PubMed Health⌕ Search

Biomedical subjects

Gary Sawers

Publications and source records attributed to Gary Sawers.

At least 19 recordsLinked to original sources
PubMed2006

The Rhizobium leguminosarum regulator IrrA affects the transcription of a wide range of genes in response to Fe availability.

We show that an unusual transcriptional regulator, called IrrA, regulates many genes in the symbiotic N2-fixing bacterium Rhizobium leguminosarum in response to iron availability. Several operons in R. leguminosarum are expressed at lower levels in cells grown in Fe-depleted compared to Fe-replete medium. These include hemA1, which encodes the haem biosynthesis enzyme amino-levulinic acid synthase; sufS2BCDS1XA, which specify enzymes for FeS cluster synthesis; rirA, a global, Fe-responsive transcriptional repressor; RL0400, which likely encodes an unusual FeS cluster scaffold; and the possible ferri-siderophore ABC transporter rrp1. Reduced expression in Fe-depleted medium was effected by IrrA, a member of the Fur super-family, which in Bradyrhizobium, the symbiont of soybeans, and in the mammalian pathogen Brucella, is unstable in Fe-replete conditions, due to an interaction with haem. The R. leguminosarum IrrA likely interacts with ICE (iron-control element) motifs, conserved sequences near the promoters of its target genes. The rirA, sufS2BCDS1XA and rrp1 genes are also known to be regulated by RirA, which represses their expression in Fe-replete medium. We present a possible model for iron-responsive gene regulation in Rhizobium, in which the IrrA and RirA regulators, working in parallel, respond to the intracellular availability of haem and, possibly, of FeS clusters respectively. Thus, these regulators may sense the physiological consequences of extraneous Fe concentrations, rather than the concentration of Fe per se, as happens in those bacteria (e.g. Escherichia coli) in which the ferric uptake regulator Fur is the global Fe-responsive gene regulator.

Bacterial Proteins↗
PubMed2005

Atomic resolution structures of resting-state, substrate- and product-complexed Cu-nitrite reductase provide insight into catalytic mechanism.

Copper-containing nitrite reductases catalyze the reduction of nitrite to nitric oxide (NO), a key step in denitrification that results in the loss of terrestrial nitrogen to the atmosphere. They are found in a wide variety of denitrifying bacteria and fungi of different physiology from a range of soil and aquatic ecosystems. Structural analysis of potential intermediates in the catalytic cycle is an important goal in understanding enzyme mechanism. Using "crystal harvesting" and substrate-soaking techniques, we have determined atomic resolution structures of four forms of the green Cu-nitrite reductase, from the soil bacterium Achromobacter cycloclastes. These structures are the resting state of the enzyme at 0.9 A, two species exhibiting different conformations of nitrite bound at the catalytic type 2 Cu, one of which is stable and also has NO present, at 1.10 A and 1.15 A, and a stable form with the product NO bound side-on to the catalytic type 2 Cu, at 1.12 A resolution. These structures provide incisive insights into the initial binding of substrate, its repositioning before catalysis, bond breakage (O-NO), and the formation of a stable NO adduct.

Achromobacter cycloclastes↗
PubMed2005

High resolution structural studies of mutants provide insights into catalysis and electron transfer processes in copper nitrite reductase.

We present high-resolution crystal structures and functional analysis of T1Cu centre mutants of nitrite reductase that perturb the redox potential and the Cys130-His129 "hard-wired" bridge through which electron transfer to the catalytic T2Cu centre occurs. These data provide insight into how activity can be altered through mutational manipulation of the electron delivery centre (T1Cu). The alteration of Cys to Ala results in loss of T1Cu and enzyme inactivation with azurin as electron donor despite the mutant enzyme retaining full nitrite-binding capacity. These data establish unequivocally that no direct transfer of electrons occurs from azurin to the catalytic type 2 Cu centre. The mutation of the axial ligand Met144 to Leu increases both the redox potential and catalytic activity, establishing that the rate-determining step of catalysis is the intermolecular electron transfer from azurin to nitrite reductase.

Achromobacter denitrificans↗
PubMed2005

Proteomic analysis reveals the wide-ranging effects of the novel, iron-responsive regulator RirA in Rhizobium leguminosarum bv. viciae.

