Cysteine desulfurases are pyridoxal 5'-phosphate-dependent homodimeric enzymes that catalyze the conversion of L-cysteine to L-alanine and sulfane sulfur via the formation of a protein-bound cysteine persulfide intermediate on a conserved cysteine residue. The enzymes are capable of donating the persulfide sulfur atoms to a variety of biosynthetic pathways for sulfur-containing biofactors, such as iron-sulfur clusters, thiamin, transfer RNA thionucleosides, biotin, and lipoic acid. The enormous advances in biochemical and structural studies of these biosynthetic pathways over the past decades provide an opportunity for detailed understanding of the nature of the excellent sulfur transfer mechanism of cysteine desulfurases.

The reductive pyrimidine catabolic pathway is absent in Escherichia coli. However, the bacterium contains an enzyme homologous to mammalian dihydropyrimidine dehydrogenase. Here, we show that E. coli dihydropyrimidine dehydrogenase is the first member of a novel NADH-dependent subclass of iron-sulfur flavoenzymes catalyzing the conversion of uracil to 5,6-dihydrouracil in vivo.The reductive pyrimidine catabolic pathway is found in most eukaryotes. It degrades uracil or thymine into ␤-amino acids through dihydropyrimidine and N-carbamoyl-␤-amino acid intermediates (Fig. 1A). These reactions are catalyzed by dihydropyrimidine dehydrogenase (DPD), dihydropyrimidinase, and ␤-alanine synthase in both mammals (16) and plants (25). ␤-Alanine, a product of the reductive pathway, not only is a substrate for the synthesis of pantothenic acid in plants and several bacteria (20, 25) but also acts as a neurotransmitter in mammals (19). Mammalian DPD, the first and rate-limiting enzyme of the reductive pyrimidine catabolic pathway, catalyzes the NADPH-dependent reduction of uracil and thymine to 5,6-dihydro derivatives (16). The mammalian enzyme is composed of two identical subunits of approximately 111 kDa. Each subunit carries one FMN, one FAD cofactor, and four [4Fe/4S] clusters and contains separate binding sites for the electron-donating cosubstrate NADPH and the electron-accepting pyrimidines (16). Bacterial enzymes for the reductive degradation of pyrimidine were originally discovered and identified 50 years ago in a uracil-fermenting bacterium, Clostridium uracilicum (1-5). In this bacterium, the reductive pyrimidine catabolic pathway is important because it enables uracil and thymine to be used as nitrogen and carbon sources for growth (Fig. 1A). Since then, the presence of DPD activity has been demonstrated in several bacterial strains, including Alcaligenes eutrophus (4, 15), Pseudomonas sp. (9,(22)(23)(24), and Escherichia coli B (21). However, little is known about the molecular characteristics of DPD in bacteria.In the course of our investigation of iron-sulfur proteins that depend on the iscS gene for iron-sulfur cluster maturation in E. coli, we found two putative iron-sulfur proteins, PreT and PreA, which exhibited ca. 30% identity to the N-and C-terminal halves, respectively, of mammalian DPD (11). It was revealed that the preT and preA genes (formerly yeiT and yeiA, respectively) form an operon structure on the genome. Bioinformatic analysis revealed that genes with sequence identities to preT and preA are also present as a putative operon in a number of bacteria, including Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, Bacillus clausii, and Clostridium tetani. Interestingly, the corresponding homologous genes, pydX and pydA, of P. aeruginosa are located in a gene cluster whose function is computationally predicted to be pyrimidine-reductive degradation (13) (Fig. 1B). In contrast, the preT-preA genes do not associate with any other putative genes for the reductive pyrimidine catabolic pat...

f Longer-and/or branched-chain polyamines are unique polycations found in thermophiles. N 4 -aminopropylspermine is considered a major polyamine in Thermococcus kodakarensis. To determine whether a quaternary branched penta-amine, N 4 -bis(aminopropyl)spermidine, an isomer of N 4 -aminopropylspermine, was also present, acid-extracted cytoplasmic polyamines were analyzed by high-pressure liquid chromatography, gas chromatography (HPLC), and gas chromatography-mass spectrometry. N 4 -bis(aminopropyl)spermidine was an abundant cytoplasmic polyamine in this species. To identify the enzyme that catalyzes N 4 -bis(aminopropyl)spermidine synthesis, the active fraction was concentrated from the cytoplasm and analyzed by linear ion trap-time of flight mass spectrometry with an electrospray ionization instrument after analysis by the MASCOT database. TK0545, TK0548, TK0967, and TK1691 were identified as candidate enzymes, and the corresponding genes were individually cloned and expressed in Escherichia coli. Recombinant forms were purified, and their N 4 -bis(aminopropyl)spermidine synthesis activity was measured. Of the four candidates, TK1691 (BpsA) was found to synthesize N 4 -bis(aminopropyl)spermidine from spermidine via N 4 -aminopropylspermidine. Compared to the wild type, the bpsA-disrupted strain DBP1 grew at 85°C with a slightly longer lag phase but was unable to grow at 93°C. HPLC analysis showed that both N 4 -aminopropylspermidine and N 4 -bis(aminopropyl)spermidine were absent from the DBP1 strain grown at 85°C, demonstrating that the branched-chain polyamine synthesized by BpsA is important for cell growth at 93°C. Sequence comparison to orthologs from various microorganisms indicated that BpsA differed from other known aminopropyltransferases that produce spermidine and spermine. BpsA orthologs were found only in thermophiles, both in archaea and bacteria, but were absent from mesophiles. These findings indicate that BpsA is a novel aminopropyltransferase essential for the synthesis of branched-chain polyamines, enabling thermophiles to grow in high-temperature environments. P olyamines are small, positively charged aliphatic molecules containing more than two amine residues present in almost all living organisms. Putrescine [4], spermidine [34], and spermine [343] are polyamines commonly observed in the cells of various living organisms, from viruses to humans (1-4). Polyamines are important in cell proliferation and cell differentiation (5, 6), as well as contributing to adaptation to various stresses (7). Interestingly, in addition to common polyamines, thermophiles contain two types of unusual polyamines as major polyamines. One type consists of long linear polyamines such as caldopentamine [3333] NH, N,. Because the relative amounts of long/branched-chain polyamines in cells of (hyper)thermophiles were found to increase as growth temperatures increased, these unique polyamines are regarded as supporting the growth of thermophilic microorganisms under high-temperature conditions (18)(19)(20). An in...