Biomedical subjects

Joseph T Jarrett

Publications and source records attributed to Joseph T Jarrett.

9 recordsLinked to original sources

PubMed2006

Role of the [2Fe-2S]2+ cluster in biotin synthase: mutagenesis of the atypical metal ligand arginine 260.

Biotin synthase (BS) is an S-adenosylmethionine (AdoMet)-dependent radical enzyme that catalyzes the addition of sulfur to dethiobiotin. Like other AdoMet radical enzymes, BS contains a [4Fe-4S] cluster that is coordinated by a highly conserved CxxxCxxC sequence motif and by the methionyl amine and carboxylate of AdoMet. The close association of the [4Fe-4S]+ cluster with AdoMet facilitates reductive cleavage of the sulfonium and the generation of transient 5'-deoxyadenosyl radicals, which are then proposed to sequentially abstract hydrogen atoms from the substrate to produce carbon radicals at C9 and C6 of dethiobiotin. BS also contains a [2Fe-2S]2+ cluster located approximately 4-5 A from dethiobiotin, and we have proposed that a bridging sulfide of this cluster quenches the substrate radicals, leading to formation of the thiophane ring of biotin. In BS from Escherichia coli, the [2Fe-2S]2+ cluster is coordinated by cysteines 97, 128, and 188, and the atypical metal ligand, arginine 260. The evolutionary conservation of an arginine guanidinium as a metal ligand suggests a novel role for this residue in tuning the reactivity or stability of the [2Fe-2S]2+ cluster. In this work, we explore the effects of mutagenesis of Arg260 to Ala, Cys, His, and Met. Although perturbations in a number of characteristics of the [2Fe-2S]2+ cluster and the proteins are noted, the reconstituted enzymes have in vitro single-turnover activities that are 30-120% of that of the wild type. Further, in vivo expression of each mutant enzyme was sufficient to sustain growth of a bioB- mutant strain on dethiobiotin-supplemented medium, suggesting the enzymes were active and efficiently reconstituted by the in vivo iron-sulfur cluster (ISC) assembly system. Although we cannot exclude an as-yet-unidentified in vivo role in cluster repair or retention, we can conclude that Arg260 is not essential for the catalytic reaction of BS.

Amino Acid Sequence↗

PubMed2005

The novel structure and chemistry of iron-sulfur clusters in the adenosylmethionine-dependent radical enzyme biotin synthase.

Biotin synthase is an adenosylmethionine-dependent radical enzyme that catalyzes the substitution of sulfur for hydrogen at the saturated C6 and C9 positions in dethiobiotin. The structure of the biotin synthase monomer is an (alpha/beta)(8) barrel that contains one [4Fe-4S](2+) cluster and one [2Fe-2S](2+) cluster that encapsulate the substrates AdoMet and dethiobiotin. The air-sensitive [4Fe-4S](2+) cluster and the reductant-sensitive [2Fe-2S](2+) cluster have unique coordination environments that include close proximity to AdoMet and DTB, respectively. The relative positioning of these components, as well as several conserved protein residues, suggests at least two potential catalytic mechanisms that incorporate sulfur from either the [2Fe-2S](2+) cluster or a cysteine persulfide into the biotin thiophane ring. This review summarizes an accumulating consensus regarding the physical and spectroscopic properties of each FeS cluster, and discusses possible roles for the [4Fe-4S](2+) cluster in radical generation and the [2Fe-2S](2+) cluster in sulfur incorporation.

Binding Sites↗

PubMed2005

Biotin synthase: enzyme or reactant?

Biotin synthase catalyzes formation of a thiophane ring through a radical mechanism that is difficult to reconstitute in vitro. Choi-Rhee and Cronan measure a turnover of 20-60 equivalents of biotin in vivo, but also find that turnover renders the protein susceptible to proteolytic destruction.

Biotin↗

PubMed2004

Crystal structure of biotin synthase, an S-adenosylmethionine-dependent radical enzyme.

The crystal structure of biotin synthase from Escherichia coli in complex with S-adenosyl-L-methionine and dethiobiotin has been determined to 3.4 angstrom resolution. This structure addresses how "AdoMet radical" or "radical SAM" enzymes use Fe4S4 clusters and S-adenosyl-L-methionine to generate organic radicals. Biotin synthase catalyzes the radical-mediated insertion of sulfur into dethiobiotin to form biotin. The structure places the substrates between the Fe4S4 cluster, essential for radical generation, and the Fe2S2 cluster, postulated to be the source of sulfur, with both clusters in unprecedented coordination environments.

Amino Acid Motifs↗

PubMed2003

Control of adenosylmethionine-dependent radical generation in biotin synthase: a kinetic and thermodynamic analysis of substrate binding to active and inactive forms of BioB.