The wide-ranging effects of RirA, a novel Fe-responsive regulator of gene expression in Rhizobium leguminosarum bv. viciae, were monitored on 2D gels. Approximately 100 proteins were expressed at higher levels in a RirA(-) mutant, compared to wild type. These included the products of the sufS(2)BCDS(1)XA operon, which probably specifies the synthesis of [FeS] clusters. Using lac fusions, this operon was confirmed to be regulated by RirA in response to Fe availability. Genes for some ABC transporters, and a protein that may be involved in making a phenazine-like molecule, were also repressed by Fe in a RirA-dependent way. Strikingly, at least 17 proteins were reduced in abundance in the RirA(-) mutant. These included three ABC transporters, a GatB-like enzyme involved in tRNA modification, and a protein that may confer bacteriocin resistance. As judged by lac reporter fusions, this apparently positive control by RirA was probably due to post-transcriptional effects, in at least some cases. Therefore, although RirA shows no sequence similarity to Fur or DtxR, it functions as a wide-ranging, Fe-responsive regulator.

Bacterial Proteins↗
PubMed2005

Synthesis of the H-cluster framework of iron-only hydrogenase.

The metal-sulphur active sites of hydrogenases catalyse hydrogen evolution or uptake at rapid rates. Understanding the structure and function of these active sites--through mechanistic studies of hydrogenases, synthetic assemblies and in silico models--will help guide the design of new materials for hydrogen production or uptake. Here we report the assembly of the iron-sulphur framework of the active site of iron-only hydrogenase (the H-cluster), and show that it functions as an electrocatalyst for proton reduction. Through linking of a di-iron subsite to a {4Fe4S} cluster, we achieve the first synthesis of a metallosulphur cluster core involved in small-molecule catalysis. In addition to advancing our understanding of the natural biological system, the availability of an active, free-standing analogue of the H-cluster may enable us to develop useful electrocatalytic materials for application in, for example, reversible hydrogen fuel cells. (Platinum is currently the preferred electrocatalyst for such applications, but is expensive, limited in availability and, in the long term, unsustainable.).

Binding Sites↗
PubMed2004

Insights into redox partner interactions and substrate binding in nitrite reductase from Alcaligenes xylosoxidans: crystal structures of the Trp138His and His313Gln mutants.

Dissimilatory nitrite reductase catalyses the reduction of nitrite to nitric oxide within the key biological process of denitrification. We present biochemical and structural results on two key mutants, one postulated to be important for the interaction with the partner protein and the other for substrate entry. Trp138, adjacent to one of the type-1 Cu ligands, is one of the residues surrounding a small depression speculated to be important in complex formation with the physiological redox partners, azurin I and II. Our data reveal that the Trp138His mutant is fully active using methyl viologen as an artificial electron donor, but there is a large decrease in activity using azurin I. These observations together with its crystal structure at a high resolution of 1.6 A confirm the importance of Trp138 in electron transfer and thus in productive interaction with azurin. A "hydrophobic pocket" on the protein surface has been identified as the channel through which nitrite may be guided to the catalytic type-2 Cu site. Glu133 and His313 at the opening of the pocket are conserved among most blue and green copper nitrite reductases (CuNiRs). The failure to soak the substrate into our high-resolution crystal form of native and mutant CuNiRs has been linked to the observation of an extraneous poly(ethylene glycol) (PEG) molecule interacting with His313. We present the crystal structure of His313Gln and the substrate-bound mutant at high resolutions of 1.65 and 1.72 A, respectively. The observation of the substrate-bound structure for the His313Gln mutant and inhibitory studies with PEG establishes the role of the hydrophobic pocket as the port of substrate entry.

Alcaligenes↗
PubMed2004

Met144Ala mutation of the copper-containing nitrite reductase from Alcaligenes xylosoxidans reverses the intramolecular electron transfer.

