The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs) that are structurally related to eukaryotic kinases. To gain insight into the role of Ser/Thr phosphorylation in this major global pathogen, we used a phosphoproteomic approach to carry out an extensive analysis of protein phosphorylation in M. tuberculosis. We identified more than 500 phosphorylation events in 301 proteins that are involved in a broad range of functions. Bioinformatic analysis of quantitative in vitro kinase assays on peptides containing a subset of these phosphorylation sites revealed a dominant motif shared by six of the M. tuberculosis STPKs. Kinase assays on a second set of peptides incorporating targeted substitutions surrounding the phosphoacceptor validated this motif and identified additional residues preferred by individual kinases. Our data provide insight into processes regulated by STPKs in M. tuberculosis and create a resource for understanding how specific phosphorylation events modulate protein activity. The results further provide the potential to predict likely cognate STPKs for newly identified phosphoproteins.signal transduction | phosphorylation motif | phosphoproteomics A key feature of all living cells is the ability to sense environmental signals and implement adaptive changes. These inputs propagate through complex signal transduction networks whose activity is often regulated by reversible protein phosphorylation. Although Ser/Thr/Tyr protein phosphorylation-based signaling in eukaryotes has been intensively studied, the extent to which this mechanism is used in prokaryotes has only recently begun to be appreciated (1). The number of protein kinases in prokaryotes varies widely. Although many bacteria have only a few or none of these enzymes, some cyanobacteria and streptomycetes have dozens of them (2). Bacteria that do possess Ser/Thr or Tyr kinases often have complex lifestyles and depend on these kinases to regulate critical processes, such as stress adaptation, development, and virulence (2).Mycobacterium tuberculosis is an extraordinarily versatile pathogen that can exist in distinct states in the host, leading to asymptomatic latent tuberculosis (TB) infection in which bacteria are thought to be dormant, or active TB disease in which the organisms are actively replicating. To achieve these different physiologic states M. tuberculosis requires mechanisms to sense a wide range of signals from the host and to coordinately regulate multiple cellular processes. In most bacterial pathogens, the predominant phosphorylation-based signal transduction mechanism is the two-component system. The M. tuberculosis genome, however, encodes 11 Ser/Thr protein kinases (STPKs) and an equal number of two-component system sensor kinases, suggesting that these two phospho-based signaling systems are of comparable importance in this organism (3).Knowledge of the substrates of each of the M. tuberculosis STPKs is essential for understanding their function; however, only a small number of kinase-sub...

The structures and mechanism of action of many terpene cyclases are known, but there are no structures of diterpene cyclases. Here, we propose structural models based on bioinformatics, site-directed mutagenesis, domain swapping, enzyme inhibition and spectroscopy that help explain the nature of diterpene cyclase structure, function, and evolution. Bacterial diterpene cyclases contain ∼20 α-helices and the same conserved “QW” and DxDD motifs as in triterpene cyclases, indicating the presence of a βγ barrel structure. Plant diterpene cyclases have a similar catalytic motif and βγ-domain structure together with a third, α-domain, forming an αβγ structure, and in H+-initiated cyclases, there is an EDxxD-like Mg2+/diphosphate binding motif located in the γ-domain. The results support a new view of terpene cyclase structure and function and suggest evolution from ancient (βγ) bacterial triterpene cyclases to (βγ) bacterial and thence to (αβγ) plant diterpene cyclases.

Summary The fragile X syndrome, a common form of inherited intellectual disability, is caused by loss of the fragile X mental retardation protein FMRP. FMRP is present predominantly in the cytoplasm where it regulates translation of proteins important for synaptic function. We identify FMRP as a chromatin binding protein that functions in the DNA damage response (DDR). Specifically, we show that FMRP binds chromatin through its tandem Tudor (Agenet) domain in vitro, and associates with chromatin in vivo. We also demonstrate that FMRP participates in the DDR in a chromatin binding-dependent manner. The DDR machinery is known to play important roles in developmental processes such as gametogenesis. We show that FMRP occupies meiotic chromosomes and regulates the dynamics of DDR machinery during mouse spermatogenesis. These findings suggest that nuclear FMRP regulates genomic stability at the chromatin interface, and may impact gametogenesis and some developmental aspects of the fragile X syndrome.