Expression, purification, crystallization and preliminary X-ray studies of<i>Lactobacillus jensenii</i>enolase

Harris, Paul T.; Raghunathan, K.; Spurbeck, Rachel R.; Arvidson, Cindy Grove; Arvidson, Dennis N.

doi:10.1107/s1744309110022748

Cited by 4 publications

(4 citation statements)

References 15 publications

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“…The third catalytic loop, residues 250-269, is missing in its entirety due to disorder. This was also noticed in structures derived from initial datasets of crystals grown by dehydrating the enolase in Tris buffer [25].…”

Section: Catalytic Loops and Comparisons With Plasminogen Binding Enomentioning

confidence: 58%

“…The cloning, expression, purification, and enzymatic activity assays of L. gasseri enolase were performed as previously reported [3,25]. Briefly, a construct containing enolase with a non-cleavable C-terminal His 6 -tag was expressed in Escherichia coli, and purified employing nickel affinity chromatography followed by anion exchange chromatography.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…Crystallization screens were conducted as described [25] at a concentration of 2-4 mg/ml. Crystals found and sieved by the melt test [26] were reproduced by hanging drop method with 3 ll of protein solution mixed with an equal volume of reservoir solution drawn from the 400 ll present in the well of a 24-well VDX crystallization plate (Hampton Research) at 25°C.…”

Section: Crystallization and Data Collectionmentioning

confidence: 99%

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Crystal structure of an efficacious gonococcal adherence inhibitor: An enolase from Lactobacillus gasseri

Raghunathan¹,

Harris²,

Spurbeck³

et al. 2014

FEBS Letters

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a b s t r a c tEnolases are highly conserved metalloenzymes ubiquitous to cellular metabolism. While these enzymes share a large degree of sequence and structural similarity, they have been shown to possess a wide range of moonlighting functions. Recent studies showed that an enolase from Lactobacillus gasseri impedes the ability of Neisseria gonorrhoeae to adhere to epithelial cells. We present the crystal structure of this enolase, the first from Lactobacillus, with one of its Mg 2+ cofactors. Determined using molecular replacement to 2.08 Å, the structure has a flexible and surface exposed catalytic loop containing lysines, and may play a role in the inhibitory function. Structured summary of protein interactions:Eno and eno bind by X-ray crystallography (View interaction)

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Section: Catalytic Loops and Comparisons With Plasminogen Binding Enomentioning

confidence: 58%

Section: Protein Expression and Purificationmentioning

confidence: 99%

Section: Crystallization and Data Collectionmentioning

confidence: 99%

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Crystal structure of an efficacious gonococcal adherence inhibitor: An enolase from Lactobacillus gasseri

Raghunathan¹,

Harris²,

Spurbeck³

et al. 2014

FEBS Letters

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“…F66, 938-940] is corrected.Sequencing of the 16S rDNA from the Lactobacillus strain used as the source of the enolase gene used for the studies reported in the article by Harris et al (2010) has shown that what was reported as Lactobacillus jensenii 25258 is in fact Lactobacillus gasseri 33323. Further studies of this crystallized enolase will properly attribute the enzyme to L. gasseri.…”

mentioning

confidence: 99%

Expression, purification, crystallization and preliminary X-ray studies ofLactobacillus jenseniienolase. Corrigendum

Harris¹,

Raghunathan²,

Spurbeck³

et al. 2013

Acta Crystallogr F Struct Biol Cryst Commun

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The article by Harris et al. [(2010) Acta Cryst. F66, 938-940] is corrected.Sequencing of the 16S rDNA from the Lactobacillus strain used as the source of the enolase gene used for the studies reported in the article by Harris et al. (2010) has shown that what was reported as Lactobacillus jensenii 25258 is in fact Lactobacillus gasseri 33323. Further studies of this crystallized enolase will properly attribute the enzyme to L. gasseri. We sincerely apologize for any confusion arising from this unfortunate error.

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A Moonlighting Enolase from Lactobacillus gasseri does not Require Enzymatic Activity to Inhibit Neisseria gonorrhoeae Adherence to Epithelial Cells

Spurbeck

Harris

Raghunathan

et al. 2015

Probiotics & Antimicro. Prot.

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Enolases are generally thought of as cytoplasmic enzymes involved in glycolysis and gluconeogenesis. However, several bacteria have active forms of enolase associated with the cell surface and these proteins are utilized for functions other than central metabolism. Recently, a surface-associated protein produced by Lactobacillus gasseri ATCC 33323 with homology to enolase was found to inhibit the adherence of the sexually transmitted pathogen, Neisseria gonorrhoeae, to epithelial cells in culture. Here, we show that the protein is an active enolase in vitro. A recombinantly expressed, C-terminal His-tagged version of the protein, His6-Eno3, inhibited gonococcal adherence. Assays utilizing inhibitors of enolase enzymatic activity showed that this inhibitory activity required the substrate-binding site to be in an open conformation; however, the enolase enzymatic activity of the protein was not necessary for inhibition of gonococcal adherence. An L. gasseri strain carrying an insertional mutation in eno3 was viable, indicating that eno3 is not an essential gene in L. gasseri 33323. This observation, along with the results of the enzyme assays, is consistent with reports that this strain encodes more than one enolase. Here we show that the three L. gasseri genes annotated as encoding an enolase are expressed. The L. gasseri eno3 mutant exhibited reduced, but not abolished, inhibition of gonococcal adherence, which supports the hypothesis that L. gasseri inhibition of gonococcal adherence is a multifactorial process.

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Expression, purification, crystallization and preliminary X-ray studies ofLactobacillus jenseniienolase

Cited by 4 publications

References 15 publications

Crystal structure of an efficacious gonococcal adherence inhibitor: An enolase from Lactobacillus gasseri

Crystal structure of an efficacious gonococcal adherence inhibitor: An enolase from Lactobacillus gasseri

Expression, purification, crystallization and preliminary X-ray studies ofLactobacillus jenseniienolase. Corrigendum

A Moonlighting Enolase from Lactobacillus gasseri does not Require Enzymatic Activity to Inhibit Neisseria gonorrhoeae Adherence to Epithelial Cells

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