B. subtilis ykuD protein at 2.0 A resolution: insights into the structure and function of a novel, ubiquitous family of bacterial enzymes

Abstract

The crystal structure of the product of the Bacillus subtilis ykuD gene was solved by the multiwavelength anomalous dispersion (MAD) method and refined using data to 2.0 A resolution. The ykuD protein is a representative of a distinctly prokaryotic and ubiquitous family found among both pathogenic and nonpathogenic Gram-positive and Gram-negative bacteria. The deduced amino acid sequence reveals the presence of an N-terminal LysM domain, which occurs among enzymes involved in cell wall metabolism, and a novel, putative catalytic domain with a highly conserved His/Cys-containing motif of hitherto unknown structure. As the wild-type protein did not crystallize, a double mutant was designed (Lys117Ala/Gln118Ala) to reduce excess surface conformational entropy. As expected, the structure of the LysM domain is similar to the NMR structure reported for an analogous domain from Escherichia coli murein transglycosylase MltD. The molecular model also shows that the 112-residue-long C-terminal domain has a novel tertiary fold consisting of a beta-sandwich with two mixed sheets, one containing five strands and the other, six strands. The two beta-sheets form a cradle capped by an alpha-helix. This domain contains a putative catalytic site with a tetrad of invariant His123, Gly124, Cys139, and Arg141. The stereochemistry of this active site shows similarities to peptidotransferases and sortases, and suggests that the enzymes of the ykuD family may play an important role in cell wall biology.

Fig. 1

Sequence alignment of the fingerprint motif in pathogenic and nonpathogenic bacteria (Φ is a hydrophobic residue and X denote any amino acid in consensus sequence).

Fig. 2

The molecular architecture of ykuD. (A) A stereodiagram showing the head-to-tail noncrystallographic dimer of ykuD molecules; the two molecules are labeled A and B. (B) A comparison of the NMR structure of the LysM domain from murein transglycosylase D (PDB entry: 1E0G), orange, and the LysM domain of ykuD, blue; for details see text. (C) The tertiary structure of the ErfK/YbiS/YhnG domain of ykuD with the fingerprint conserved stretch shown in red; the side-chains of His123, Gly124, Ser136, Cys139, and Arg141 are shown in full. (D) A schematic representation of the teriary fold of the ErfK/YbiS/YhnG domain.

Fig. 3

The ykuD putative active site involved in a crystal contact. Side-chains of select residues, as well as Gly124, are shown in full; sulfate ions are represented by spheres: red (oxygen) and green (sulfur).

Fig. 4

A comparison of the catalytic site of ykuD with other enzymes. (A) The ykuD catalytic triad with the side-chain of Cys139 rotated to achieve the best stereochemistry; H-bonds are shown by dotted lines, putative oxyanion hole forming backbone amides are denoted by asterisks. (B) Trypsin (PDB entry: 1YYY), with oxyanion hole indicated by asterisks. (C) Comparison of the ykuD and sortase A (PDB entry: 1T2O ) putative Cys-Arg dyads; ykuD residues are shown in light colors and the labels indicating the sortase A amino acids are in italics.