Pulse radiolysis has been employed to investigate the intramolecular electron transfer (ET) between the type 1 (T1) and type 2 (T2) copper sites in the Met144Ala Alcaligenes xylosoxidans nitrite reductase (AxCuNiR) mutant. This mutation increases the reduction potential of the T1 copper center. Kinetic results suggest that the change in driving force has a dramatic influence on the reactivity: The T2Cu(II) is initially reduced followed by ET to T1Cu(II). The activation parameters have been determined and are compared with those of the wild-type (WT) AxCuNiR. The reorganization energy of the T2 site in the latter enzyme was calculated to be 1.6+/-0.2 eV which is two-fold larger than that of the T1 copper center in the WT protein.

Alcaligenes↗
PubMed2004

mRNA secondary structure modulates translation of Tat-dependent formate dehydrogenase N.

Formate dehydrogenase N (FDH-N) of Escherichia coli is a membrane-bound enzyme comprising FdnG, FdnH, and FdnI subunits organized in an (alphabetagamma)3 configuration. The FdnG subunit carries a Tat-dependent signal peptide, which localizes the protein complex to the periplasmic side of the membrane. We noted that substitution of the first arginine (R5) in the twin arginine signal sequence of FdnG for a variety of other amino acids resulted in a dramatic (up to 60-fold) increase in the levels of protein synthesized. Bioinformatic analysis suggested that the mRNA specifying the first 17 codons of fdnG forms a stable stem-loop structure. A detailed mutational analysis has demonstrated the importance of this mRNA stem-loop in modulating FDH-N translation.

Artificial Gene Fusion↗
PubMed2003

The ECF sigma factor RpoI of R. leguminosarum initiates transcription of the vbsGSO and vbsADL siderophore biosynthetic genes in vitro.

When complexed with Escherichia coli RNA polymerase core enzyme, purified RpoI protein of Rhizobium leguminosarum initiated transcription in vitro from promoters of the vbsADL and vbsGSO operons, which are needed to synthesise the siderophore vicibactin. There is a single transcription initiation site for rpoI, regardless of whether the cells are grown in Fe-replete or Fe-depleted media, but levels of rpoI mRNA were reduced, though not abolished, in the presence of Fe. Unlike PvdS, a similar Pseudomonas sigma factor needed to transcribe genes involved in pyoverdine synthesis, RpoI transcribes vbsADL and vbsGSO in the absence of the cognate siderophore. The RpoI sigma factor is not required for transcription of rpoI.

Bacterial Proteins↗
PubMed2003

Crystallization and preliminary X-ray analysis of the E. coli hypothetical protein TdcF.

Crystals of the hypothetical protein TdcF (subunit MW = 14 007) from Escherichia coli were grown by vapour diffusion. The protein crystallizes in space group P2(1)2(1)2, with unit-cell parameters a = 72.67, b = 86.22, c = 62.62 A. Native data to a resolution of 2.35 A were collected from a single crystal at 100 K on a rotating-anode X-ray generator. Preliminary analysis of these data indicated that the asymmetric unit corresponded to a trimer, which was supported by a convincing molecular-replacement solution using the YjgF trimer as the probe structure.

Amino Acid Sequence↗
PubMed2003

Atomic resolution structures of native copper nitrite reductase from Alcaligenes xylosoxidans and the active site mutant Asp92Glu.

We provide the first atomic resolution (<1.20 A) structure of a copper protein, nitrite reductase, and of a mutant of the catalytically important Asp92 residue (D92E). The atomic resolution where carbon-carbon bonds of the peptide become clearly resolved, remains a key goal of structural analysis. Despite much effort and technological progress, still very few structures are known at such resolution. For example, in the Protein Data Bank (PDB) there are some 200 structures of copper proteins but the highest resolution structure is that of amicyanin, a small (12 kDa) protein, which has been resolved to 1.30 A. Here, we present the structures of wild-type copper nitrite reductase (wtNiR) from Alcaligenes xylosoxidans (36.5 kDa monomer), the "half-apo" recombinant native protein and the D92E mutant at 1.04, 1.15 and 1.12A resolutions, respectively. These structures provide the basis from which to build a detailed mechanism of this important enzyme.

Alcaligenes↗
PubMed2003

Characterization of transcriptional regulation of Shewanella frigidimarina Fe(III)-induced flavocytochrome c reveals a novel iron-responsive gene regulation system.

