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Differentiation in African trypanosomes (Trypanosoma brucei) entails passage between a mammalian host, where parasites exist as a proliferative slender form or a G0-arrested stumpy form, and the tsetse fly. Stumpy forms arise at the peak of each parasitaemia and are committed to differentiation to procyclic forms that inhabit the tsetse midgut. We have identified a protein tyrosine phosphatase (TbPTP1) that inhibits trypanosome differentiation. Consistent with a tyrosine phosphatase, recombinant TbPTP1 exhibits the anticipated substrate and inhibitor profile, and its activity is impaired by reversible oxidation. TbPTP1 inactivation in monomorphic bloodstream trypanosomes by RNA interference or pharmacological inhibition triggers spontaneous differentiation to procyclic forms in a subset of committed cells. Consistent with this observation, homogeneous populations of stumpy forms synchronously differentiate to procyclic forms when tyrosine phosphatase activity is inhibited. Our data invoke a new model for trypanosome development in which differentiation to procyclic forms is prevented in the bloodstream by tyrosine dephosphorylation. It may be possible to use PTP1B inhibitors to block trypanosomatid transmission.

Research has been conducted on the low-molecular weight protein tyrosine phosphatases (LTP1). An X-ray crystallographic structure of a wild-type LTP1 from Saccharomyces cerevisiae is described.

Research was conducted to examine the structure of the S19A mutant of bovine protein tyrosine phosphatase, a member of the class of low-molecular weight protein tyrosine phosphatases found to be ubiquitous in mammalian cells, at 2.2 angstrom resolution. Results indicate that in the crystal, the protein forms a dimer in which Tyr131 and Tyr132 from one monomer insert into the active site of the other monomer. The significance for regulation and substrate specificity of hydrogen bond interactions that take place across the dimer interface was also analyzed.

Research was conducted to determine the role of lysine 267 in the catalytic function of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-Coa) reductase of Pseudomonas mevalonii. Site-directed mutagenesis results show that while replacement of lysine by alanine, histidine, arginine, or carboxyamidomethylcysteine rendered the enzyme inactive, aminoethylcysteine substitution led to high catalytic activity.

X-ray diffraction analysis was used to examine the molecular structure of the complex found between the minor groove binding drug netropsin and dodecamer d(CGCAAATTTGCG). The results reveal a refinement of 19.8% of the R-factor and a revolution of 2.2-angstroms. Five out of six of the A.T base pairs from A5. T20 to T9.A16 were covered by netropsin from its structural position in the narrow minor groove of the B-DNA fragment. Netropsin is permitted to bind in a position with maximum contact with the DNA by the long six A.T base pair. Hydrogen bonds with O2 of thymines and N3 of adenines are created by the binding.

Abstract Background Phosphoinositide metabolism is essential to membrane dynamics and impinges on many cellular processes, including phagocytosis. Modulation of phosphoinositide metabolism is important for pathogenicity and virulence of many human pathogens, allowing them to survive and replicate in the host cells. Phosphoinositide phosphatases from bacterial pathogens are therefore key players in this modulation and constitute attractive targets for chemotherapy. MptpB, a virulence factor from Mycobacterium tuberculosis, has phosphoinositide phosphatase activity and a distinct active site P-loop signature HCXXGKDR that shares characteristics with eukaryotic lipid phosphatases and protein tyrosine phosphatases. We used this P-loop signature as a "diagnostic motif" to identify related putative phosphatases with phosphoinositide activity in other organisms. Results We found more than 200 uncharacterised putative phosphatase sequences with the conserved signature in bacteria, with some related examples in fungi and protozoa. Many of the sequences identified belong to recognised human pathogens. Interestingly, no homologues were found in any other organisms including Archaea, plants, or animals. Phylogenetic analysis revealed that these proteins are unrelated to classic eukaryotic lipid phosphatases. However, biochemical characterisation of those from Listeria monocytogenes and Leishmania major, demonstrated that, like MptpB, they have phosphatase activity towards phosphoinositides. Mutagenesis studies established that the conserved Asp and Lys in the P-loop signature (HCXXGKDR) are important in catalysis and substrate binding respectively. Furthermore, we provide experimental evidence that the number of basic residues in the P-loop is critical in determining activity towards poly-phosphoinositides. Conclusion This new family of enzymes in microorganisms shows distinct sequence and biochemical characteristics to classic eukaryotic lipid phosphatases and they have no homologues in humans. This study provides a foundation for examining the biological role of this new family of phosphatases and their potential as pharmaceutical targets against infectious diseases.

