Kinetic and Mutational Studies of Three NifS Homologs from Escherichia coli: Mechanistic Difference between L-Cysteine Desulfurase and L-Selenocysteine Lyase Reactions

Abstract

We have purified three NifS homologs from Escherichia coli, CSD, CsdB, and IscS, that appear to be involved in iron-sulfur cluster formation and/or the biosynthesis of selenophosphate. All three homologs catalyze the elimination of Se and S from L-selenocysteine and L-cysteine, respectively, to form L-alanine. These pyridoxal 5'-phosphate enzymes were inactivated by abortive transamination, yielding pyruvate and a pyridoxamine 5'-phosphate form of the enzyme. The enzymes showed non-Michaelis-Menten behavior for L-selenocysteine and L-cysteine. When pyruvate was added, they showed Michaelis-Menten behavior for L-selenocysteine but not for L-cysteine. Pyruvate significantly enhanced the activity of CSD toward L-selenocysteine. Surprisingly, the enzyme activity toward L-cysteine was not increased as much by pyruvate, suggesting the presence of different rate-limiting steps or reaction mechanisms for L-cysteine desulfurization and the degradation of L-selenocysteine. We substituted Ala for each of Cys358 in CSD, Cys364 in CsdB, and Cys328 in IscS, residues that correspond to the catalytically essential Cys325 of Azotobacter vinelandii NifS. The enzyme activity toward L-cysteine was almost completely abolished by the mutations, whereas the activity toward L-selenocysteine was much less affected. This indicates that the reaction mechanism of L-cysteine desulfurization is different from that of L-selenocysteine decomposition, and that the conserved cysteine residues play a critical role only in L-cysteine desulfurization.

Mammalian frataxin directly enhances sulfur transfer of NFS1 persulfide to both ISCU and free thiols

“…These data confirm the results of the single-turnover kinetics. The reduction step is also the rate-limiting reaction of the catalytic cycle of other group I cysteine desulfurases, which suggests that this might be a common feature of this class of enzymes 43,44,49 . Taken together, our results thus point to a unique molecular function of FXN as an enhancer of sulfur transfer to ISCU and small free thiols (Fig.…”

“…L-cysteine and DTT were reported to inhibit the cysteine desulfurase Slr0387 from Synechocystis sp. PCC 6803 and NifS from E. coli 43,44 . Therefore, we hypothesized that the saturation behaviours observed by other groups with NFS1 could be due to inhibition caused by L-cysteine itself at ratios above 100 equivalents.…”

The SufE Protein and the SufBCD Complex Enhance SufS Cysteine Desulfurase Activity as Part of a Sulfur Transfer Pathway for Fe-S Cluster Assembly in Escherichia coli

“…Strains and Media-(His) 6 -SufE and SufABCDSE were expressed in TOP10 (Invitrogen), and (His) 6 -SufA and (His) 6 -SufS were expressed in BL21(DE3)plysS (Invitrogen). All of the bacterial growth was in Lennox Broth (10 g of tryptone, 5 g of yeast extract, 5 g of NaCl/liter).…”

“…SufA was PCR-amplified using two primers (5Ј-GTTGCTTCAGAATTCCGAGACATAGTACCGCC-3Ј and 5Ј-GAGG-TAAATCGATGGATCCGCATTCAGGAAC-3Ј), digested with EcoRI and BamHI, and cloned into the corresponding sites of pRSETB to generate pGSO167. Plasmids were designed such that (His) 6 -SufA and (His) 6 SufS were fused with a (His) 6 -tag at their N termini while (His) 6 -SufE was fused with a Myc and (His) 6 -tag at the C terminus. Mutation of SufE Cys 51 to Ser was performed using the pGSO165 construct, two mutagenic primers (5Ј-CAAAATAGCATTCAGGGCAGCCAGAGT-CAGGTGTGG-3Ј and 5Ј-CCACACCTGACTCTGGCTGCCCTGAAT-GCTATTTTG-3Ј), and the QuikChange site-directed Mutagenesis kit (Stratagene) according to the manufacturer's protocol to generate pGSO168.…”

Methanococcus vannielii Selenium Metabolism: Purification and N‐terminal Amino Acid Sequences of a Novel Selenium‐binding Protein and Selenocysteine Lyase

“…The three different E. coli gene products that exhibit cysteine/selenocysteine lyase activities use both amino acids as substrate (17,19) and only one of these enzymes has relatively higher selenocysteine lyase activity. An additional factor that binds to this lyase may be required for specific delivery of selenium in E. coli.…”