PubMed Health⌕ Search

Biomedical subjects

Robert P Hausinger

Publications and source records attributed to Robert P Hausinger.

28 records · Page 2Linked to original sources
PubMed2003

Intrinsic tryptophan fluorescence as a probe of metal and alpha-ketoglutarate binding to TfdA, a mononuclear non-heme iron dioxygenase.

2,4-Dichlorophenoxyacetic acid (2,4-D)/alpha-ketoglutarate (alphaKG) dioxygenase, TfdA, couples the oxidative decarboxylation of alphaKG to the oxidation of the herbicide 2,4-D using a mononuclear non-heme Fe(II) active site. The intrinsic tryptophan fluorescence associated with the four Trp residues in TfdA allows for the use of fluorescence spectroscopy to monitor the binding of iron and alphaKG to the enzyme. The fluorescence spectrum of TfdA is quenched by 50-85% upon addition of Fe(II) or alphaKG, allowing determination of their binding affinities (K(d)=7.45+/-0.61 and 3.35+/-0.35 microM, respectively). Cu, Zn, Mn, Co, Mg, and Ca dictations also quench the TfdA fluorescence with affinities similar to that of Fe(II), whereas monovalent cations such as Na, K, and Li do not. H114A and D116A mutant forms of TfdA, lacking either a histidine or aspartate metallocenter ligand, exhibit weaker affinity for both Fe(II) and alphaKG based on the fluorescence changes. Trp256 is predicted to lie within 5 A of the metal and alphaKG binding sites; however, its substitution by Phe or Leu has negligible effects on the Fe(II)- and alphaKG-dependent fluorescence quenching. Because Trp195 is predicted to be quite distant ( approximately 15 A) from the active site, we conclude that some combination of Trp113 and Trp248 serves as the reporter that senses metal and cofactor binding to TfdA.

Binding Sites↗
PubMed2003

Nickel uptake and utilization by microorganisms.

Nickel is an essential nutrient for selected microorganisms where it participates in a variety of cellular processes. Many microbes are capable of sensing cellular nickel ion concentrations and taking up this nutrient via nickel-specific permeases or ATP-binding cassette-type transport systems. The metal ion is specifically incorporated into nickel-dependent enzymes, often via complex assembly processes requiring accessory proteins and additional non-protein components, in some cases accompanied by nucleotide triphosphate hydrolysis. To date, nine nickel-containing enzymes are known: urease, NiFe-hydrogenase, carbon monoxide dehydrogenase, acetyl-CoA decarbonylase/synthase, methyl coenzyme M reductase, certain superoxide dismutases, some glyoxylases, aci-reductone dioxygenase, and methylenediurease. Seven of these enzymes have been structurally characterized, revealing distinct metallocenter environments in each case.

Acetyl Coenzyme A↗
PubMed2003

Metal ion dependence of recombinant Escherichia coli allantoinase.

Allantoinase is a suspected dinuclear metalloenzyme that catalyzes the hydrolytic cleavage of the five-member ring of allantoin (5-ureidohydantoin) to form allantoic acid. Recombinant Escherichia coli allantoinase purified from overproducing cultures amended with 2.5 mM zinc, 1 mM cobalt, or 1 mM nickel ions was found to possess approximately 1.4 Zn, 0.0 Co, 0.0 Ni, and 0.4 Fe; 0.1 Zn, 1.0 Co, 0.0 Ni, and 0.2 Fe; and 0.0 Zn, 0.0 Co, 0.6 Ni, and 0.1 Fe per subunit, respectively, whereas protein obtained from nonamended cultures contains near stoichiometric levels of iron. We conclude that allantoinase is incompletely activated in the recombinant cells, perhaps due to an insufficiency of a needed accessory protein. Enzyme isolated from nonsupplemented cultures possesses very low activity (k(cat) = 34.7 min(-1)) compared to the zinc-, cobalt-, and nickel-containing forms of allantoinase (k(cat) values of 5,000 and 28,200 min(-1) and 200 min(-1), respectively). These rates and corresponding K(m) values (17.0, 19.5, and 80 mM, respectively) are significantly greater than those that have been reported previously. Absorbance spectroscopy of the cobalt species reveals a band centered at 570 nm consistent with five-coordinate geometry. Dithiothreitol is a competitive inhibitor of the enzyme, with significant K(i) differences for the zinc and cobalt species (237 and 795 micro M, respectively). Circular dichroism spectroscopy revealed that the zinc enzyme utilizes only the S isomer of allantoin, whereas the cobalt allantoinase prefers the S isomer, but also hydrolyzes the R isomer at about 1/10 the rate. This is the first report for metal content of allantoinase from any source.

