A RecA / RAD51 homologue from a hyperthermophilic archaeon retains the major RecA domain only

 A gene encoding a RecA/RAD51 homologue from a hyperthermophilic archaeon, Pyrococcus sp. KOD1 (Pk), was cloned, sequenced and expressed in Escherichia coli. The deduced 210-amino acid sequence was compared to homologues from bacteria (RecA), eukaryotes (RAD51, DMC1) and archaea (RadA). The entire protein from Pk (Pk-REC) basically corresponds to the essential central domain of its counterparts and lacks the two smaller RecA subdomains at the N- and C-termini. The sequence comparison suggests that Pk-REC represents a common prototype of RecA, RAD51, DMC1 and RadA, with higher enzymatic activity. Recombinant Pk-REC was fully active and complemented the ultraviolet light sensitivity of an E. coli recA mutant strain. Received: 11 June 1996 / Accepted: August 31 1996     

Parkinson's disease-associated mutant LRRK2 phosphorylates Rab7L1 and modifies trans-Golgi morphology

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the major genetic cause of autosomal-dominantly inherited Parkinson's disease. LRRK2 is implicated in the regulation of intracellular trafficking, neurite outgrowth and PD risk in connection with Rab7L1, a putative interactor of LRRK2. Recently, a subset of Rab GTPases have been reported as substrates of LRRK2. Here we examine the kinase activity of LRRK2 on Rab7L1 in situ in cells. Phos-tag analyses and metabolic labeling assays revealed that LRRK2 readily phosphorylates Golgi-localized wild-type Rab7L1 but not mutant forms that are distributed in the cytoplasm. In vitro assays demonstrated direct phosphorylation of Rab7L1 by LRRK2. Subsequent screening using Rab7L1 mutants harboring alanine-substitution for every single Ser/Thr residue revealed that Ser72 is a major phosphorylation site, which was confirmed by using a phospho-Ser72-specific antibody. Moreover, LRRK2 pathogenic Parkinson mutants altogether markedly enhanced the phosphorylation at Ser72. The modulation of Ser72 phosphorylation in Rab7L1 resulted in an alteration of the morphology and distribution of the trans-Golgi network. These data collectively support the involvement of Rab7L1 phosphorylation in the LRRK2-mediated cellular and pathogenetic mechanisms.     

Cysteine desulphurase plays an important role in environmental adaptation of the hyperthermophilic archaeon Thermococcus kodakarensis

The sulphur atoms of sulphur‐containing cofactors that are essential for numerous cellular functions in living organisms originate from L‐cysteine via cysteine desulphurase (CSD) activity. However, many (hyper)thermophilic archaea, which thrive in solfataric fields and are positioned near the root of the evolutionary tree of life, lack CSD orthologues. The existence of CSD orthologues in a subset of (hyper)thermophilic archaea is of interest with respect to the evolution of sulphur‐trafficking systems for the cofactors. This study demonstrates that the disruption of the csd gene of Thermococcus kodakarensis, a facultative elemental sulphur (S⁰)‐reducing hyperthermophilic archaeon, encoding Tk‐CSD, conferred a growth defect evident only in the absence of S⁰, and that growth can be restored by the addition of S⁰, but not sulphide. We show that the csd gene is not required for biosynthesis of thiamine pyrophosphate or molybdopterin, irrespective of the presence or absence of S⁰, but is necessary for iron‐sulphur cluster biosynthesis in the absence of S⁰. Recombinant form of Tk‐CSD expressed in Escherichia coli was obtained and it was found to catalyse the desulphuration of L‐cysteine. The obtained data suggest that hyperthermophiles might benefit from a capacity for CSD‐dependent iron‐sulphur cluster biogenesis, which allows them to thrive outside solfataric environments.     

The O6-methylguanine-DNA methyltransferase from the hyperthermophilic archaeon Pyrococcus sp. KOD1: a thermostable repair enzyme

The enzyme O⁶-methylguanine-DNA methyltransferase (MGMT) is the most common form of cellular defense against the biological effects of O⁶-methylguanine (O⁶-MeG) in DNA. Based on PCR amplification using primers derived from conserved amino acid sequences of MGMTs from 11 species, we isolated the DNA region coding for MGMT from the hyperthermophilic archaeon Pyrococcus sp. KOD1. The MGMT gene from KOD1 (mgtk) comprises 522 nucleotides, encoding 174 amino acid residues; its product shows considerable similarity to the corresponding mammalian, yeast and bacterial enzymes, especially around putative methyl acceptor sites. Phylogenetic analysis of MGMTs showed that archaeal MGMTs were grouped with their bacterial counterparts. The location of the MGMT gene on the KOD1 chromosome was also determined. The cloned KOD1 MGMT gene was overexpressed using the T7 RNA polymerase expression system, and the recombinant protein was purified by ammonium sulfate fractionation, heat treatment, ion-exchange chromatography and gel filtration chromatography. The purified recombinant protein was assayed for its enzyme activity by monitoring transfer of [³H]methyl groups from the substrate DNA to the MGMT protein; the activity was found to be stable at 90°?C for at least 30?min. When the mgtk gene was placed under the control of the lac promoter and expressed in the methyltransferase-deficient Escherichia coli strain KT233 (Δada, Δogt) cells, a MGMT was produced. The enzyme was functional in vivo and complemented the mutant phenotype, making the cells resistant to the cytotoxic properties of the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine.     

Host—vector systems in thermophilic Bacilli and their applications

New host—vector systems in thermophilic bacteria have been developed. Using these systems, a few structural genes for extra-cellular enzymes from both a mesophile and a thermophile have been cloned and expressed in a thermophile at elevated temperatures.     

The synthesis and biodistribution of [11C]metformin as a PET probe to study hepatobiliary transport mediated by the multi-drug and toxin extrusion transporter 1 (MATE1) in vivo

In order to develop a new positron emission tomography (PET) probe to study hepatobiliary transport mediated by the multi-drug and toxin extrusion transporter 1 (MATE1), ¹¹C-labelled metformin was synthesized and then evaluated as a PET probe. [¹¹C]Metformin ([¹¹C]4) was synthesized in three steps, from [¹¹C]methyl iodide. Evaluation by small animal PET of [¹¹C]4 showed that there was increased concentrations of [¹¹C]4 in the livers of mice pre-treated with pyrimethamine, a potential inhibitor of MATEs, inhibiting the hepatobiliary excretion of metformin. Radiometabolite analysis showed that [¹¹C]4 was not degraded in vivo during the PET scan. Biodistribution studies were undertaken and the organ distributions were extrapolated into a standard human model. In conclusion, [¹¹C]4 may be useful as a PET probe to non-invasively study the in vivo function of hepatobiliary transport and drug–drug interactions, mediated by MATE1 in future clinical investigations.