Biotin synthase (BS) is an AdoMet-dependent radical enzyme that catalyzes the insertion of sulfur into saturated C6 and C9 atoms in the substrate dethiobiotin. To facilitate sulfur insertion, BS catalyzes the reductive cleavage of AdoMet to methionine and 5'-deoxyadenosyl radicals, which then abstract hydrogen atoms from the C6 and C9 positions of dethiobiotin. The enzyme from Escherichia coli is purified as a dimer that contains one [2Fe-2S]2+ cluster per monomer and can be reconstituted in vitro to contain an additional [4Fe-4S]2+ cluster per monomer. Since each monomer contains each type of cluster, the dimeric enzyme could contain one active site per monomer, or could contain a single active site at the dimer interface. To address these possibilities, and to better understand the manner in which biotin synthase controls radical generation and reactivity, we have examined the binding of AdoMet and DTB to reconstituted biotin synthase. We find that both the [2Fe-2S]2+ cluster and the [4Fe-4S]2+ cluster must be present for tight substrate binding. Further, substrate binding is highly cooperative, with the affinity for AdoMet increasing >20-fold in the presence of DTB, while DTB binds only in the presence of AdoMet. The stoichiometry of binding is ca. 2:1:1 AdoMet:DTB:BS dimer, suggesting that biotin synthase has a single functional active site per dimer. AdoMet binding, either in the presence or in the absence of DTB, leads to a decrease in the magnitude of the UV-visible absorption band at approximately 400 nm that we attribute to changes in the coordination environment of the [4Fe-4S]2+ cluster. Using these spectral changes as a probe, we have examined the kinetics of AdoMet and DTB binding, and propose an ordered binding mechanism that is followed by a conformational change in the enzyme-substrate complex. This kinetic analysis suggests that biotin synthase is evolved to bind AdoMet both weakly and slowly in the absence of DTB, while both the rate of binding and the affinity for AdoMet are increased in the presence of DTB. Cooperative binding of AdoMet and DTB may be an important mechanism for limiting the production of 5'-deoxyadenosyl radicals in the absence of the correct substrate.

Anaerobiosis↗

PubMed2003

The generation of 5'-deoxyadenosyl radicals by adenosylmethionine-dependent radical enzymes.

Adenosylmethionine-dependent radical enzymes provide a novel mechanism for generating the highly oxidizing 5'-deoxyadenosyl radical in an anaerobic reducing environment. Recent studies suggest a unique covalent interaction between adenosylmethionine and a catalytic iron-sulfur cluster that may promote inner-sphere electron transfer to the sulfonium, resulting in the reductive cleavage of a C[bond]S and the generation of a 5'-deoxyadenosyl radical. The utilization of this radical as a catalytic and stoichiometric oxidant in many different enzyme reactions reflects the broad diversity of radical enzymes throughout biology.

Deoxyadenosines↗

PubMed2002

Thermal inactivation of reduced ferredoxin (flavodoxin):NADP+ oxidoreductase from Escherichia coli.

Ferredoxin (flavodoxin):NADP+ oxidoreductase (FNR) is an essential enzyme that supplies electrons from NADPH to support flavodoxin-dependent enzyme radical generation and enzyme activation. FNR is a monomeric enzyme that contains a non-covalently bound FAD cofactor. We report that reduced FNR from Escherichia coli is subject to inactivation due to unfolding of the protein and dissociation of the FADH(2) cofactor at 37 degrees C. The inactivation rate is temperature-dependent in a manner that parallels the thermal unfolding of the protein and is slowed by binding of ferredoxin or flavodoxin. Understanding factors that minimize inactivation is critical for utilizing FNR as an accessory protein for S-adenosyl-L-methionine-dependent radical enzymes and manipulating FNR as an electron source for biotechnology applications.

Enzyme Stability↗

PubMed2002

Electron acceptor specificity of ferredoxin (flavodoxin):NADP+ oxidoreductase from Escherichia coli.

Reduced flavodoxin I (Fld1) is required in Escherichia coli for reductive radical generation in AdoMet-dependent radical enzymes and reductive activation of cobalamin-dependent methionine synthase. Ferredoxin (Fd) and flavodoxin II (Fld2) are also present, although their precise roles have not been ascertained. Ferredoxin (flavodoxin):NADP+ oxidoreductase (FNR) was discovered in E. coli as an NADPH-dependent reductant of Fld1 that facilitated generation of active methionine synthase in vitro; FNR and Fld1 will also supply electrons for the reductive cleavage of AdoMet essential for generating protein or substrate radicals in pyruvate formate-lyase, class III ribonucleotide reductase, biotin synthase, and, potentially, lipoyl synthase. As part of ongoing efforts to understand the various redox pathways that will support AdoMet-dependent radical enzymes in E. coli, we have examined the relative specificity of E. coli FNR for Fd, Fld1, and Fld2. While FNR will reduce all three proteins, Fd is the kinetically and thermodynamically preferred partner. Fd binds to FNR with high affinity (K(d)<or=0.5 microM) and is reduced under single-turnover conditions with k(obs)=2.3s(-1) and under steady state conditions with k(cat)=0.15s(-1). Fld1 and Fld2 behave similarly with respect to FNR, with affinities approximately 4- to 7-fold weaker and reduction rates that are 10- to 100-fold slower than those for Fd. Surprisingly we find that Fld1 and Fld2 can obtain electrons from reduced Fd at rates that are comparable to those obtained with reduced FNR. Thus we propose that the primary electron acceptor for E. coli FNR is Fd, while Fld1 can obtain electrons slowly either from FNR or via Fd as a mediator.

Base Sequence↗

PubMed2002

Evidence from Mössbauer spectroscopy for distinct [2Fe-2S](2+) and [4Fe-4S](2+) cluster binding sites in biotin synthase from Escherichia coli.

Biotin synthase is an AdoMet-dependent radical enzyme that catalyzes the insertion of an FeS cluster-derived sulfur atom into dethiobiotin. The dimeric enzyme is purified containing one [2Fe-2S]2+ cluster per monomer, but it is most active when reconstituted with an additional [4Fe-4S]2+ cluster per monomer. Using Mössbauer spectroscopy coupled with differential reconstitution of each cluster with 57Fe, we show that the reconstituted enzyme has approximately 1:1 [2Fe-2S]2+ and [4Fe-4S]2+ clusters and that the [4Fe-4S]2+ cluster is assembled at an alternate site not previously occupied by the [2Fe-2S]2+ cluster. These data suggest that biotin synthase is evolved to simultaneously accommodate two different clusters with unique roles in catalysis.

Bacterial Proteins↗