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The Salmonella typhimurium ' yeaZ' gene ( StyeaZ) encodes an essential protein of unknown function ( StYeaZ), which has previously been annotated as a putative homolog of the Pasteurella haemolytica M22 O-sialoglycoprotein endopeptidase Gcp. YeaZ has also recently been reported as the first example of an RPF from a gram-negative bacterial species. To further characterize the properties of StYeaZ and the widely occurring MK-M22 family, we describe the purification, biochemical analysis, crystallization, and structure determination of StYeaZ. The crystal structure of StYeaZ reveals a classic two-lobed actin-like fold with structural features consistent with nucleotide binding. However, microcalorimetry experiments indicated that StYeaZ neither binds polyphosphates nor a wide range of nucleotides. Additionally, biochemical assays show that YeaZ is not an active O-sialoglycoprotein endopeptidase, consistent with the lack of the critical zinc binding motif. We present a detailed comparison of YeaZ with available structural homologs, the first reported structural analysis of an MK-M22 family member. The analysis indicates that StYeaZ has an unusual orientation of the A and B lobes which may require substantial relative movement or interaction with a partner protein in order to bind ligands. Comparison of the fold of YeaZ with that of a known RPF domain from a gram-positive species shows significant structural differences and therefore potentially distinctive RPF mechanisms for these two bacterial classes. Proteins 2006. © 2006 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Salmonella typhimurium YegS is a protein conserved in many prokaryotes. Although the function of YegS is not definitively known, it has been annotated as a potential diacylglycerol or sphingosine kinase based on sequence similarity with eukaryotic enzymes of known function. To further characterize YegS, we report its purification, biochemical analysis, crystallization, and structure determination. The crystal structure of YegS reveals a two-domain fold related to bacterial polyphosphate/ATP NAD kinases, comprising a central cleft between an N-terminal alpha/beta domain and a C-terminal two-layer beta-sandwich domain; conserved structural features are consistent with nucleotide binding within the cleft. The N-terminal and C-terminal domains of YegS are however counter-rotated, relative to the polyphosphate/ATP NAD kinase archetype, such that the potential nucleotide binding site is blocked. There are also two Ca2+ binding sites and two hydrophobic clefts, one in each domain of YegS. Analysis of mutagenesis data from eukaryotic homologues of YegS suggest that the N-terminal cleft may bind activating lipids while the C-terminal cleft may bind the lipid substrate. Microcalorimetry experiments showed interaction between recombinant YegS and Mg2+, Ca2+, and Mn2+ ions, with a weaker interaction also observed with polyphosphates and ATP. However, biochemical assays showed that recombinant YegS is endogenously neither an active diacylglycerol nor sphingosine kinase. Thus although the bioinformatics analysis and structure of YegS indicate that many of the ligand recognition determinants for lipid kinase activity are present, the absence of such activity may be due to specificity for a different lipid substrate or the requirement for activation by an, as yet, undetermined mechanism. In this regard the specific interaction of YegS with the periplasmic chaperone OmpH, which we demonstrate from pulldown experiments, may be of significance. Such an interaction suggests that YegS can be translocated to the periplasm and directed to the outer-membrane, an environment that may be required for enzyme activity., (2007 Wiley-Liss, Inc.)