Functional role of highly conserved residues of the N-terminal tail and first transmembrane segment of a P4-ATPase

The P4 family of P-type ATPases (P4-ATPases) plays an important role in maintaining phospholipid asymmetry in eukaryotic cell membranes. Leishmania miltefosine transporter (LMT) is a plasma membrane (PM) P4-ATPase that catalyses translocation into the parasite of the leishmanicidal drug miltefosine as well as phosphatidylcholine and phosphatidylethanolamine analogues. In the present study, we analysed the role, in LMT, of a series of highly conserved amino acids previously undescribed in the N-terminal region of P4-ATPases. Seven residues were identified and, according to an LMT structural model, five were located in the cytosolic N-terminal tail (Asn58, Ile60, Lys64, Tyr65 and Phe70) and the other two (Pro72 and Phe79) in the first transmembrane segment (TM1). Alanine-scanning mutagenesis analysis showed that N58A, Y65A and F79A mutations caused a considerable reduction in the LMT translocase activity. These mutations did not affect protein expression levels. We generated additional mutations in these three residues to assess the influence of the conservation degree on LMT translocase activity. Some of these mutations reduced expression levels without affecting the interaction between LMT and its CDC50 subunit, LRos3. Conserved and non-conserved mutations in the invariant residue Asn58 drastically reduced the translocase activity. Consequently, Asn58 may be necessary to achieve optimal catalytic LMT activity as previously described for the potentially equivalent Asn39 of the sarco/endoplasmic reticulum Ca2+-ATPase isoform 1a (SERCA1a). Additionally, conservation of a hydrophobic residue at position 79 is crucial for LMT stability.

Renormalization of massless Feynman amplitudes in configuration space

A systematic study of recursive renormalization of Feynman amplitudes is carried out both in Euclidean and in Minkowski configuration spaces. For a massless quantum field theory (QFT), we use the technique of extending associate homogeneous distributions to complete the renormalization recursion. A homogeneous (Poincaré covariant) amplitude is said to be convergent if it admits a (unique covariant) extension as a homogeneous distribution. For any amplitude without subdivergences — i.e. for a Feynman distribution that is homogeneous off the full (small) diagonal — we define a renormalization invariant residue. Its vanishing is a necessary and sufficient condition for the convergence of such an amplitude. It extends to arbitrary — not necessarily primitively divergent — Feynman amplitudes. This notion of convergence is finer than the usual power counting criterion and includes cancellation of divergences.

Structure and function of the transketolase from Mycobacterium tuberculosis and comparison with the human enzyme

The transketolase (TKT) enzyme in Mycobacterium tuberculosis represents a novel drug target for tuberculosis treatment and has low homology with the orthologous human enzyme. Here, we report on the structural and kinetic characterization of the transketolase from M. tuberculosis (TBTKT), a homodimer whose monomers each comprise 700 amino acids. We show that TBTKT catalyses the oxidation of donor sugars xylulose-5-phosphate and fructose-6-phosphate as well as the reduction of the acceptor sugar ribose-5-phosphate. An invariant residue of the TKT consensus sequence required for thiamine cofactor binding is mutated in TBTKT; yet its catalytic activities are unaffected, and the 2.5 Å resolution structure of full-length TBTKT provides an explanation for this. Key structural differences between the human and mycobacterial TKT enzymes that impact both substrate and cofactor recognition and binding were uncovered. These changes explain the kinetic differences between TBTKT and its human counterpart, and their differential inhibition by small molecules. The availability of a detailed structural model of TBTKT will enable differences between human and M. tuberculosis TKT structures to be exploited to design selective inhibitors with potential antitubercular activity.

Genetic Analysis of the Invariant Residue G791 in Escherichia coli 16S rRNA Implicates RelA in Ribosome Function

ABSTRACT Previous studies identified G791 in Escherichia coli 16S rRNA as an invariant residue for ribosome function. In order to establish the functional role of this residue in protein synthesis, we searched for multicopy suppressors of the mutant ribosomes that bear a G-to-U substitution at position 791. We identified relA, a gene whose product has been known to interact with ribosomes and trigger a stringent response. Overexpression of RelA resulted in the synthesis of approximately 1.5 times more chloramphenicol acetyltransferase (CAT) protein than could be synthesized by the mutant ribosomes in the absence of RelA overexpression. The ratio of mutant rRNA to the total ribosome pool was not changed, and the steady-state level of CAT mRNA was decreased by RelA overexpression. These data confirmed that the phenotype of RelA as a multicopy suppressor of the mutant ribosome did not result from the enhanced synthesis of mutant rRNA or CAT mRNA from the plasmid. To test whether the phenotype of RelA was related to the stringent response induced by the increased cellular level of (p)ppGpp, we screened for mutant RelA proteins whose overexpression enhances CAT protein synthesis by the mutant ribosomes as effectively as wild-type RelA overexpression and then screened for those whose overexpression does not produce sufficiently high levels of (p)ppGpp to trigger the stringent response under the condition of amino acid starvation. Overexpression of the isolated mutant RelA proteins resulted in the accumulation of (p)ppGpp in cells, which was amounted to approximately 18.2 to 38.9% of the level of (p)ppGpp found in cells that overexpress the wild-type RelA. These findings suggest that the function of RelA as a multicopy suppressor of the mutant ribosome does not result from its (p)ppGpp synthetic activity. We conclude that RelA has a previously unrecognized role in ribosome function.