The bacterium Shewanella frigidimarina can grow anaerobically by utilizing Fe(III) as a respiratory electron acceptor. This results in the synthesis of a number of periplasmic c-type cytochromes, which are absent when the organism is grown in the absence of added Fe(III). One cytochrome, IfcA, is synthesized when Fe(III) is present as the sole respiratory electron acceptor or when it is present in combination with oxygen, fumarate, or nitrate. The ifcA gene was thus selected for a study of iron-responsive gene regulation of respiratory proteins in S. frigidimarina. The monocistronic ifcA gene clusters with two other monocistronic genes, ifcO, encoding a putative outer membrane porin, and ifcR, encoding a putative transcriptional regulator of the LysR superfamily. Analysis of transcription of all three genes under a range of growth conditions in the wild type and an ifcR insertion mutant and analysis of a strain that constitutively expresses ifcR revealed that iron regulation is exerted at the level of ifcR transcription. In the presence of Fe(III) IfcR is synthesized and acts positively to regulate expression of ifcO and ifcA. Control of Fe(III) respiration by this novel regulatory system differs markedly from Fur-mediated regulation of iron assimilation, in which Fur serves as an Fe(II)-activated repressor.

Bacterial Proteins↗
PubMed2002

Alcaligenes xylosoxidans dissimilatory nitrite reductase: alanine substitution of the surface-exposed histidine 139l ligand of the type 1 copper center prevents electron transfer to the catalytic center.

Nitrite reductase of Alcaligenes xylosoxidans contains three blue type 1 copper centers with a function in electron transfer and three catalytic type 2 copper centers. The mutation H139A, in which the solvent-exposed histidine ligand of the type 1 copper ion was changed to alanine, resulted in the formation of a colorless protein containing 4.4 Cu atoms per trimer. The enzyme was inactive with reduced azurin as the electron donor, and in contrast to the wild-type enzyme, no EPR features assignable to type 1 copper centers were observed. Instead, the EPR spectrum of the H139A enzyme, with parameters of g(1) = 2.347 and A(1) = 10 mT, was typical of type 2 copper centers. On the addition of nitrite, the EPR features developed spectral features with increased rhombicity, with g(1) = 2.29 and A(1) = 11 mT, arising from the type 2 catalytic site. As assessed by visible spectroscopy, ferricyanide (E degree = +430 mV) was unable to oxidize the H139A enzyme, and this required a 30-fold excess of K(2)IrCl(6) (E degree = +867 mV). Oxidation resulted in the EPR spectrum developing additional axial features with g(1) = 2.20 and A(1) = 9.5 mT, typical of type 1 copper centers. The oxidized enzyme after separation from the excess of K(2)IrCl(6) by gel filtration was a blue-green color with absorbance maxima at 618 and 420 nm. The instability of the protein prevented the precise determination of the midpoint potential, but these properties indicate that it is in the range 700-800 mV, an increase of at least approximately 470 mV compared with the native enzyme. This high potential, which is consistent with a trigonal planar geometry of the Cu ion, effectively prevents azurin-mediated electron transfer from the type 1 center to the catalytic type 2 Cu site. However, with dithionite as reductant, 20% of the activity of the wild-type enzyme was observed, indicating that the direct reduction of the catalytic site by dithionite can occur. When CuSO(4) was added to the crude extract before isolation of the enzyme, the Cu content of the purified H139A enzyme increased to 5.7 Cu atoms per trimer. The enzyme remained colorless, and the activity with dithionite as a donor was not significantly increased. The additional copper in such preparations was associated with an axial type 2 Cu EPR signal with g(1) = 2.226 and A(1) = 18 mT, and which were not changed by the addition of nitrite, consistent with the activity data.

Alanine↗
PubMed2002

Biochemical and crystallographic studies of the Met144Ala, Asp92Asn and His254Phe mutants of the nitrite reductase from Alcaligenes xylosoxidans provide insight into the enzyme mechanism.

