Structure of RizA, an<scp>L</scp>-amino-acid ligase from<i>Bacillus subtilis</i>

Kagawa, Wataru; Arai, Tomomi; Ishikura, Shun; Kino, Kuniki; Kurumizaka, Hitoshi

doi:10.1107/s2053230x15012698

Cited by 14 publications

(24 citation statements)

References 29 publications

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“…5A; see also Fig. S11A to C in the supplemental material) based on that of RizA (PDB 4WD3), a well-defined ligase in the rhizociticin pathway from Bacillus subtilis (20). According to this model, PolG contains three independent domains (designated A, B, and C domains) ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…According to this model, PolG contains three independent domains (designated A, B, and C domains) ( Fig. 5A), which are conserved in the L-amino acid ligases, including RizA, BL0025, and BacD (20). Furthermore, in silico analysis showed that the five conserved residues deduced for ATP binding in RizA could also be discerned in PolG (Lys110, Lys150, Glu267, Glu188, and Glu280) ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…S8 in the supplemental material). While the conserved ATP-binding sites are shared by a number of ATP-dependent ligases, their function is as yet undefined (20). To determine if these sites are essential for PolG activity, we individually mutated them and purified the resultant PolG variants.…”

Section: Resultsmentioning

confidence: 99%

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An ATP-Dependent Ligase with Substrate Flexibility Involved in Assembly of the Peptidyl Nucleoside Antibiotic Polyoxin

Gong

Jing

et al. 2018

Appl Environ Microbiol

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Polyoxin (POL) is an unusual peptidyl nucleoside antibiotic, in which the peptidyl moiety and nucleoside skeleton are linked by an amide bond. However, their biosynthesis remains poorly understood. Here, we report the deciphering of PolG as an ATP-dependent ligase responsible for the assembly of POL. A mutant is capable of accumulating multiple intermediates, including the peptidyl moiety (carbamoylpolyoxamic acid [CPOAA]) and the nucleoside skeletons (POL-C and the previously overlooked thymine POL-C). We further demonstrate that PolG employs an ATP-dependent mechanism for amide bond formation and that the generation of the hybrid nucleoside antibiotic POL-N is also governed by PolG. Finally, we determined that the deduced ATP-binding sites are functionally essential for PolG and that they are highly conserved in a number of related ATP-dependent ligases. These insights have allowed us to propose a catalytic mechanism for the assembly of peptidyl nucleoside antibiotic via an acyl-phosphate intermediate and have opened the way for the combinatorial biosynthesis/pathway engineering of this group of nucleoside antibiotics. POL is well known for its remarkable antifungal bioactivities and unusual structural features. Actually, elucidation of the POL assembly logic not only provides the enzymatic basis for further biosynthetic understanding of related peptidyl nucleoside antibiotics but also contributes to the rational generation of more hybrid nucleoside antibiotics via synthetic biology strategy.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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An ATP-Dependent Ligase with Substrate Flexibility Involved in Assembly of the Peptidyl Nucleoside Antibiotic Polyoxin

Gong

Jing

et al. 2018

Appl Environ Microbiol

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“…LALs contain the ATP-grasp fold, which is composed of three conserved domains referred to as the A-domain (N-terminal domain), the B-domain (central domain) and the C-domain (Cterminal domain). These three domains commonly grasp the ATP molecule, and also provide binding sites for the Mg2 + ion and the amino-acid substrate [54].…”

Section: Discussionmentioning

confidence: 99%

Pan-Genome-Wide Analysis of Pantoea Ananatis Identified Genes Linked to Pathogenicity in Onion

Agarwal¹,

Choudhary²,

Stice³

et al. 2020

Preprint

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Background: Pantoea ananatis is a member of a Pantoea spp. complex that causes center rot of onion, which significantly affects onion yield and quality. This pathogen does not have typical virulence factors like type II or type III secretion systems but appears to require a biosynthetic gene-cluster, HiVir/PASVIL (located chromosomally), for a phosphonate secondary metabolite, and the onion-virulence regions, OVR (localized on a megaplasmid), for onion pathogenicity and virulence, respectively. Results: We conducted a deep pan-genome-wide association study (pan-GWAS) to predict additional genes associated with pathogenicity in P. ananatis using a panel of diverse strains (n = 81). We utilized a red-onion scale necrosis assay as an indicator of pathogenicity. Based on this assay, we differentiated pathogenic (n = 51) - vs. non-pathogenic (n = 30)-strains phenotypically. Pan-GWAS revealed a large core genome of 3,153 genes and a flexible accessory genome of ≤5,065 genes. Phylogenomic analysis using pan-GWAS and presence and absence variants (PAVs) distinguished red-scale necrosis inducing (pathogenic) strains from non-scale necrosis inducing (non-pathogenic) strains of P. ananatis. The pan-GWAS also predicted 42 genes, including 14 from the previously identified HiVir/PASVIL cluster associated with pathogenicity, and 28 novel genes that were not previously associated with pathogenicity in onion. Of the 28 novel genes identified, eight have annotated functions of site-specific tyrosine kinase, N-acetylmuramoyl-L-alanine amidase, TraR/DksA family transcriptional regulator, and HTH-type transcriptional regulator. The remaining 20 genes are currently hypothetical. Conclusions: This is the first report of using pan-GWAS on P. ananatis for the prediction of novel genes contributing to pathogenicity in onion, which will be utilized for further functional analyses. Pan-genomic differences (using PAVs) differentiated onion pathogenic from non-pathogenic strains of P. ananatis, which has been difficult to achieve using single or multiple gene-based phylogenetic analyses. The pan-genome analysis also allowed us to evaluate the presence and absence of HiVir/PASVIL genes and 11 megaplasmid-borne OVR-A genes regarded as the ‘alt’ cluster that aid in P. ananatis colonization in onion bulbs. We concluded that HiVir/PASVIL genes are associated with pathogenic P. ananatis strains and the alt gene cluster alone is not responsible for pathogenicity on onion. The pan-genome also provides clear evidence of constantly evolving accessory genes in P. ananatis that may contribute to host-range expansion and niche-adaptation.

