Dennis H. Flint

Assembly of Iron-Sulfur Clusters IDENTIFICATION OF AN iscSUA-hscBA-fdx GENE CLUSTER FROM AZOTOBACTER VINELANDII

An enzyme having the samel-cysteine desulfurization activity previously described for the NifS protein was purified from a strain ofAzotobacter vinelandii deleted for the nifSgene. This protein was designated IscS to indicate its proposed role in iron-sulfur cluster assembly. Like NifS, IscS is a pyridoxal-phosphate containing homodimer. Information gained from microsequencing of oligopeptides obtained by tryptic digestion of purified IscS was used to design a strategy for isolation and DNA sequence analysis of a 7,886-base pair A. vinelandii genomic segment that includes the iscS gene. The iscS gene is contained within a gene cluster that includes homologs to nifU and another gene contained within the major nif cluster ofA. vinelandii previously designated orf6. These genes have been designated iscU and iscA,respectively. Information available from complete genome sequences ofEscherichia coli and Hemophilus influenzaereveals that they also encode iscSUA gene clusters. A wide conservation of iscSUA genes in nature and evidence that NifU and NifS participate in the mobilization of iron and sulfur for nitrogenase-specific iron-sulfur cluster formation suggest that the products of the iscSUA genes could play a general role in the formation or repair of iron-sulfur clusters. The proposal that IscS is involved in mobilization of sulfur for iron-sulfur cluster formation in A. vinelandii is supported by the presence of acysE-like homolog in another gene cluster located immediately upstream from the one containing the iscSUAgenes. O-Acetylserine synthase is the product of thecysE gene, and it catalyzes the rate-limiting step in cysteine biosynthesis. A similar cysE-like gene is also located within the nif gene cluster of A. vinelandii. The likely role of such cysE-like gene products is to increase the cysteine pool needed for iron-sulfur cluster formation. Another feature of the iscSUA gene cluster region from A. vinelandii is that E. coli genes previously designated as hscB,hscA, and fdx are located immediately downstream from, and are probably co-transcribed with, theiscSUA genes. The hscB, hscA, andfdx genes are also located adjacent to theiscSUA genes in both E. coli and H. influenzae. The E. coli hscA and hscBgene products have previously been shown to bear primary sequence identity when respectively compared with the dnaK anddnaJ gene products and have been proposed to be members of a heat-shock-cognate molecular chaperone system of unknown function. The close proximity and apparent co-expression of iscSUAand hscBA in A. vinelandii indicate that the proposed chaperone function of the hscBA gene products could be related to the maturation of iron-sulfur cluster-containing proteins. Attempts to place non-polar insertion mutations within eitherA. vinelandii iscS or hscA revealed that such mutations could not be stably maintained in the absence of the corresponding wild-type allele. These results reveal a very strong selective pressure against the maintenance of A. vinelandii iscS or hscA knock-out mutations and suggest that such mutations are either lethal or highly deleterious. In contrast toiscS or hscA, a strain having a polar insertion mutation within the cysE-like gene was readily isolated and could be stably maintained. These results show that thecysE-like gene located upstream from iscS is not essential for cell growth and that the cysE-like gene and the iscSUA-hscBA-fdx genes are contained within separate transcription units.

Studies on the Synthesis of the Fe-S Cluster of Dihydroxy-acid Dehydratase in Escherichia coli Crude Extract ISOLATION OF O-ACETYLSERINE SULFHYDRYLASES A AND B AND β-CYSTATHIONASE BASED ON THEIR ABILITY TO MOBILIZE SULFUR FROM CYSTEINE AND TO PARTICIPATE IN Fe-S CLUSTER SYNTHESIS

