Structure analysis of the Staphylococcus aureus UDP-N-acetyl-mannosamine dehydrogenase Cap5O involved in capsular polysaccharide biosynthesis.

Bacterial UDP-sugar dehydrogenases are part of the biosynthesis pathway of extracellular polysaccharides. These compounds act as important virulence factors by protecting the cell from opsonophagocytosis and complement-mediated killing. In Staphylococcus aureus, the protein Cap5O catalyzes the oxidation of UDP-N-acetyl-mannosamine to UDP-N-acetyl-mannosaminuronic acid. Cap5O is crucial for the production of serotype 5 capsular polysaccharide that prevents the interaction of bacteria with both phagocytic and nonphagocytic eukaryotic cells. However, details of its catalytic mechanism remain unknown. We thus crystallized Cap5O and solved the first structure of an UDP-N-acetyl-mannosamine dehydrogenase. This study revealed that the catalytic cysteine makes a disulfide bond that has never been observed in other structurally characterized members of the NDP-sugar dehydrogenase family. Biochemical and mutagenesis experiments demonstrated that the formation of this disulfide bridge regulates the activity of Cap5O. We also identified two arginine residues essential for Cap5O activity. Previous data suggested that Cap5O is activated by tyrosine phosphorylation, so we characterized the phosphorylation site and examined the underlying regulatory mechanism.

Identification of structural and molecular determinants of the tyrosine-kinase Wzc and implications in capsular polysaccharide export.

Capsular polysaccharides are well-established virulence factors of pathogenic bacteria. Their biosynthesis and export are regulated within the transmembrane polysaccharide assembly machinery by the autophosphorylation of atypical tyrosine-kinases, named BY-kinases. However, the accurate functioning of these tyrosine-kinases remains unknown. Here, we report the crystal structure of the non-phosphorylated cytoplasmic domain of the tyrosine-kinase Wzc from Escherichia coli in complex with ADP showing that it forms a ring-shaped octamer. Mutational analysis demonstrates that a conserved EX(2) RX(2) R motif involved in subunit interactions is essential for polysaccharide export. We also elucidate the role of a putative internal regulatory tyrosine and we show that BY-kinases from proteobacteria autophosphorylate on their C-terminal tyrosine cluster via a single-step intermolecular mechanism. This structure-function analysis also allows us to demonstrate that two different parts of a conserved basic region called the RK-cluster are essential for polysaccharide export and for kinase activity respectively. Based on these data, we revisit the dichotomy made between BY-kinases from proteobacteria and firmicutes and we propose a unique process of oligomerization and phosphorylation. We also reassess the function of BY-kinases in the capsular polysaccharide assembly machinery.

Structural basis for the regulation mechanism of the tyrosine kinase CapB from Staphylococcus aureus.

Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function.

Identification of the idiosyncratic bacterial protein tyrosine kinase (BY-kinase) family signature.

MOTIVATION: Most of the protein tyrosine kinases found in bacteria have been recently classified in a new family, termed BY-kinase. Indeed, they share no sequence homology with their eukaryotic counterparts and have no known eukaryotic homologues. They are involved in several biological functions (e.g. capsule biosynthesis, antibiotic resistance, virulence mechanism). Thus, they can be considered interesting therapeutic targets to develop new drugs to treat infectious diseases. However, their identification is rendered difficult due to slow progress in their structural characterization and comes most often from biochemical experiments. Moreover BY-kinase sequences are related to many other bacterial proteins involved in several biological functions (e.g. ParA family proteins). Accordingly, their annotations in generalist databases, sequence analysis and classification remain partial and inhomogeneous and there is no bioinformatics resource dedicated to these proteins. RESULTS: The combination of similarity search with sequence-profile alignment, pattern matching and sliding window computation to detect the tyrosine cluster was used to identify BY-kinase sequences in UniProt Knowledgebase. Cross-validations with keywords searches, pattern matching with several patterns and checking of motifs conservation in multiple sequence alignments were performed. Our pipeline identified 640 sequences as BY-kinases and allowed the definition of a PROSITE pattern that is the signature of the BY-kinases. The sequences identified by our pipeline as BY-kinases share a good sequence similarity with BY-kinases that have already been biochemically characterized, and they all bear the characteristic motifs of the catalytic domain, including the three Walker-like motifs followed by a tyrosine cluster. AVAILABILITY: http://bykdb.ibcp.fr

Tyrosine-kinases in bacteria: from a matter of controversy to the status of key regulatory enzymes.

