymology - Fe-S Cluster Enzymes Part A ◽

10.1016/bs.mie.2017.06.045 ◽

2017 ◽

pp. 55-82 ◽

Cited By ~ 2

Author(s):

Christine Wachnowsky ◽

James A. Cowan

Keyword(s):

In Vitro Studies ◽

Iron Sulfur Cluster ◽

Sulfur Cluster ◽

Cellular Iron ◽

Iron Sulfur ◽

Cluster Biosynthesis

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Iron–sulfur cluster biosynthesis

Biochemical Society Transactions ◽

10.1042/bst0361112 ◽

2008 ◽

Vol 36 (6) ◽

pp. 1112-1119 ◽

Cited By ~ 112

Author(s):

Sibali Bandyopadhyay ◽

Kala Chandramouli ◽

Michael K. Johnson

Keyword(s):

Scaffold Proteins ◽

Cluster Assembly ◽

Cysteine Desulfurase ◽

Iron Sulfur Cluster ◽

Iron Sulfur ◽

Eukaryotic Organisms ◽

Almost All ◽

Cluster Biosynthesis

Iron–sulfur (Fe–S) clusters are present in more than 200 different types of enzymes or proteins and constitute one of the most ancient, ubiquitous and structurally diverse classes of biological prosthetic groups. Hence the process of Fe–S cluster biosynthesis is essential to almost all forms of life and is remarkably conserved in prokaryotic and eukaryotic organisms. Three distinct types of Fe–S cluster assembly machinery have been established in bacteria, termed the NIF, ISC and SUF systems, and, in each case, the overall mechanism involves cysteine desulfurase-mediated assembly of transient clusters on scaffold proteins and subsequent transfer of pre-formed clusters to apo proteins. A molecular level understanding of the complex processes of Fe–S cluster assembly and transfer is now beginning to emerge from the combination of in vivo and in vitro approaches. The present review highlights recent developments in understanding the mechanism of Fe–S cluster assembly and transfer involving the ubiquitous U-type scaffold proteins and the potential roles of accessory proteins such as Nfu proteins and monothiol glutaredoxins in the assembly, storage or transfer of Fe–S clusters.

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Endophytic Actinomycetes: A Novel Source of Potential Acyl Homoserine Lactone Degrading Enzymes

BioMed Research International ◽

10.1155/2013/782847 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 21

Author(s):

Surang Chankhamhaengdecha ◽

Suphatra Hongvijit ◽

Akkaraphol Srichaisupakit ◽

Pattra Charnchai ◽

Watanalai Panbangred

Keyword(s):

Pathogenic Bacteria ◽

Sequence Similarity ◽

Soft Rot ◽

Quorum Quenching ◽

Homoserine Lactone ◽

Amide Bond ◽

Side Chain ◽

Signal Molecules ◽

Endophytic Actinomycetes

Several Gram-negative pathogenic bacteria employN-acyl-L-homoserine lactone (HSL) quorum sensing (QS) system to control their virulence traits. Degradation of acyl-HSL signal molecules by quorum quenching enzyme (QQE) results in a loss of pathogenicity in QS-dependent organisms. The QQE activity of actinomycetes in rhizospheric soil and inside plant tissue was explored in order to obtain novel strains with high HSL-degrading activity. Among 344 rhizospheric and 132 endophytic isolates, 127 (36.9%) and 68 (51.5%) of them, respectively, possessed the QQE activity. The highest HSL-degrading activity was at151.30±3.1 nmole/h/mL from an endophytic actinomycetes isolate, LPC029. The isolate was identified asStreptomycesbased on16S rRNAgene sequence similarity. The QQE from LPC029 revealed HSL-acylase activity that was able to cleave an amide bond of acyl-side chain in HSL substrate as determined by HPLC. LPC029 HSL-acylase showed broad substrate specificity from C6- to C12-HSL in which C10HSL is the most favorable substrate for this enzyme. In anin vitropathogenicity assay, the partially purified HSL-acylase efficiently suppressed soft rot of potato caused byPectobacterium carotovorumssp.carotovorumas demonstrated. To our knowledge, this is the first report of HSL-acylase activity derived from an endophyticStreptomyces.

