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N A Larsen

Publications and source records attributed to N A Larsen.

At least 19 recordsLinked to original sources
PubMed2006

Structural characterization of enterobactin hydrolase IroE.

The proliferation of many pathogenic bacteria is limited by the scarcity of soluble iron in their environment. Many of these bacteria scavenge iron by synthesizing and exporting small molecule siderophores that chelate iron. Iron-bound siderophores are subsequently imported for metabolic processing. Three related serine hydrolases have been characterized biochemically in this pathway: Fes, IroD, and IroE. Here, we report the crystal structure of IroE from uropathogenic Escherichia coli CFT073. The native structure and a complex with diisopropyl fluorophosphonate (DFP, a potent serine hydrolase inhibitor) were determined at 2.3 and 1.4 A resolution, respectively. IroE has the typical alpha/beta-hydrolase fold with an atypical catalytic dyad composed of Ser 189 and His 287. Mutation of either residue was detrimental to catalysis. In addition, rather than the typical oxyanion hole composed of backbone amides, IroE employs the atypical guanidinium moiety of Arg 130. Asp 90 anchors Arg 130 in the active site, and mutation of either residue was likewise detrimental to catalysis. We also compare the structure of IroE to the structure of Fes from Shigella flexneri (PDB entry 2B20). Both enzymes have similar active sites, but Fes has an additional amino-terminal lid domain. These lid domains are proposed to confer specificity to these related hydrolases.

Amino Acid Substitution↗
PubMed2004

Crystallographic and biochemical analysis of cocaine-degrading antibody 15A10.

Catalytic antibody 15A10 hydrolyzes the benzoyl ester of cocaine to form the nonpsychoactive metabolites benzoic acid and ecgonine methylester. Here, we report biochemical and structural studies that characterize the catalytic mechanism. The crystal structure of the cocaine-hydrolyzing monoclonal antibody (mAb) 15A10 has been determined at 2.35 A resolution. The binding pocket is fairly shallow and mainly hydrophobic but with a cluster of three hydrogen-bond donating residues (TrpL96, AsnH33, and TyrH35). Computational docking of the transition state analogue (TSA) indicates that these residues are appropriately positioned to coordinate the phosphonate moiety of the TSA and, hence, form an oxyanion hole. Tyrosine modification of the antibody with tetranitromethane reduced hydrolytic activity to background level. The contribution from these and other residues to catalysis and TSA binding was explored by site-directed mutagenesis of 15A10 expressed in a single chain fragment variable (scFv) format. The TyrH35Phe mutant had 4-fold reduced activity, and TrpL96Ala, TrpL96His, and AsnH33Ala mutants were all inactive. Comparison with an esterolytic antibody D2.3 revealed a similar arrangement of tryptophan, asparagine, and tyrosine residues in the oxyanion hole that stabilizes the transition state for ester hydrolysis. Furthermore, the crystal structure of the bacterial cocaine esterase (cocE) also showed that the cocE employs a tyrosine hydroxyl in the oxyanion hole. Thus, the biochemical and structural data are consistent with the catalytic antibody providing oxyanion stabilization as its major contribution to catalysis.

Amino Acid Sequence↗
PubMed2002

Chlamydia pneumoniae DNA in peripheral blood mononuclear cells in dialysis patients, renal transplant recipients and healthy controls.