Invariant Leu Preceding Turn Motif Phosphorylation Site Controls the Interaction of Protein Kinase C with Hsp70

Heat shock proteins play important roles in regulating signal transduction in cells by associating with, and stabilizing, diverse signaling molecules, including protein kinases. Previously, we have shown that heat shock protein Hsp70 associates with protein kinase C (PKC) via an interaction that is triggered by dephosphorylation at the turn phosphorylation motif. Here we have identified an invariant residue in the carboxyl terminus of PKC that mediates the binding to Hsp70. Specifically, we show that Hsp70 binds to Leu (Leu-640) immediately preceding the conserved turn motif autophosphorylation site (Thr-641) in PKC βII. Co-immunoprecipitation experiments reveal that mutation of Leu-640 to Gly decreases the interaction of Hsp70 with PKC βII. This weakened interaction between Hsp70 and the mutant PKCs results in accumulation of dephosphorylated PKC in the detergent-insoluble fraction of cells. In addition, the Hsp70-binding mutant is considerably more sensitive to down-regulation compared with WT PKC: disruption of Hsp70 binding leads to accelerated dephosphorylation and enhanced ubiquitination of mutant PKC upon phorbol ester treatment. Last, pulse-chase experiments demonstrate that Hsp70 preferentially binds the species of mature PKC that has become dephosphorylated compared with the newly synthesized protein that has yet to be phosphorylated. Thus, Hsp70 binds a hydrophobic residue preceding the turn motif, protecting PKC from down-regulation and sustaining the signaling lifetime of the kinase.

Methionine Regeneration and Aspartate Aminotransferase in Parasitic Protozoa

ABSTRACT Aspartate aminotransferases have been cloned and expressed fromCrithidia fasciculata, Trypanosoma brucei brucei, Giardia intestinalis, andPlasmodium falciparum and have been found to play a role in the final step of methionine regeneration from methylthioadenosine. All five enzymes contain sequence motifs consistent with membership in the Ia subfamily of aminotransferases; the crithidial and giardial enzymes and one trypanosomal enzyme were identified as cytoplasmic aspartate aminotransferases, and the second trypanosomal enzyme was identified as a mitochondrial aspartate aminotransferase. The plasmodial enzyme contained unique sequence substitutions and appears to be highly divergent from the existing members of the Ia subfamily. In addition, the P. falciparum enzyme is the first aminotransferase found to lack the invariant residue G197 (P. K. Mehta, T. I. Hale, and P. Christen, Eur. J. Biochem. 214:549–561, 1993), a feature shared by sequences discovered in P. vivax and P. berghei. All five enzymes were able to catalyze aspartate-ketoglutarate, tyrosine-ketoglutarate, and amino acid-ketomethiobutyrate aminotransfer reactions. In the latter, glutamate, phenylalanine, tyrosine, tryptophan, and histidine were all found to be effective amino donors. The crithidial and trypanosomal cytosolic aminotransferases were also able to catalyze alanine-ketoglutarate and glutamine-ketoglutarate aminotransfer reactions and, in common with the giardial aminotransferase, were able to catalyze the leucine-ketomethiobutyrate aminotransfer reaction. In all cases, the kinetic constants were broadly similar, with the exception of that of the plasmodial enzyme, which catalyzed the transamination of ketomethiobutyrate significantly more slowly than aspartate-ketoglutarate aminotransfer. This result obtained with the recombinant P. falciparum aminotransferase parallels the results seen for total ketomethiobutyrate transamination in malarial homogenates; activity in the latter was much lower than that in homogenates from other organisms. Total ketomethiobutyrate transamination in Trichomonas vaginalis and G. intestinalis homogenates was extensive and involved lysine-ketomethiobutyrate enzyme activity in addition to the aspartate aminotransferase activity. The methionine production in these two species could be inhibited by the amino-oxy compounds canaline and carboxymethoxylamine. Canaline was also found to be an uncompetitive inhibitor of the plasmodial aspartate aminotransferase, with aKi of 27 μM.

Stabilization of the oxy form of tyrosinase by a single conservative amino acid substitution

Asp-208 of Streptomyces glaucescens tyrosinase (an invariant residue in the CuB-binding region of tyrosinases and haemocyanins) was conservatively substituted by glutamic acid. Although having little effect on spectroscopic or kinetic properties of the enzyme, the mutation greatly decreased the lability of Cu-bound O2. A rationalization for these results is given, based on the crystal structure of Panuliris interruptus haemocyanin in the conserved CuB-binding region.

β-lactamase I from Bacillus cereus. Structure and site-directed mutagenesis

The sequence of the gene for beta-lactamase I from Bacillus cereus 569/H has been redetermined. Oligonucleotide-directed mutagenesis has been carried out, and the effects of the changes on the ampicillin-resistance of Escherichia coli TG1 expressing the mutant genes have been studied. Lysine-73, close to the active-site serine-70 and a highly-conserved residue, has been converted into arginine. This change had a large effect on activity, but did not abolish it. An even larger effect was found in the mutant in which glutamate-166 had been converted into glutamine; this had little or no activity. On the other hand, the conversion of glutamate-168 into aspartate gave fully active enzyme. Glutamate-166 is an invariant residue, but glutamate-168 is not. Alanine-123 has been replaced by cysteine, to give active enzyme; this change forms part of the plan to introduce a disulphide bond into the enzyme.