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Biofilm-Constructing Variants of Paraburkholderia phytofirmans PsJN Outcompete the Wild-Type Form in Free-Living and Static Conditions but Not In Planta

Applied and Environmental Microbiology ◽

10.1128/aem.02670-18 ◽

2019 ◽

Vol 85 (11) ◽

Cited By ~ 1

Author(s):

Marine Rondeau ◽

Qassim Esmaeel ◽

Jérôme Crouzet ◽

Pauline Blin ◽

Isabelle Gosselin ◽

...

Keyword(s):

Wild Type ◽

In Planta ◽

Free Living ◽

Iron Sulfur Cluster ◽

Content Type ◽

Iron Sulfur ◽

Type Form ◽

Static Conditions ◽

Wild Type Form

ABSTRACT Members of the genus Burkholderia colonize diverse ecological niches. Among the plant-associated strains, Paraburkholderia phytofirmans PsJN is an endophyte with a broad host range. In a spatially structured environment (unshaken broth cultures), biofilm-constructing specialists of P. phytofirmans PsJN colonizing the air-liquid interface arose at high frequency. In addition to forming a robust biofilm in vitro and in planta on Arabidopsis roots, those mucoid phenotypic variants display a reduced swimming ability and modulate the expression of several microbe-associated molecular patterns (MAMPs), including exopolysaccharides (EPS), flagellin, and GroEL. Interestingly, the variants induce low PR1 and PDF1.2 expression compared to that of the parental strain, suggesting a possible evasion of plant host immunity. We further demonstrated that switching from the planktonic to the sessile form did not involve quorum-sensing genes but arose from spontaneous mutations in two genes belonging to an iron-sulfur cluster: hscA (encoding a cochaperone protein) and iscS (encoding a cysteine desulfurase). A mutational approach validated the implication of these two genes in the appearance of variants. We showed for the first time that in a heterogeneous environment, P. phytofirmans strain PsJN is able to rapidly diversify and coexpress a variant that outcompete the wild-type form in free-living and static conditions but not in planta. IMPORTANCE Paraburkholderia phytofirmans strain PsJN is a well-studied plant-associated bacterium known to induce resistance against biotic and abiotic stresses. In this work, we described the spontaneous appearance of mucoid variants in PsJN from static cultures. We showed that the conversion from the wild-type (WT) form to variants (V) correlates with an overproduction of EPS, an enhanced ability to form biofilm in vitro and in planta, and a reduced swimming motility. Our results revealed also that these phenotypes are in part associated with spontaneous mutations in an iron-sulfur cluster. Overall, the data provided here allow a better understanding of the adaptive mechanisms likely developed by P. phytofirmans PsJN in a heterogeneous environment.

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Physical and Functional Interactions of a Monothiol Glutaredoxin and an Iron Sulfur Cluster Carrier Protein with the Sulfur-donating Radical S-Adenosyl-l-methionine Enzyme MiaB

Journal of Biological Chemistry ◽

10.1074/jbc.m113.460360 ◽

2013 ◽

Vol 288 (20) ◽

pp. 14200-14211 ◽

Cited By ~ 20

Author(s):

Sylvain Boutigny ◽

Avneesh Saini ◽

Edward E. K. Baidoo ◽

Natasha Yeung ◽

Jay D. Keasling ◽

...

Keyword(s):

Protein Interactions ◽

Carrier Protein ◽

Cluster Assembly ◽

Carrier Proteins ◽

Iron Sulfur Cluster ◽

Iron Sulfur ◽

Initial Cluster ◽

Initial Assembly

The biosynthesis of iron sulfur (FeS) clusters, their trafficking from initial assembly on scaffold proteins via carrier proteins to final incorporation into FeS apoproteins, is a highly coordinated process enabled by multiprotein systems encoded in iscRSUAhscBAfdx and sufABCDSE operons in Escherichia coli. Although these systems are believed to encode all factors required for initial cluster assembly and transfer to FeS carrier proteins, accessory factors such as monothiol glutaredoxin, GrxD, and the FeS carrier protein NfuA are located outside of these defined systems. These factors have been suggested to function both as shuttle proteins acting to transfer clusters between scaffold and carrier proteins and in the final stages of FeS protein assembly by transferring clusters to client FeS apoproteins. Here we implicate both of these factors in client protein interactions. We demonstrate specific interactions between GrxD, NfuA, and the methylthiolase MiaB, a radical S-adenosyl-l-methionine-dependent enzyme involved in the maturation of a subset of tRNAs. We show that GrxD and NfuA physically interact with MiaB with affinities compatible with an in vivo function. We furthermore demonstrate that NfuA is able to transfer its cluster in vitro to MiaB, whereas GrxD is unable to do so. The relevance of these interactions was demonstrated by linking the activity of MiaB with GrxD and NfuA in vivo. We observe a severe defect in in vivo MiaB activity in cells lacking both GrxD and NfuA, suggesting that these proteins could play complementary roles in maturation and repair of MiaB.