Dissimilatory nitrite reductase catalyses the reduction of nitrite (NO(2)(-)) to nitric oxide (NO). Copper-containing nitrite reductases contain both type 1 and type 2 Cu sites. Electron transfer from redox partners is presumed to be mediated via the type 1 Cu site and used at the catalytic type 2 Cu centre along with the substrate nitrite. At the type 2 Cu site, Asp92 has been identified as a key residue in substrate utilisation, since it hydrogen bonds to the water molecule at the nitrite binding site. We have also suggested that protons enter the catalytic site via Asp92, through a water network that is mediated by His254. The role of these residues has been investigated in the blue copper nitrite reductase from Alcaligenes xylosoxidans (NCIMB 11015) by a combination of point mutation, enzymatic activity measurement and structure determination.In addition, it has been suggested that the enzyme operates via an ordered mechanism where an electron is transferred to the type 2 Cu site largely when the second substrate nitrite is bound and that this is controlled via the lowering of the redox potential of the type 2 site when it is loaded with nitrite. Thus, a small perturbation of the type 1 Cu site should result in a significant effect on the activity of the enzyme. For this reason a mutation of Met144, which is the weakest ligand of the type 1 Cu, is investigated. The structures of H254F, D92N and M144A have been determined to 1.85 A, 1.9 A and 2.2 A resolution, respectively. The D92N and H254F mutants have negligible or no activity, while the M144A mutant has 30 % activity of the native enzyme. Structural and spectroscopic data show that the loss of activity in H254F is due to the catalytic site being occupied by Zn while the loss/reduction of activity in D92N/M144A are due to structural reasons. The D92N mutation results in the loss of the Asp92 hydrogen bond to the Cu-ligated water. Therefore, the ligand is no longer able to perform proton abstraction. Even though the loss of activity in H254F is due to lack of catalytic Cu, the mutation does cause the disruption of the water network, confirming its key role in proton channel. The structure of the H254F mutant is the first case where full occupancy Zn at the type 2 Cu site is observed, but despite the previously noted similarity of this site to the carbonic anhydrase catalytic site, no carbonic anhydrase activity is observed. The H254F and D92N mutant structures provide, for the first time, observation of surface Zn sites which may act as a Zn sink and prevent binding of Zn at the catalytic Cu site in the native enzyme.

Alcaligenes↗
PubMed2002

RirA, an iron-responsive regulator in the symbiotic bacterium Rhizobium leguminosarum.

Mutations in a Rhizobium leguminosarum gene, rirA (rhizobial iron regulator), caused high-level, constitutive expression of at least eight operons whose transcription is normally Fe-responsive and whose products are involved in the synthesis or uptake of siderophores, or in the uptake of haem or of other iron sources. Close homologues of RirA exist in other rhizobia and in the pathogen Brucella; many other bacteria have deduced proteins with more limited sequence similarity. None of these homologues had been implicated in Fe-mediated gene regulation. Transcription of rirA itself is about twofold higher in cells grown in Fe-replete than in Fe-deficient growth media. Mutations in rirA reduced growth rates in Fe-replete and -depleted medium, but did not appear to affect symbiotic N(2) fixation.

Amino Acid Sequence↗
PubMed2002

Expression of the Escherichia coli yfiD gene responds to intracellular pH and reduces the accumulation of acidic metabolic end products.

The YfiD protein of Escherichia coli has been reported to be an acid-inducible protein. Here it is shown that expression of a yfiD::lac reporter fusion is enhanced up to 3 small middle dot5-fold during acidic growth. The anaerobic transcription factor FNR was confirmed as the major regulator of yfiD expression, and ArcA was found to enhance anaerobic yfiD expression, probably by displacing a repressing FNR dimer in the -93 small middle dot5 region of the promoter. Moreover, the pyruvate sensor PdhR was shown to act as a minor anaerobic repressor of yfiD expression. On the basis of its strong homology to the C-terminal region of pyruvate formate-lyase (PFL) it was predicted that YfiD would be a radical-containing enzyme. The YfiD radical was found to be introduced by the PFL-activase enzyme, but unlike PFL, AdhE did not deactivate radicalized YfiD. The extent of radical activation of YfiD was enhanced by low intracellular pH, and thus it was concluded that both yfiD expression and YfiD activity are affected by growth at low pH. The yfiD mutant strain JRG4033 excreted increased levels of organic acids compared to the parental strain when grown in chemostat culture under oxygen-starved conditions, consistent with the acid-inducibility of yfiD expression and the recently reported ability of YfiD to rescue the activity of oxygenolytically cleaved PFL.

Acetyltransferases↗