Andreas Pich

Tungstate Uptake by a Highly Specific ABC Transporter inEubacterium acidaminophilum

The Gram-positive anaerobeEubacterium acidaminophilum contains at least two tungsten-dependent enzymes: viologen-dependent formate dehydrogenase and aldehyde dehydrogenase.185W-Labeled tungstate was taken up by this organism with a maximum rate of 0.53 pmol min− 1mg− 1 of protein at 36 °C. The uptake was not affected by equimolar amounts of molybdate. The genestupABC coding for an ABC transporter specific for tungstate were cloned in the downstream region of genes encoding a tungsten-containing formate dehydrogenase. The substrate-binding protein, TupA, of this putative transporter was overexpressed inEscherichia coli, and its binding properties toward oxyanions were determined by a native polyacrylamide gel retardation assay. Only tungstate induced a shift of TupA mobility, suggesting that only this anion was specifically bound by TupA. If molybdate and sulfate were added in high molar excess (>1000-fold), they were also slightly bound by TupA. The K d value for tungstate was determined to be 0.5 μm. The genes encoding the tungstate-specific ABC transporter exhibited highest similarities to putative transporters from Methanobacterium thermoautotrophicum, Haloferax volcanii, Vibrio cholerae, and Campylobacter jejuni. These five transporters represent a separate phylogenetic group of oxyanion ABC transporters as evident from analysis of the deduced amino acid sequences of the binding proteins. Downstream of the tupABCgenes, the genes moeA, moeA-1,moaA, and a truncated moaC have been identified by sequence comparison of the deduced amino acid sequences. They should participate in the biosynthesis of the pterin cofactor that is present in molybdenum- and tungsten-containing enzymes except nitrogenase.

Identification of D-proline reductase from Clostridium sticklandii as a selenoenzyme and indications for a catalytically active pyruvoyl group derived from a cysteine residue by cleavage of a proprotein.

Highly active d-proline reductase was obtained from Clostridium sticklandiiby a modified purification scheme. The cytoplasmic enzyme had a molecular mass of about 870 kDa and was composed of three subunits with molecular masses of 23, 26, and 45 kDa. The 23-kDa subunit contained a carbonyl group at its N terminus, which could either be labeled with fluorescein thiosemicarbazide or removed byo-phenylenediamine; thus, N-terminal sequencing became feasible for this subunit. l-[14C]proline was covalently bound to the 23-kDa subunit if proline racemase and NaBH4 were added. Selenocysteine was detected in the 26-kDa subunit, which correlated with an observed selenium content of 10.6 g-atoms in d-proline reductase. No other non-proteinaceous cofactor was identified in the enzyme. A 4.8-kilobase pair (kb)EcoRI fragment was isolated and sequenced containing the two genes prdA and prdB. prdA coding for a 68-kDa protein was most likely translated as a proprotein that was posttranslationally cleaved at a threonine-cysteine site to give the 45-kDa subunit and most probably a pyruvoyl-containing 23-kDa subunit. The gene prdB encoded the 26-kDa subunit and contained anin frame UGA codon for selenocysteine insertion.prdA and prdB were transcribed together on a transcript of 4.5 kb; prdB was additionally transcribed as indicated by a 0.8-kb mRNA species.

Proline biosynthesis from L-ornithine in Clostridium sticklandii: purification of delta1-pyrroline-5-carboxylate reductase, and sequence and expression of the encoding gene, proC

Clostridium sticklandii utilizes combinations of amino acids for growth by Stickland reactions. Proline is an efficient electron acceptor in these reactions and is reduced to 5-aminovalerate. Proline can be partly synthesized from ornithine by the action of ornithine aminotransferase and delta1-pyrroline-5-carboxylate (PCA) reductase. Both enzymes were present in crude extracts of C. sticklandii in sufficient activity of 0.93 nkat (mg protein)(-1) and 4.3 nkat (mg protein)(-1), respectively, whereas enzymes involved in proline biosynthesis from glutamate were not detected. PCA reductase was purified to homogeneity in a three-step procedure involving ammonium sulfate precipitation, affinity chromatography with Procion Red and gel filtration on Sephadex GF200. The homogeneous enzyme was most likely an octamer of 230 kDa with a subunit size of 25 kDa as obtained by SDS-PAGE and 28.9 kDa as calculated from the sequence. Apparent Km values for PCA and NADH were 0.19 mM and 0.025 mM, respectively. The enzyme also catalysed in vitro the reverse reaction, the oxidation of proline, at alkaline pH values above 8 and higher substrate concentrations (apparent Km values: 1.55 mM for proline and 10.5 mM for NAD at pH 10.0). Studies with growing cells of C. sticklandii and [15N]proline revealed that proline is not oxidized in vivo because 15N was solely detected by HPLC-MS in 5-aminovalerate as the product of proline reduction. The proC gene encoding PCA reductase of C. sticklandii was cloned, sequenced and heterologously expressed in Escherichia coli. The enzyme exhibited high homologies to PCA reductases from different sources. Thus, C. sticklandii is able to synthesize the electron acceptor proline from ornithine (a degradation product of arginine) by action of ornithine aminotransferase and PCA reductase.

Molecular analysis of the grd operon coding for genes of the glycine reductase and of the thioredoxin system from Clostridium sticklandii.

Abstract. A probe based on the sequence of the gene encoding selenoprotein A of glycine reductase of Clostridium sticklandii was used to obtain clones of adjacent DNA that encoded the other components of glycine reductase, proteins B and C, in addition to thioredoxin and thioredoxin reductase. The genes of the thioredoxin system and the glycine reductase were shown to be transcribed together, confirming an operon structure. In addition, a gene (grdX) encoding a 13.7-kDa protein of unknown function seemed to be associated with the reductase genes. Four potential promoters were identified by mapping the 5′-end of the mRNAs. The sequence of promoter P1 was shown to be similar to the σ70 promoter consensus sequence. The other three promoters were similar to each other, but not to known promoter consensus sequences. The transcripts starting at each of the four promoters were terminated to about 80% at a predicted loop structure downstream of grdB; the remaining transcripts continued through this structure and covered the genes encoding both subunits of protein C and bmpA, a gene that was also expressed monocistronically.

Partial purification of an iron‐dependent L‐serine dehydratase from Clostridium sticklandii

An oxygen‐sensitive and highly unstable L‐serine dehydratase was partially purified from the Grampositive anaerobe Clostridium sticklandii. The final active preparation contained five proteins of 27, 30, 44.5, 46, and 58 kDa as judged by SDS‐PAGE. The N‐terminal sequence of the 30 kDa subunit showed some similarity to the α‐subunits of the iron‐containing L‐serine dehydratases from Clostridium propionicum and Peptostreptococcus asaccharolyticus. Oxygen‐inactivated L‐serine dehydratase from C. sticklandii was reactivated by incubation with Fe2+ under reducing conditions. Furthermore, the enzyme was inactivated by iron‐chelating substances like phenanthroline and EDTA. Pyridoxal‐5‐phosphate (PLP) did not stimulate the activity, and known inhibitors of PLP‐containing enzymes such as NaBH4 had no effect on the activity of L‐serine dehydratase from C. sticklandii.