A comprehensive in silico analysis of sortase superfamily.

Sortases are cysteine transpeptidases that assemble surface proteins and pili in their cell envelope. Encoded by all Gram-positive bacteria, few Gram-negative bacteria and archaea, sortases are currently divided into six classes (A-F). Due to the steep increase in bacterial genome data in recent years, the number of sortase homologues have also escalated rapidly. In this study, we used protein sequence similarity networks to explore the taxonomic diversity of sortases and also to evaluate the current classification of these enzymes. The resultant data suggest that sortase classes A, B, and D predominate in Firmicutes and classes E and F are enriched in Actinobacteria, whereas class C is distributed in both Firmicutes and Actinobacteria except Streptomyces family. Sortases were also observed in various Gram-negatives and euryarchaeota, which should be recognized as novel classes of sortases. Motif analysis around the catalytic cysteine was also performed and suggested that the residue at 2nd position from cysteine may help distinguish various sortase classes. Moreover, the sequence analysis indicated that the catalytic arginine is highly conserved in almost all classes except sortase F in which arginine is replaced by asparagine in Actinobacteria. Additionally, class A sortases showed higher structural variation as compared to other sortases, whereas inter-class comparisons suggested structures of class C and D2 exhibited best similarities. A better understanding of the residues highlighted in this study should be helpful in elucidating their roles in substrate binding and the sortase function, and successively could help in the development of strong sortase inhibitors.

Bacterial GCN5-Related N-Acetyltransferases: From Resistance to Regulation.

The GCN5-related N-acetyltransferases family (GNAT) is an important family of proteins that includes more than 100000 members among eukaryotes and prokaryotes. Acetylation appears as a major regulatory post-translational modification and is as widespread as phosphorylation. N-Acetyltransferases transfer an acetyl group from acetyl-CoA to a large array of substrates, from small molecules such as aminoglycoside antibiotics to macromolecules. Acetylation of proteins can occur at two different positions, either at the amino-terminal end (αN-acetylation) or at the ε-amino group (εN-acetylation) of an internal lysine residue. GNAT members have been classified into different groups on the basis of their substrate specificity, and in spite of a very low primary sequence identity, GNAT proteins display a common and conserved fold. This Current Topic reviews the different classes of bacterial GNAT proteins, their functions, their structural characteristics, and their mechanism of action.

Sorting sortases: a nomenclature proposal for the various sortases of Gram-positive bacteria.

Bacterial surface proteins constitute a diverse group of molecules with important functions, such as adherence, invasion, signaling and interaction with the host immune system or the environment. In Gram-positive bacteria, many surface proteins are anchored to the cell wall envelope by an enzyme named sortase, which recognizes a conserved carboxylic sorting motif. The sequence of the prototype staphylococcal SrtA has been widely used to identify homologs in bacterial genomes, revealing a profusion of sortases in almost all Gram-positive bacteria, often with more than one sortase-like protein per genome [M.J. Pallen, A.C. Lam, M. Antonio, K. Dunbar, Trends Microbiol. 9 (2001) 97-102]. In light of increasing reports on the identification and/or characterization of paralogous sortase genes, a classification of sortases now appears necessary. This report provides an analysis of sixty-one sortases from complete Gram-positive genomes, and suggests the existence of four structural groups of sortases. We propose the classification of sortases into 4 classes designated A, B, C and D. This classification should help to discriminate between sortases in the future.

A comparative genome analysis identifies distinct sorting pathways in gram-positive bacteria.

Surface proteins in gram-positive bacteria are frequently required for virulence, and many are attached to the cell wall by sortase enzymes. Bacteria frequently encode more than one sortase enzyme and an even larger number of potential sortase substrates that possess an LPXTG-type cell wall sorting signal. In order to elucidate the sorting pathways present in gram-positive bacteria, we performed a comparative analysis of 72 sequenced microbial genomes. We show that sortase enzymes can be partitioned into five distinct subfamilies based upon their primary sequences and that most of their substrates can be predicted by making a few conservative assumptions. Most bacteria encode sortases from two or more subfamilies, which are predicted to function nonredundantly in sorting proteins to the cell surface. Only approximately 20% of sortase-related proteins are most closely related to the well-characterized Staphylococcus aureus SrtA protein, but nonetheless, these proteins are responsible for anchoring the majority of surface proteins in gram-positive bacteria. In contrast, most sortase-like proteins are predicted to play a more specialized role, with each anchoring far fewer proteins that contain unusual sequence motifs. The functional sortase-substrate linkage predictions are available online (http://www.doe-mbi.ucla.edu/Services/Sortase/) in a searchable database.

Metalloid tolerance based on phytochelatins is not functionally equivalent to the arsenite transporter Acr3p.

Active transport of metalloids by Acr3p and Ycf1p in Saccharomyces cerevisiae and chelation by phytochelatins in Schizosaccharomyces pombe, nematodes, and plants represent distinct strategies of metalloid detoxification. In this report, we present results of functional comparison of both resistance mechanisms. The S. pombe and wheat phytochelatin synthase (PCS) genes, when expressed in S. cerevisiae, mediate only modest resistance to arsenite and thus cannot functionally compensate for Acr3p. On the other hand, we show for the first time that phytochelatins also contribute to antimony tolerance as PCS fully complement antimonite sensitivity of ycf1Delta mutant. Remarkably, heterologous expression of PCS sensitizes S. cerevisiae to arsenate, while ACR3 confers much higher arsenic resistance in pcsDelta than in wild-type S. pombe. The analysis of PCS and ACR3 homologues distribution in various organisms and our experimental data suggest that separation of ACR3 and PCS genes may lead to the optimal tolerance status of the cell.

Structure of crystalline D-Tyr-tRNA(Tyr) deacylase. A representative of a new class of tRNA-dependent hydrolases.

Cell growth inhibition by several d-amino acids can be explained by an in vivo production of d-aminoacyl-tRNA molecules. Escherichia coli and yeast cells express an enzyme, d-Tyr-tRNA(Tyr) deacylase, capable of recycling such d-aminoacyl-tRNA molecules into free tRNA and d-amino acid. Accordingly, upon inactivation of the genes of the above deacylases, the toxicity of d-amino acids increases. Orthologs of the deacylase are found in many cells. In this study, the crystallographic structure of dimeric E. coli d-Tyr-tRNA(Tyr) deacylase at 1.55 A resolution is reported. The structure corresponds to a beta-barrel closed on one side by a beta-sheet lid. This barrel results from the assembly of the two subunits. Analysis of the structure in relation with sequence homologies in the orthologous family suggests the location of the active sites at the carboxy end of the beta-strands. The solved structure markedly differs from those of all other documented tRNA-dependent hydrolases.

Evidence for essential histidines in human pituitary glutaminyl cyclase.

Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the formation of the pyroglutamyl residue present at the amino terminus of numerous secretory peptides and proteins. Treatment with diethyl pyrocarbonate inactivated recombinant human QC with the apparent modification of three essential histidine residues. Comparisons of the protein sequences of QC from a variety of eukaryotic species show four completely conserved histidine residues. Mutation of each of these residues to glutamine resulted in two mutant enzymes that were inactive (H140Q and H330Q), suggesting a role in catalysis, and two that exhibited increased Km values (H307Q and H319Q), suggesting a role in substrate binding. Consistent with these results is the prediction that QC possesses a zinc aminopeptidase domain in which the four histidines identified here are present in the active site. Mammalian glutaminyl cyclases may, therefore, have structural and catalytic similarities to a family of bacterial zinc aminopeptidases.