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.
Protein crystallization screens frequently yield salt crystals as well as protein crystals. A simple method for determining whether a crystal is composed of salt or macromolecules is suggested. A drop containing one or more crystals is transferred to a glass cover slip and the cover slip is then passed through the flame of a Bunsen burner. Macromolecule crystals are destroyed by this treatment, while salt crystals generally remain. The test can be performed after other commonly used tests such as crushing and staining.
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)
Recombinant Lactobacillus jensenii enolase fused to a C-terminal noncleavable His tag was expressed in Escherichia coli, purified and crystallized by sittingdrop vapor diffusion. A complete data set was collected to 3.25 Å resolution. The crystals belonged to space group I4, with unit-cell parameters a = b = 145.31, c = 99.79 Å . There were two protein subunits in the asymmetric unit, which gave a Matthews coefficient V M of 2.8 Å 3 Da À1 , corresponding to 55.2% solvent content.
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