Allantoin↗
PubMed2002

Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage.

Methylating agents generate cytotoxic and mutagenic DNA damage. Cells use 3-methyladenine-DNA glycosylases to excise some methylated bases from DNA, and suicidal O(6)-methylguanine-DNA methyltransferases to transfer alkyl groups from other lesions onto a cysteine residue. Here we report that the highly conserved AlkB protein repairs DNA alkylation damage by means of an unprecedented mechanism. AlkB has no detectable nuclease, DNA glycosylase or methyltransferase activity; however, Escherichia coli alkB mutants are defective in processing methylation damage generated in single-stranded DNA. Theoretical protein fold recognition had suggested that AlkB resembles the Fe(ii)- and alpha-ketoglutarate-dependent dioxygenases, which use iron-oxo intermediates to oxidize chemically inert compounds. We show here that purified AlkB repairs the cytotoxic lesions 1-methyladenine and 3-methylcytosine in single- and double-stranded DNA in a reaction that is dependent on oxygen, alpha-ketoglutarate and Fe(ii). The AlkB enzyme couples oxidative decarboxylation of alpha-ketoglutarate to the hydroxylation of these methylated bases in DNA, resulting in direct reversion to the unmodified base and the release of formaldehyde.

Adenine↗
PubMed2002

Probing the 2,4-dichlorophenoxyacetate/alpha-ketoglutarate dioxygenase substrate-binding site by site-directed mutagenesis and mechanism-based inactivation.

TfdA is an Fe(II)- and alpha-ketoglutarate- (alphaKG-) dependent dioxygenase that hydroxylates the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) producing a hemiacetal that spontaneously decomposes to 2,4-dichlorophenol and glyoxylate. On the basis of a recently published TfdA structural model [Elkins et al. (2002) Biochemistry 41, 5185-5192], His214, Lys71, Arg278, and the backbone amide of Ser117 are suggested to bind the 2,4-D carboxylate; Lys95 and possibly Lys71 are hypothesized to interact with the 2,4-D ether atom; and Arg274 and Thr141 are suspected to bind alphaKG. TfdA variants with substitutions at these and other positions were purified and characterized in order to explore the roles of these residues in catalysis. The K71L, K71Q, K95L, K95Q, R274Q, R274L, and R278Q variants exhibited significantly increased 2,4-D K(m), alphaKG K(m), and alphaKG K(d) values, consistent with their proposed roles in substrate binding. A protease-sensitive site was successfully eliminated in the R78Q variant, which also exhibited decreased affinity for 2,4-D. In contrast, the Y81F, Y126F, T141V, Y169F, and Y244F variants showed only modest changes in their kinetics. An observed 4-fold lower K(m) of the K95L variant compared to wild-type protein with the alternative substrate 2,4-dichlorocinnamic acid provided additional evidence for an interaction between Lys95 and the 2,4-D ether atom. Phenylpropiolic acid was identified as a mechanism-based inactivator of the enzyme [K(i) = 38.1 +/- 6.0 microM and k(inact)(max) = 2.3 +/- 0.1 min(-1)]. This acetylenic compound covalently modifies a peptide (166-AEHYALNSR-174) that is predicted to form one side of the substrate-binding pocket. The K95L variant of TfdA was not inactivated by phenylpropiolic acid, providing added support that Lys95 is present at the active site. These results support the identity of suspected substrate-binding residues derived from structural modeling studies and extend our understanding of the oxidative chemistry carried out by TfdA.

Alkylation↗
PubMed2002

X-ray crystal structure of Escherichia coli taurine/alpha-ketoglutarate dioxygenase complexed to ferrous iron and substrates.