When considering protein phosphorylation in bacteria, phosphorylation of aspartic acid and histidine residues mediated by the two-component systems is the first to spring to mind. And yet other phosphorylation systems have been described in bacteria in the past 20 years including eukaryotic-like serine/threonine kinases and more recently tyrosine-kinases. Among the latter, a peculiar type is widespread among bacteria, but not in higher organisms. These enzymes possess unique structural features defining thus a new family of enzymes termed Bacterial tyrosine kinases (BY-kinases). BY-kinases have been shown to be mainly involved in polysaccharide production, but their ability to phosphorylate endogenous substrates indicates that they participate in the regulation of other functions of the bacterial cell. Recent advances in mass spectrometry based phosphoproteomics provided lists of many new phosphotyrosine-proteins, indicating that BY-kinases may be involved in regulating a large array of other cellular functions. One may expect that in a near future, tyrosine phosphorylation will turn out to be one of the key regulatory processes in the bacterial cell and will yield new insights into the understanding of its physiology.

Comparative analysis of the Tyr-kinases CapB1 and CapB2 fused to their cognate modulators CapA1 and CapA2 from Staphylococcus aureus.

A particular class of tyrosine-kinases sharing no structural similarity with eukaryotic tyrosine-kinases has been evidenced in a large array of bacterial species. These bacterial tyrosine-kinases are able to autophosphorylate on a C-terminal tyrosine-rich motif. Their autophosphorylation has been shown to play a crucial role in the biosynthesis or export of capsular polysaccharide. The analysis of the first crystal structure of the staphylococcal tyrosine kinase CapB2 associated with the activating domain of the transmembrane modulator CapA1 had brought conclusive explanation for both the autophosphorylation and activation processes. In order to explain why CapA1 activates CapB2 more efficiently than its cognate transmembrane modulator CapA2, we solved the crystal structure of CapA2B2 and compared it with the previously published structure of CapA1B2. This structural analysis did not provide the expected clues about the activation discrepancy observed between the two modulators. Staphylococcus aureus also encodes for a CapB2 homologue named CapB1 displaying more than 70% sequence similarity and being surprisingly nearly unable to autophosphorylate. We solved the crystal structure of CapA1B1 and carefully compare it with the structure of CapA1B2. The active sites of both proteins are highly conserved and the biochemical characterization of mutant proteins engineered to test the importance of small structural discrepancies identified between the two structures did not explain the inactivity of CapB1. We thus tested if CapB1 could phosphorylate other protein substrates or hydrolyze ATP. However, no activity could be detected in our in vitro assays. Taken together, these data question about the biological role of the homologous protein pairs CapA1/CapB1 and CapA2/CapB2 and we discuss about several possible interpretations.

Tyrosine phosphorylation of the UDP-glucose dehydrogenase of Escherichia coli is at the crossroads of colanic acid synthesis and polymyxin resistance.

BACKGROUND: In recent years, an idiosyncratic new class of bacterial enzymes, named BY-kinases, has been shown to catalyze protein-tyrosine phosphorylation. These enzymes share no structural and functional similarities with their eukaryotic counterparts and, to date, only few substrates of BY-kinases have been characterized. BY-kinases have been shown to participate in various physiological processes. Nevertheless, we are at a very early stage of defining their importance in the bacterial cell. In Escherichia coli, two BY-kinases, Wzc and Etk, have been characterized biochemically. Wzc has been shown to phosphorylate the UDP-glucose dehydrogenase Ugd in vitro. Not only is Ugd involved in the biosynthesis of extracellular polysaccharides, but also in the production of UDP-4-amino-4-deoxy-L-arabinose, a compound that renders E. coli resistant to cationic antimicrobial peptides. METHODOLOGY/PRINCIPAL FINDINGS: Here, we studied the role of Ugd phosphorylation. We first confirmed in vivo the phosphorylation of Ugd by Wzc and we demonstrated that Ugd is also phosphorylated by Etk, the other BY-kinase identified in E. coli. Tyrosine 71 (Tyr71) was characterized as the Ugd site phosphorylated by both Wzc and Etk. The regulatory role of Tyr71 phosphorylation on Ugd activity was then assessed and Tyr71 mutation was found to prevent Ugd activation by phosphorylation. Further, Ugd phosphorylation by Wzc or Etk was shown to serve distinct physiological purposes. Phosphorylation of Ugd by Wzc was found to participate in the regulation of the amount of the exopolysaccharide colanic acid, whereas Etk-mediated Ugd phosphorylation appeared to participate in the resistance of E. coli to the antibiotic polymyxin. CONCLUSIONS/SIGNIFICANCE: Ugd phosphorylation seems to be at the junction between two distinct biosynthetic pathways, illustrating the regulatory potential of tyrosine phosphorylation in bacterial physiology.