In spite of rapid advances in microbial genomics and an expanding set of complete prokaryotic genomes, a significant portion of bacterial genes code for nonannotated proteins of unknown structure and function. As the search for novel antibiotic targets intensifies in the face of an advancing threat of revival in infectious diseases, structural characterization of such orphan proteins or entire families of bacterial proteins is of particular importance.
One such structurally uncharacterized, ubiquitous family of exclusively prokaryotic proteins comprises putative enzymes containing the so-called ErfK/YbiS/YhnG domain (Pfam03734.8) as identified by the SMART database.1
These proteins are found in a vast variety of bacteria, ranging from pathogenic Bacillus anthracis
, Vibrio cholera
, Clostridium tetani
, and others, to nonpathogenic Escherichia coli
, Thermotoga maritima
, Bacillus subtilis
, and so forth. All ErfK/YbiS/YhnG domains contain a highly conserved stretch of amino acids (ΦGΦHGTX10
(S/T)XGCΦR(M/L), where Φ is a hydrophobic residue and X
denotes any amino acid). The presence of absolutely conserved His and Cys residues is suggestive of a catalytic function, probably as hydrolases or transferases (). Many of these proteins also contain the so-called LysM domain, which appears to have a peptidoglycan-binding function and is found in enzymes involved in cell wall degradation, and other proteins that could be associated with bacterial cell walls.2
Interestingly, homologous domains have also been discovered in a family of plant transmembrane receptor kinases, which are involved in the recognition of bacteria secreting lipochitin oligosaccharide signal molecules.3
Only one structural study of a LysM domain has been reported to date.2
The association of the ErfK/YbiS/YhnG domain with the LysM module suggests that the former may also play a role in cell wall degradation or, more specifically, in peptidoglycan metabolism. This is interesting because such unique prokaryotic proteins constitute a particularly suitable target for antibiotics.
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).
The B. subtilis
ykuD protein was selected as one of the targets for high-throughput structure determination by the Midwest Center for Structural Genomics (MCSG). It expressed well in E. coli
but failed to crystallize despite extensive screening. We have therefore applied the recently proposed approach of surface conformational entropy reduction to generate on the protein’s surface patches that would be able to mediate crystal contacts.4
Accordingly, Lys117 and Gln118 were mutated to alanines, and the purified double mutant yielded high-quality crystals.
In this article we describe the details of the ykuD structure. Aside from the expected LysM module, the structure reveals a novel tertiary fold for the C-terminal ErfK/YbiS/YhnG domain. The stereochemical features of the amino acids in the fingerprint, conserved stretch (i.e., of His123, Gly124, Cys139, and Arg131), confirm that the ErfK domain is very likely to have catalytic properties. Based on the crystal structure, we propose a possible catalytic mechanism for the enzyme and speculate on the nature of the substrate. Our work sets the stage for functional and biochemical characterization of a novel and probably important family of ubiquitous bacterial enzymes.