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Mitochondrial Arabidopsis thaliana TRXo Isoforms Bind an Iron–Sulfur Cluster and Reduce NFU Proteins In Vitro

Antioxidants ◽

10.3390/antiox7100142 ◽

2018 ◽

Vol 7 (10) ◽

pp. 142 ◽

Cited By ~ 8

Author(s):

Flavien Zannini ◽

Thomas Roret ◽

Jonathan Przybyla-Toscano ◽

Tiphaine Dhalleine ◽

Nicolas Rouhier ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Protein Interactions ◽

Function Analysis ◽

Active Form ◽

Three Dimensional ◽

Dimensional Structure ◽

Regulation Mechanism ◽

Iron Sulfur Cluster ◽

Iron Sulfur

In plants, the mitochondrial thioredoxin (TRX) system generally comprises only one or two isoforms belonging to the TRX h or o classes, being less well developed compared to the numerous isoforms found in chloroplasts. Unlike most other plant species, Arabidopsis thaliana possesses two TRXo isoforms whose physiological functions remain unclear. Here, we performed a structure–function analysis to unravel the respective properties of the duplicated TRXo1 and TRXo2 isoforms. Surprisingly, when expressed in Escherichia coli, both recombinant proteins existed in an apo-monomeric form and in a homodimeric iron–sulfur (Fe-S) cluster-bridged form. In TRXo2, the [4Fe-4S] cluster is likely ligated in by the usual catalytic cysteines present in the conserved Trp-Cys-Gly-Pro-Cys signature. Solving the three-dimensional structure of both TRXo apo-forms pointed to marked differences in the surface charge distribution, notably in some area usually participating to protein–protein interactions with partners. However, we could not detect a difference in their capacity to reduce nitrogen-fixation-subunit-U (NFU)-like proteins, NFU4 or NFU5, two proteins participating in the maturation of certain mitochondrial Fe-S proteins and previously isolated as putative TRXo1 partners. Altogether, these results suggest that a novel regulation mechanism may prevail for mitochondrial TRXs o, possibly existing as a redox-inactive Fe-S cluster-bound form that could be rapidly converted in a redox-active form upon cluster degradation in specific physiological conditions.

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DNA Microarray Analysis of the Hyperthermophilic Archaeon Pyrococcus furiosus: Evidence for a New Type of Sulfur-Reducing Enzyme Complex

Journal of Bacteriology ◽

10.1128/jb.183.24.7027-7036.2001 ◽

2001 ◽

Vol 183 (24) ◽

pp. 7027-7036 ◽

Cited By ~ 70

Author(s):

Gerrit J. Schut ◽

Jizhong Zhou ◽

Michael W. W. Adams

Keyword(s):