It has been suggested that infection with Chlamydia pneumoniae plays a role in the development and maintenance of atherosclerosis based on differences in the prevalence of antibodies against Chlamydia pneumoniae in patients with and without atherosclerotic lesions and on the presence of bacteria in atherosclerotic lesions. It is well known that patients undergoing chronic dialysis treatment and renal transplant recipients have a considerably increased risk of cardiovascular disease. In this study it is hypothesized that patients with these conditions have a higher prevalence of Chlamydia pneumoniae DNA in the white cells of the peripheral blood. Blood samples from 196 dialysis patients, 114 renal transplant recipients and 342 healthy controls were analysed with an in-house nested polymerase chain reaction (nPCR) and tested for the presence of Chlamydia pneumoniae DNA. The prevalence of Chlamydia pneumoniae DNA was significantly higher in dialysis patients (16.3%) than in healthy controls (8.5%, p < 0.01), whereas no significant difference was found between the prevalence in renal transplant recipients (9.6%) and healthy controls. The prevalence was not related to gender or age in either group, and it was the same in diabetics and non-diabetics. Dialysis patients have a higher prevalence of Chlamydia pneumoniae DNA than healthy controls. The lower prevalence of Chlamydia pneumoniae DNA in renal transplant recipients than in dialysis patients may be due to selection of dialysis patients with few or no cardiovascular complications for renal transplantation. Our results are consistent with the hypothesis that Chlamydia pneumoniae is associated with the pathogenesis of atherosclerosis.

Adult↗
PubMed2001

Structural basis for a disfavored elimination reaction in catalytic antibody 1D4.

Murine antibody 1D4 selectively catalyzes a highly disfavored beta-elimination reaction. Crystal structures of unliganded 1D4 and 1D4 in complex with a transition-state analog (TSA) have elucidated a possible general base mode of catalysis. The structures of the unliganded and liganded Fabs were determined to 1.80 and 1.85 A resolution, respectively. The structure of the complex reveals a binding pocket with high shape complementarity to the TSA, which is recruited to coerce the substrate into the sterically demanding, eclipsed conformation that is required for catalysis. A histidine residue and two water molecules are likely involved in the catalysis. The structure supports either a concerted E2 or stepwise E1cB-like mechanism for elimination. Finally, the liganded 1D4 structure shows minor conformational rearrangements in CDR H2, indicative of induced-fit binding of the hapten. 1D4 has pushed the boundaries of antibody-mediated catalysis into the realm of disfavored reactions and, hence, represents an important milestone in the development of this technology.

Animals↗
PubMed2001

Antibody catalysis of the oxidation of water.

Recently we reported that antibodies can generate hydrogen peroxide (H2O2) from singlet molecular oxygen (1O2*). We now show that this process is catalytic, and we identify the electron source for a quasi-unlimited generation of H2O2. Antibodies produce up to 500 mole equivalents of H2O2 from 1O2*, without a reduction in rate, and we have excluded metals or Cl- as the electron source. On the basis of isotope incorporation experiments and kinetic data, we propose that antibodies use H2O as an electron source, facilitating its addition to 1O2* to form H2O3 as the first intermediate in a reaction cascade that eventually leads to H2O2. X-ray crystallographic studies with xenon point to putative conserved oxygen binding sites within the antibody fold where this chemistry could be initiated. Our findings suggest a protective function of immunoglobulins against 1O2* and raise the question of whether the need to detoxify 1O2* has played a decisive role in the evolution of the immunoglobulin fold.

Animals↗
PubMed2001

Crystal structure of a cocaine-binding antibody.

Murine monoclonal antibody GNC92H2 was elicited by active immunization with a cocaine immunoconjugate and binds free cocaine with excellent specificity and moderate affinity. Improvement of affinity, as well as humanization of GNC92H2, would be advantageous in immunopharmacotherapy for cocaine addiction, and for emergency cases of drug overdose. Toward this end, the crystal structure of an engineered murine-human chimeric Fab of GNC92H2 complexed with cocaine was determined at 2.3 A resolution. Structural analysis reveals a binding pocket with high shape and charge complementarity to the cocaine framework, which explains the specificity for cocaine, as opposed to the pharmacologically inactive cocaine metabolites. Importantly, the structure provides a foundation for mutagenesis to enhance the binding affinity for cocaine and potent cocaine derivatives, such as cocaethylene, and for additional humanization of the antibody.

Animals↗
PubMed2000

Using antibody catalysis to study the outcome of multiple evolutionary trials of a chemical task.

