Structural investigation on WlaRG from <i>Campylobacter jejuni</i>: A sugar aminotransferase

Dow, G.T.; Gilbert, Michel; Thoden, James B.; Holden, Hazel M.

doi:10.1002/pro.3109

Cited by 12 publications

(11 citation statements)

References 41 publications

(83 reference statements)

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“…The gene encoding SAMN03097714_1080 (PA1080c) from P. ananatis strain NFR11 was synthesized by Integrated DNA Technologies and placed into pET28T3G, a laboratory pET28b(+) vector that had been previously modified to incorporate a TEV protease cleavage recognition site after the N‐terminal polyhistidine tag …”

Section: Methodsmentioning

confidence: 99%

Investigation of a sugar N‐formyltransferase from the plant pathogen Pantoea ananatis

2019

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Pantoea ananatis is a Gram-negative bacterium first recognized in 1928 as the causative agent of pineapple rot in the Philippines. Since then various strains of the organism have been implicated in the devastation of agriculturally important crops. Some strains, however, have been shown to function as non-pathogenic plant growth promoting organisms. To date, the factors that determine pathogenicity or lack thereof between the various strains are not well understood. All P. ananatis strains contain lipopolysaccharides, which differ with respect to the identities of their associated sugars. Given our research interest on the presence of the unusual sugar, 4-formamido-4,6-dideoxy-Dglucose, found on the lipopolysaccharides of Campylobacter jejuni and Francisella tularensis, we were curious as to whether other bacteria have the appropriate biosynthetic machinery to produce these unique carbohydrates. Four enzymes are typically required for their biosynthesis: a thymidylyltransferase, a 4,6-dehydratase, an aminotransferase, and an N-formyltransferase. Here, we report that the gene SAMN03097714_1080 from the P. ananatis strain NFR11 does, indeed, encode for an N-formyltransferase, hereafter referred to as PA1080c. Our kinetic analysis demonstrates that PA1080c displays classical Michaelis-Menten kinetics with dTDP-4-amino-4,6-dideoxy-D-glucose as the substrate and N 10 -formyltetrahydrofolate as the carbon source. In addition, the X-ray structure of PA1080c, determined to 1.7 Å resolution, shows that the enzyme adopts the molecular architecture observed for other sugar N-formyltransferases. Analysis of the P. ananatis NFR11 genome suggests that the three other enzymes necessary for N-formylated sugar biosynthesis are also present. Intriguingly, those strains of P. ananatis that are non-pathogenic apparently do not contain these genes.

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Section: Methodsmentioning

confidence: 99%

Investigation of a sugar N‐formyltransferase from the plant pathogen Pantoea ananatis

2019

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“…Relevant refinement statistics are provided in Table 2. The aminotransferase structure was solved with PHASER (20) using PDB entry 5U1Z as a starting model 56 . The ketoisomerase structure was determined with PHASER (20) using PDB entry 5TPU as a starting model 30 …”

Section: Methodsmentioning

confidence: 99%

“…The aminotransferase structure was solved with PHASER (20) using PDB entry 5U1Z as a starting model. 56 The ketoisomerase structure was determined with PHASER (20) using PDB entry 5TPU as a starting model. 30 4.5 | Synthesis of dTDP-4-keto-6-deoxy-Dglucose A buffered solution of 50 mM dTDP-D-glucose was prepared in 50 mM HEPPS (pH 8.0).…”

Section: Structure Solution and Model Refinementmentioning

confidence: 99%

Investigation of the enzymes required for the biosynthesis of an unusual formylated sugar in the emerging human pathogen Helicobacter canadensis

et al. 2021

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It is now well established that the Gram-negative bacterium, Helicobacter pylori, causes gastritis in humans. In recent years, it has become apparent that the so-called non-pylori Helicobacters, normally infecting pigs, cats, and dogs, may also be involved in human pathology via zoonotic transmission. Indeed, more than 30 species of non-pylori Helicobacters have been identified thus far. One such organism is Helicobacter canadensis, an emerging pathogen whose

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“…Primers were designed that incorporated NdeI and XhoI restriction sites. The PCR product was digested with NdeI and XhoI and ligated into pET28T3G, a laboratory pET28b(+) vector that had been previously modified to incorporate a TEV protease cleavage recognition site after the N‐terminal polyhistidine tag …”

Section: Methodsmentioning

confidence: 99%

The three‐dimensional structure of NeoB: An aminotransferase involved in the biosynthesis of neomycin

2018

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The aminoglycoside antibiotics, discovered as natural products in the 1940s, demonstrate a broad antimicrobial spectrum. Due to their nephrotoxic and ototoxic side effects, however, their widespread clinical usage has typically been limited to the treatment of serious infections. Neomycin B, first isolated from strains of Streptomyces in 1948, is one such drug that was approved for human use by the U.S. Food and Drug Administration in 1964. Only within the last 11 years has the biochemical pathway for its production been elaborated, however. Here we present the three-dimensional architecture of NeoB from Streptomyces fradiae, which is a pyridoxal 5'-phosphate or PLP-dependent aminotransferase that functions on two different substrates in neomycin B biosynthesis. For this investigation, four high resolution X-ray structures of NeoB were determined in various complexed states. The overall fold of NeoB is that typically observed for members of the "aspartate aminotransferase" family with the exception of an additional three-stranded antiparallel β-sheet that forms part of the subunit-subunit interface of the dimer. The manner in which the active site of NeoB accommodates quite different substrates has been defined by this investigation. In addition, during the course of this study, we also determined the structure of the aminotransferase GenB1 to high resolution. GenB1 functions as an aminotransferase in gentamicin biosynthesis. Taken together, the structures of NeoB and GenB1, presented here, provide the first detailed descriptions of aminotransferases that specifically function on aldehyde moieties in aminoglycoside biosynthesis.

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Structural investigation on WlaRG from Campylobacter jejuni: A sugar aminotransferase

Cited by 12 publications

References 41 publications

Investigation of a sugar N‐formyltransferase from the plant pathogen Pantoea ananatis

Investigation of a sugar N‐formyltransferase from the plant pathogen Pantoea ananatis

Investigation of the enzymes required for the biosynthesis of an unusual formylated sugar in the emerging human pathogen Helicobacter canadensis

The three‐dimensional structure of NeoB: An aminotransferase involved in the biosynthesis of neomycin

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