Abstract Background The genomes of the three parasitic protozoa Trypanosoma cruzi, Trypanosoma brucei and Leishmania major are the main subject of this study. These parasites are responsible for devastating human diseases known as Chagas disease, African sleeping sickness and cutaneous Leishmaniasis, respectively, that affect millions of people in the developing world. The prevalence of these neglected diseases results from a combination of poverty, inadequate prevention and difficult treatment. Protein phosphorylation is an important mechanism of controlling the development of these kinetoplastids. With the aim to further our knowledge of the biology of these organisms we present a characterisation of the phosphatase complement (phosphatome) of the three parasites. Results An ontology-based scan of the three genomes was used to identify 86 phosphatase catalytic domains in T. cruzi, 78 in T. brucei, and 88 in L. major. We found interesting differences with other eukaryotic genomes, such as the low proportion of tyrosine phosphatases and the expansion of the serine/threonine phosphatase family. Additionally, a large number of atypical protein phosphatases were identified in these species, representing more than one third of the total phosphatase complement. Most of the atypical phosphatases belong to the dual-specificity phosphatase (DSP) family and show considerable divergence from classic DSPs in both the domain organisation and sequence features. Conclusion The analysis of the phosphatome of the three kinetoplastids indicates that they possess orthologues to many of the phosphatases reported in other eukaryotes, including humans. However, novel domain architectures and unusual combinations of accessory domains, suggest distinct functional roles for several of the kinetoplastid phosphatases, which await further experimental exploration. These distinct traits may be exploited in the selection of suitable new targets for drug development to prevent transmission and spread of the diseases, taking advantage of the already extensive knowledge on protein phosphatase inhibitors.

Biophysical and structural studies of protein kinase monopolar spindle 1 (Mps1) catalytic domain was carried out in the presence of ATP and the aspecific model kinase inhibitor staurosporine to understand the nucleotide binding site architecture of Mps1. The results for the structure of the Mps1 ATP-binding site provide new biophysical insights into Mps1-ligand interactions that would be useful for the design of specific Mps1 inhibitors, including those employing a therapeutically validated quinazoline template.

The characterization of HMG-CoA reductase (HMGR) from Listeria monocytogenes is reported and, the gene encoding the enzyme was amplified from genomic DNA and expressed in Escherichia coli as a 6x-His-tagged form, producing milligram quantities of purified recombinant enzyme. The purified enzyme exhibits dual coenzyme specificity, utilizing both NAD(H) and NADP(H) in catalysis, however it is found that catalytic efficiency using NADP(H) is approximately 200 times greater than when using NAD(H).

Summary: Ppz Ser/Thr protein phosphatases (PPases) are found only in fungi and have been proposed as potential antifungal targets. In Saccharomyces cerevisiae Ppz1 (ScPpz1) is involved in regulation of monovalent cation homeostasis. ScPpz1 is inhibited by two regulatory proteins, Hal3 and Vhs3, which have moonlighting properties, contributing to the formation of an unusual heterotrimeric PPC decarboxylase (PPCDC) complex crucial for CoA biosynthesis. Here we report the functional characterization of CnPpz1 (CNAG_03673) and two possible Hal3‐like proteins, CnHal3a (CNAG_00909) and CnHal3b (CNAG_07348) from the pathogenic fungus Cryptococcus neoformans. Deletion of CnPpz1 or CnHal3b led to phenotypes unrelated to those observed in the equivalent S. cerevisiae mutants, and the CnHal3b‐deficient strain was less virulent. CnPpz1 is a functional PPase and partially replaced endogenous ScPpz1. Both CnHal3a and CnHal3b interact with ScPpz1 and CnPpz1 in vitro but do not inhibit their phosphatase activity. Consistently, when expressed in S. cerevisiae, they poorly reproduced the Ppz1‐regulatory properties of ScHal3. In contrast, both proteins were functional monogenic PPCDCs. The CnHal3b isoform was crystallized and, for the first time, the 3D‐structure of a fungal PPCDC elucidated. Therefore, our work provides the foundations for understanding the regulation and functional role of the Ppz1‐Hal3 system in this important pathogenic fungus. We describe the structure and function of CnPpz1 and two putative Ppz1 regulators (CnHal3a and CnHal3b). CnPpz1 is relatively alike to Ppz1 phosphatases in other fungi but is not involved in virulence. CnHal3a and CnHal3b display a function in CoA biosynthesis, but only CnHal3b is important for virulence. Neither CnHal3a and CnHal3b regulate CnPpz1 and, therefore, in contrast to S. cerevisiae and C. albicans, these are not moonlighting proteins. [ABSTRACT FROM AUTHOR]