Catalytic aldolase antibodies generated by immunization with two different, but structurally related, beta-diketone haptens were cloned and sequenced to study similarities and differences between independently evolved catalysts. Kinetic and sequence analysis coupled with mutagenesis, structural, and modeling studies reveal that the defining event in the evolution of these catalysts was a somatic mutation that placed a lysine residue in a deep, yet otherwise unrefined, hydrophobic pocket. We suggest that covalent chemistries may be as readily selected from the immune repertoire as the traditional noncovalent interactions that have formed the basis of immunochemistry until this time. Further, we believe that these experiments recapitulate the defining events in the evolution of nature's enzymes, particularly as they relate to chemical mechanism, catalytic promiscuity, and gene duplication.

Amino Acid Sequence↗
PubMed1982

Nephrotoxicity of plasticizers investigated by 48 hours hypothermic perfusion of dog kidneys.

The possible nephrotoxicity of the plasticizers diethyl phthalate and di-2-ethylhexyl phthalate was tested in vitro using a 48 hours continuous pulsatile hypothermic perfusion of canine kidneys with a human albumin perfusion medium. Since polysorbate 80 was used to facilitate the solution of the plasticizers in the perfusion medium, this substance was also tested. Six groups containing 9--15 kidneys were perfused with different amounts of plasticizers and/or polysorbate 80 added. In perfusates containing polysorbate 80 either alone or with one of the two plasticizers, the LDH activity and the potassium concentrations rose significantly higher than in the control group (p less than 0.001). The kidney weight gain was also significantly greater in these groups. A "blind" histological examination of needle biopsies by light microscopy revealed no differences among the groups. Although the biochemical evidence of tissue damage was not tested by re-implantation of the kidneys, we suggest that caution should be exercised in the use of polysorbate 80 in organ perfusion systems.

Animals↗
PubMed1981

Distribution of arsenic, manganese, and selenium in the human brain in chronic renal insufficiency, Parkinson's disease, and amyotrophic lateral sclerosis.

The concentrations of arsenic, manganese and selenium/g wet tissue weight were determined in samples from 24 areas of the human brain from 3 patients with chronic renal insufficiency, 2 with Parkinson's disease and 1 with amyotrophic lateral sclerosis. The concentrations of the 3 elements were determined for each sample by neutron activation analysis with radiochemical separation. Overall arsenic concentrations were about 2.5 times higher in patients with chronic renal failure than in controls, and lower than normal in the patients with Parkinson's disease and amyotrophic lateral sclerosis. There were no obvious differences in the overall concentrations of manganese and selenium from one group to another. Even multivariate data analysis by the SIMCA method failed to reveal any significant difference in the distribution pattern of manganese and selenium in Parkinson's disease compared to normal controls.

Adult↗
PubMed1980

The significance of pH variations in human albumin perfusion media during continuous hypothermic renal perfusion.

The variation of pH in perfusate during renal machine perfusion has been regarded exclusively as an effect of kidney metabolism. During preservation of 21 canine kidneys for 48 hours in a Gambro PF-2D machine the pH was measured in the human albumin perfusion medium used, and in control samples stored a 4 degrees C. An increase in pH from 7.21 (+/- 0.03) to 7.66 (+/- 0.10) was found in the machine perfusate. In the stored perfusion medium an increase from 7.21 (+/- 0.03) to 7.43 (+/- 0.20) was measured. We conclude that the pH change in perfusate is not a reliable parameter for the functional state of the kidney unless the spontaneous pH variation in the perfusion medium is simultaneously recorded and a correction made.

Albumins↗
PubMed1979

Topographical distribution of arsenic, manganese, and selenium in the normal human brain.

The concentrations of arsenic, manganese and selenium per gram wet tissue weight were determined in samples from 24 areas of normal human brain from 5 persons with ages ranging from 15 to 81 years of age. The concentrations of the 3 elements were determined for each sample by means of neutron activation analysis with radiochemical separation. Distinct patterns of distribution were shown for each of the 3 elements. Variations between individuals were found for some but not all brain areas, resulting in coefficients of variation between individuals of about 30% for arsenic, 10% for manganese and 20% for selenium. The results seem to indicate that arsenic is associated with the lipid phase, manganese with the dry matter and selenium with the aqueous phase of brain tissue.

Adolescent↗