Taurine/alpha-ketoglutarate dioxygenase (TauD), a non-heme Fe(II) oxygenase, catalyses the conversion of taurine (2-aminoethanesulfonate) to sulfite and aminoacetaldehyde concurrent with the conversion of alpha-ketoglutarate (alphaKG) to succinate and CO(2). The enzyme allows Escherichia coli to use taurine, widely available in the environment, as an alternative sulfur source. Here we describe the X-ray crystal structure of TauD complexed to Fe(II) and both substrates, alphaKG and taurine. The tertiary structure and fold of TauD are similar to those observed in other enzymes from the broad family of Fe(II)/alphaKG-dependent oxygenases, with closest structural similarity to clavaminate synthase. Using the TauD coordinates, a model was determined for the closely related enzyme 2,4-dichlorophenoxyacetate/alphaKG dioxygenase (TfdA), supporting predictions derived from site-directed mutagenesis and other studies of that biodegradative protein. The TauD structure and TfdA model define the metal ligands and the positions of nearby aromatic residues that undergo post-translational modifications involving self-hydroxylation reactions. The substrate binding residues of TauD were identified and those of TfdA predicted. These results, along with sequence alignment information, reveal how TauD selects a tetrahedral substrate anion in preference to the planar carboxylate selected by TfdA, providing insight into the mechanism of enzyme catalysis.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2002

Non-heme iron oxygenases.

Our understanding of the biological significance and chemical properties of non-heme iron oxygenases has increased dramatically in recent years. New group members have emerged from genome sequences and biochemical analyses. Spectroscopic and crystallographic studies have provided critical insights into catalysis. Self-hydroxylation reactions, commonplace in these proteins, reveal important features of metallocenter reactivity.

Animals↗
PubMed2002

tfdA-like genes in 2,4-dichlorophenoxyacetic acid-degrading bacteria belonging to the Bradyrhizobium-Agromonas-Nitrobacter-Afipia cluster in alpha-Proteobacteria.

The 2,4-dichlorophenoxyacetate (2,4-D)/alpha-ketoglutarate dioxygenase gene (tfdA) homolog designated tfdAalpha was cloned and characterized from 2,4-D-degrading bacterial strain RD5-C2. This Japanese upland soil isolate belongs to the Bradyrhizobium-Agromonas-Nitrobacter-Afipia cluster in the alpha subdivision of the class Proteobacteria on the basis of its 16S ribosomal DNA sequence. Sequence analysis showed 56 to 60% identity of tfdAalpha to representative tfdA genes. A MalE-TfdAalpha fusion protein expressed in Escherichia coli exhibited about 10 times greater activity for phenoxyacetate than 2,4-D in an alpha-ketoglutarate- and Fe(II)-dependent reaction. The deduced amino acid sequence of TfdAalpha revealed a conserved His-X-Asp-X(146)-His-X(14)-Arg motif characteristic of the active site of group II alpha-ketoglutarate-dependent dioxygenases. The tfdAalpha genes were also detected in 2,4-D-degrading alpha-Proteobacteria previously isolated from pristine environments in Hawaii and in Saskatchewan, Canada (Y. Kamagata, R. R. Fulthorpe, K. Tamura, H. Takami, L. J. Forney, and J. M. Tiedje, Appl. Environ. Microbiol. 63:2266-2272, 1997). These findings indicate that the tfdA genes in beta- and gamma-Proteobacteria and the tfdAalpha genes in alpha-Proteobacteria arose by divergent evolution from a common ancestor.

2,4-Dichlorophenoxyacetic Acid↗
PubMedDate not supplied

FeII/alpha-ketoglutarate-dependent hydroxylases and related enzymes.

FeII/alpha-ketoglutarate (alphaKG)-dependent hydroxylases catalyze an amazing diversity of reactions that result in protein side-chain modifications, repair of alkylated DNA/RNA, biosynthesis of antibiotics and plant products, metabolism related to lipids, and biodegradation of a variety of compounds. These enzymes possess a beta-strand "jellyroll" structural fold that contains three metal-binding ligands found in a His1-X-Asp/Glu-Xn-His2 motif. The cosubstrate, alphaKG, chelates FeII using its C-2 keto group (binding opposite the Asp/Glu residue) and C-1 carboxylate (coordinating opposite either His1 or His2). Oxidative decomposition of alphaKG forms CO2 plus succinate and leads to the generation of an FeIV-oxo or other activated oxygen species that hydroxylate the primary substrate. The reactive oxygen species displays alternate reactivity in related enzymes that catalyze desaturations, ring expansions, or ring closures. Other enzymes resemble the FeII/alphaKG-dependent hydroxylases in terms of protein structure or chemical mechanism but do not utilize alphaKG as a substrate. This review describes the reactions catalyzed by this superfamily of enzymes, highlights key active site features revealed by structural studies, and summarizes results from spectroscopic and other approaches that provide insights into the chemical mechanisms.

Ferrous Compounds↗