Detection Limit ◽

Dna Microarrays ◽

Pyrococcus Furiosus ◽

Hypothetical Protein ◽

Rrna Processing ◽

Iron Sulfur Cluster ◽

Reading Frame ◽

Iron Sulfur ◽

New Type ◽

Induced Proteins

ABSTRACT DNA microarrays were constructed by using 271 open reading frame (ORFs) from the genome of the archaeon Pyrococcus furiosus. They were used to investigate the effects of elemental sulfur (S°) on the levels of gene expression in cells grown at 95°C with maltose as the carbon source. The ORFs included those that are proposed to encode proteins mainly involved in the pathways of sugar and peptide catabolism, in the metabolism of metals, and in the biosynthesis of various cofactors, amino acids, and nucleotides. The expression of 21 ORFs decreased by more than fivefold when cells were grown with S° and, of these, 18 encode subunits associated with three different hydrogenase systems. The remaining three ORFs encode homologs of ornithine carbamoyltransferase and HypF, both of which appear to be involved in hydrogenase biosynthesis, as well as a conserved hypothetical protein. The expression of two previously uncharacterized ORFs increased by more than 25-fold when cells were grown with S°. Their products, termed SipA and SipB (for sulfur-induced proteins), are proposed to be part of a novel S°-reducing, membrane-associated, iron-sulfur cluster-containing complex. Two other previously uncharacterized ORFs encoding a putative flavoprotein and a second FeS protein were upregulated more than sixfold in S°-grown cells, and these are also thought be involved in S° reduction. Four ORFs that encode homologs of proteins involved in amino acid metabolism were similarly upregulated in S°-grown cells, a finding consistent with the fact that growth on peptides is a S°-dependent process. An ORF encoding a homolog of the eukaryotic rRNA processing protein, fibrillarin, was also upregulated sixfold in the presence of S°, although the reason for this is as yet unknown. Of the 20 S°-independent ORFs that are the most highly expressed (at more than 20 times the detection limit), 12 of them represent enzymes purified from P. furiosus, but none of the products of the 34 S°-independent ORFs that are not expressed above the detection limit have been characterized. These results represent the first derived from the application of DNA microarrays to either an archaeon or a hyperthermophile.

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Development of 6′-N-Acylated Isepamicin Analogs with Improved Antibacterial Activity against Isepamicin-Resistant Pathogens

Biomolecules ◽

10.3390/biom10060893 ◽

2020 ◽

Vol 10 (6) ◽

pp. 893

Author(s):

Yeon Hee Ban ◽

Myoung Chong Song ◽

Hee Jin Kim ◽

Heejeong Lee ◽

Jae Bok Wi ◽

...

Keyword(s):

Antibacterial Activity ◽

Resistance Mechanism ◽

Amino Group ◽

Gram Negative Bacteria ◽

Enzymatic Modification ◽

Gram Negative ◽

Substrate Promiscuity ◽

Reduced Toxicity

The development of new aminoglycoside (AG) antibiotics has been required to overcome the resistance mechanism of AG-modifying enzymes (AMEs) of AG-resistant pathogens. The AG acetyltransferase, AAC(6′)-APH(2″), one of the most typical AMEs, exhibiting substrate promiscuity towards a variety of AGs and acyl-CoAs, was employed to enzymatically synthesize new 6′-N-acylated isepamicin (ISP) analogs, 6′-N-acetyl/-propionyl/-malonyl ISPs. They were all active against the ISP-resistant Gram-negative bacteria tested, and the 6′-N-acetyl ISP displayed reduced toxicity compared to ISP in vitro. This study demonstrated the importance of the modification of the 6′-amino group in circumventing AG-resistance and the potential of regioselective enzymatic modification of AG scaffolds for the development of more robust AG antibiotics.

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Two Distinct L-Lactate Dehydrogenases Play a Role in the Survival of Neisseria gonorrhoeae in Cervical Epithelial Cells

The Journal of Infectious Diseases ◽

10.1093/infdis/jiz468 ◽

2019 ◽

Vol 221 (3) ◽

pp. 449-453 ◽

Cited By ~ 1

Author(s):

Nathan H Chen ◽

Cheryl-Lynn Y Ong ◽

Jonathan O’Sullivan ◽

Ines Ibranovic ◽

Krystelle Davey ◽

...

Keyword(s):

Lactate Dehydrogenase ◽

Epithelial Cells ◽

Neisseria Gonorrhoeae ◽

Functional Role ◽

Iron Sulfur Cluster ◽

Iron Sulfur ◽

Cervical Epithelial Cells ◽

Lactate Dehydrogenases

Abstract L-lactate is an abundant metabolite in a number of niches in host organisms and represents an important carbon source for bacterial pathogens such as Neisseria gonorrhoeae. In this study, we describe an alternative, iron-sulfur cluster-containing L-lactate dehydrogenase (LutACB), that is distinct from the flavoprotein L-lactate dehydrogenase (LldD). Expression of lutACB was found to be positively regulated by iron, whereas lldD was more highly expressed under conditions of iron-limitation. The functional role of LutACB and LldD was reflected in in vitro studies of growth and in the survival of N gonorrhoeae in primary cervical epithelial cells.

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