The apoprotein of Escherichia coli dihydroxy-acid dehydratase, which contains a catalytically essential [4Fe-4S] cluster in its active form, has been used as a substrate to investigate Fe-S cluster synthesis. The inactive apoprotein could be reactivated in vitro by factors present in the crude extract of E. coli and to a much smaller extent in the presence of Fe3+, S2−, and dithiothreitol. This reactivation occurs as a result of Fe-S cluster synthesis. It is anticipated that the Fe-S cluster synthesis observed in crude extracts in vitro may involve some of the components that participate in Fe-S cluster synthesis in vivo. The origin of the sulfur used to form Fe-S clusters was investigated. Four enzymatic activities in the crude extract of E. coli were found that can provide sulfur for Fe-S cluster synthesis in vitro by mobilizing the sulfur from cysteine. The purification of the proteins responsible for three of these activities is reported in this paper. The three proteins have been identified as O-acetylserine sulfhydrylase A, O-acetylserine sulfhydrylase B, and β-cystathionase. The rate and extent of sulfide mobilization from cysteine in the reaction catalyzed by O-acetylserine sulfhydrylases A and B depend on the presence of nucleophiles that can add to the aminoacrylate formed on the enzyme following the removal of sulfide from cysteine. A new amino acid is formed when the nucleophiles add to the aminoacrylate. Sulfur mobilization by β-cystathionase does not require a nucleophile, and the reaction is a minor variation on the cleavage of β-cystathionine, with pyruvate, ammonia, and sulfide being the products. Once sulfur is mobilized by these enzymes, its efficient use in Fe-S cluster synthesis seems to be affected by the presence of yet unidentified factors present in crude extract. In crude extract and partially purified preparations from E. coli where these factors are present, the rapidity with which Fe-S clusters are formed and the efficiency with which sulfur is used imply an orderly controlled formation of Fe-S clusters that is generally typified by enzymatic reactions.

Purification and characterization of biotin synthases.

Publisher Summary This chapter describes the purification and characterization of biotin synthases. The final step in the biotin biosynthetic pathway consists of the addition of a sulfur atom between the methyl and methylene carbon atoms adjacent to the imidazolinone ring of dethiobiotin to form the vitamin biotin. The Escherichia coli ( E. Coli ) bioB gene encodes a protein that catalyzes this reaction. This gene has been cloned and sequenced. Highly homologous genes have been identified, cloned, and sequenced from Bacillus sphaericus , Saccharomyces cerevisiae , and Arabidopsis thaliana . The Bacillus and yeast genes are known to encode proteins that catalyze the biotin synthase reaction. Elemental analysis, ultraviolate (UV)–visible spectroscopy, and electron paramagnetic resonance (EPR) studies reveal that the 82-kDa form of E. coli biotin synthase is a homodimer containing one [2Fe–2S] cluster per monomer. A defined mixture of components that supports biotin synthase activity is described. The defined in vitro reaction mixture that has been developed is adapted from a system that utilizes cell-flee extract of E. coli . Although biotin synthase is not oxygen labile, the activity assay is usually performed anaerobically to avoid oxidative chemical reactions that can occur in the presence of some of the reaction components.

Activation of Iron and Sulfur for Nitrogenase Metallocluster Formation

Work in our laboratories has involved the use of genetic, biochemical, and biophysical approaches to analyze the assembly and catalytic mechanism of nitrogenase. Azotobacter vinelandii has been used for these studies because it produces copious amounts of the catalytic components of nitrogenase - the Fe protein and the MoFe protein - and because it is amenable to sophisticated genetic manipulation. Groundwork in our laboratories, and in the laboratory of Paul Bishop, involved the isolation and nucleotide sequence analysis of all, or most, of the A. vinelandii genes directly involved in nitrogenase catalysis. Work in Bishop’s laboratory ultimately led to the remarkable discovery and characterization of two “alternative” nitrogenases, a Vanadium-dependent and Iron-only nitrogenase. We, on the other hand, have concentrated on the characterization of the “traditional” Molybdenum-dependent enzyme. It is worth noting that some - but not all - of the gene products required for maturation of the Mo-dependent enzyme are also required for maturation of the alternative nitrogenases (Kennedy, Dean, 1992). How the expression of these various genes is controlled to permit the accumulation of the appropriate form of nitrogenase -under the appropriate conditions - is a fascinating question currently under study in several laboratories.