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“…LALs contain the ATP-grasp fold, which is composed of three conserved domains referred to as the A-domain (N-terminal domain), the B-domain (central domain) and the Cdomain (C-terminal domain). These three domains commonly grasp the ATP molecule, and also provide binding sites for the Mg2+ ion and the amino-acid substrate [54].…”

Section: Discussionmentioning

confidence: 99%

Pan-genome-wide analysis ofPantoea ananatisidentified genes linked to pathogenicity in onion

Agarwal

Choudhary

Stice

et al. 2020

Preprint

View full text Add to dashboard Cite

Pantoea ananatis is a member of a Pantoea spp. complex that causes center rot of onion, which significantly affects onion yield and quality. This pathogen does not have typical virulence factors like type II or type III secretion systems but appears to require a biosynthetic gene-cluster, HiVir/PASVIL (located chromosomally), for a phosphonate secondary metabolite, and the onion-virulence regions, OVR (localized on a megaplasmid), for onion pathogenicity and virulence, respectively. We conducted a deep pan-genome-wide association study (pan-GWAS) to predict additional genes associated with pathogenicity in P. ananatis using a panel of diverse strains (n = 81). We utilized a red-onion scale necrosis assay as an indicator of pathogenicity. Based on this assay, we differentiated pathogenic (n = 51)- vs. non-pathogenic (n = 30)-strains phenotypically. Pan-GWAS revealed a large core genome of 3,153 genes and a flexible accessory genome of ≤5,065 genes. Phylogenomic analysis using pan-GWAS and presence and absence variants (PAVs) distinguished red-scale necrosis inducing (pathogenic) strains from non-scale necrosis inducing (non-pathogenic) strains of P. ananatis. The pan-GWAS also predicted 42 genes, including 14 from the previously identified HiVir/PASVIL cluster associated with pathogenicity, and 28 novel genes that were not previously associated with pathogenicity in onion. Of the 28 novel genes identified, eight have annotated functions of site-specific tyrosine kinase, N-acetylmuramoyl-L-alanine amidase, TraR/DksA family transcriptional regulator, and HTH-type transcriptional regulator. The remaining 20 genes are currently hypothetical. This is the first report of using pan-GWAS on P. ananatis for the prediction of novel genes contributing to pathogenicity in onion, which will be utilized for further functional analyses. Pan-genomic differences (using PAVs) differentiated onion pathogenic from non-pathogenic strains of P. ananatis, which has been difficult to achieve using single or multiple gene-based phylogenetic analyses. The pan-genome analysis also allowed us to evaluate the presence and absence of HiVir/PASVIL genes and 11 megaplasmid-borne OVR-A genes regarded as the ‘alt’ cluster that aid in P. ananatis colonization in onion bulbs. We concluded that HiVir/PASVIL genes are associated with pathogenic P. ananatis strains and the alt gene cluster alone is not responsible for pathogenicity on onion. The pan-genome also provides clear evidence of constantly evolving accessory genes in P. ananatis that may contribute to host-range expansion and niche-adaptation.Author summaryPantoea ananatis is a major bacterial pathogen that causes center rot of onion and diseases of a number of other plant species. In order to understand the genome architecture and identify genes responsible for pathogenicity in onion, a pan-genome analysis was performed. We used 81 strains of P. ananatis collected over 20 years from different regions of the state of Georgia, USA. The pan-genome study identified a core genome with a conserved set of genes and an accessory genome that displayed variation among strains. We conducted pan-GWAS (pan-genome-wide association study) using presence and absence variants (PAVs) in the genomes and associated onion-pathogenic phenotypes based on a red-onion scale necrosis assay. The study resulted in identification of genes, including a cluster of chromosomal HiVir/PASVIL genes, that are significantly associated with the onion pathogenic phenotype. In addition, we identified 28 novel genes, a majority of which (n = 20) have hypothetical functions. We concluded and further substantiated earlier findings that a cluster of genes is responsible for pathogenicity on onion. The pan-genome analysis also allowed us to evaluate the presence and absence of HiVir/PASVIL genes and 11 megaplasmid-borne OVR-A genes regarded as the ‘alt’ cluster that aid in bacterial colonization of onion bulbs by P. ananatis strains. We concluded that HiVir/PASVIL genes are associated with onion-pathogenic strains, and the alt gene cluster alone is not responsible for pathogenicity on onion. This study also provides potential evidence of constantly evolving accessory genes in P. ananatis which may help in host range expansion and adaptation to diverse niches.

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Structure of RizA, anL-amino-acid ligase fromBacillus subtilis

Cited by 14 publications

References 29 publications

An ATP-Dependent Ligase with Substrate Flexibility Involved in Assembly of the Peptidyl Nucleoside Antibiotic Polyoxin

An ATP-Dependent Ligase with Substrate Flexibility Involved in Assembly of the Peptidyl Nucleoside Antibiotic Polyoxin

Pan-Genome-Wide Analysis of Pantoea Ananatis Identified Genes Linked to Pathogenicity in Onion

Pan-genome-wide analysis ofPantoea ananatisidentified genes linked to pathogenicity in onion

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