Late surgical explantation and aortic valve replacement after transcatheter aortic valve implantation.

Transcatheter aortic valve implantation (TAVI) in patients with bicuspid aortic valve disease is associated with higher rates of paravalvular aortic regurgitation, which may require subsequent surgical correction. We report a case of successful late surgical CoreValve explantation 1,389 days after TAVI in a patient with bicuspid aortic valve stenosis and McArdle's disease who developed severe paravalvular aortic regurgitation. We confirm that neoendothelialization and incorporation of the nitinol cage into the aortic wall had occurred at nearly 4 years postimplantation, although explantation with careful endarterectomy could still be performed without requiring simultaneous aortic root replacement.

Fibronectin-binding proteins are required for biofilm formation by community-associated methicillin-resistant Staphylococcus aureus strain LAC.

Community-associated methicillin-resistant Staphylococcus aureus of the USA300 lineage is emerging as an important cause of medical device-related infection. However, few factors required for biofilm accumulation by USA300 strains have been identified, and the processes involved are poorly understood. Here, we identify S. aureus proteins required for the USA300 isolate LAC to form biofilm. A mutant with a deletion of the fnbA and fnbB genes did not express the fibronectin-binding proteins FnBPA and FnBPB and lacked the ability to adhere to fibronectin or to form biofilm. Biofilm formation by the mutant LAC∆fnbAfnbB could be restored by expression of FnBPA or FnBPB from a plasmid demonstrating that both of these proteins can mediate biofilm formation when expressed by LAC. Expression of FnBPA and FnBPB increased bacterial aggregation suggesting that fibronectin-binding proteins can promote the accumulation phase of biofilm. Loss of fibronectin-binding proteins reduced the initial adherence of bacteria, indicating that these proteins are also involved in primary attachment. In summary, these findings improve our understanding of biofilm formation by the USA300 strain LAC by demonstrating that the fibronectin-binding proteins are required.

Detection of HSV type-1 and type-2 IgG using an in vitro PCA based homogeneous immunoassay.

Enzyme immunoassays (EIAs) are widely used in the clinical laboratory and research institutes for the detection of biologically relevant analytes. Almost all EIAs are heterogeneous in nature and require multiple steps of process. In contrast, homogeneous immunoassays (HA) offer a simplified one-step approach with a number of potential advantages over contemporary heterogeneous EIAs such as higher throughput and greater clinical utility. Utilizing TEM-1 beta-lactamase as a reporter enzyme, we have developed HAs based on in vitro protein fragment complementation (PCA) for the detection of antibodies and potentially be used for antigens or other biomarkers. In this proof-of-principle study we demonstrate the successful in vitro differentiation of anti-herpes simplex virus (HSV) type-1 and type-2 Immunoglobulin G (IgG) in human serum with high sensitivity and specificity.

Structure and mechanism of inhibition of plant acetohydroxyacid synthase.

Plants and microorganisms synthesize valine, leucine and isoleucine via a common pathway in which the first reaction is catalysed by acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This enzyme is of substantial importance because it is the target of several herbicides, including all members of the popular sulfonylurea and imidazolinone families. However, the emergence of resistant weeds due to mutations that interfere with the inhibition of AHAS is now a worldwide problem. Here we summarize recent ideas on the way in which these herbicides inhibit the enzyme, based on the 3D structure of Arabidopsis thaliana AHAS. This structure also reveals important clues for understanding how various mutations can lead to herbicide resistance.

Thiamin nutrition and catalysis-induced instability of thiamin diphosphate.

Thiamin (vitamin B1) is required in animal diets because it is the precursor of the enzyme cofactor, thiamin diphosphate. Unlike other B vitamins, the dietary thiamin requirement is proportional to non-fat energy intake but there is no obvious biochemical reason for this relationship. In the present communication we show for two enzymes that the cofactor undergoes a slow destruction during catalysis, which may explain the interdependence of thiamin and energy intakes.

Herbicide-binding sites revealed in the structure of plant acetohydroxyacid synthase.

The sulfonylureas and imidazolinones are potent commercial herbicide families. They are among the most popular choices for farmers worldwide, because they are nontoxic to animals and highly selective. These herbicides inhibit branched-chain amino acid biosynthesis in plants by targeting acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This report describes the 3D structure of Arabidopsis thaliana AHAS in complex with five sulfonylureas (to 2.5 A resolution) and with the imidazolinone, imazaquin (IQ; 2.8 A). Neither class of molecule has a structure that mimics the substrates for the enzyme, but both inhibit by blocking a channel through which access to the active site is gained. The sulfonylureas approach within 5 A of the catalytic center, which is the C2 atom of the cofactor thiamin diphosphate, whereas IQ is at least 7 A from this atom. Ten of the amino acid residues that bind the sulfonylureas also bind IQ. Six additional residues interact only with the sulfonylureas, whereas there are two residues that bind IQ but not the sulfonylureas. Thus, the two classes of inhibitor occupy partially overlapping sites but adopt different modes of binding. The increasing emergence of resistant weeds due to the appearance of mutations that interfere with the inhibition of AHAS is now a worldwide problem. The structures described here provide a rational molecular basis for understanding these mutations, thus allowing more sophisticated AHAS inhibitors to be developed. There is no previously described structure for any plant protein in complex with a commercial herbicide.

How an enzyme answers multiple-choice questions.

Acetohydroxyacid synthase (AHAS) is the first common enzyme in the pathway for the biosynthesis of branched-chain amino acids. Interest in the enzyme has escalated over the past 20 years since it was discovered that AHAS is the target of the sulfonylurea and imidazolinone herbicides. However, several questions regarding the reaction mechanism have remained unanswered, particularly the way in which AHAS "chooses" its second substrate. A new method for the detection of reaction intermediates enables calculation of the microscopic rate constants required to explain this phenomenon.

Elucidating the specificity of binding of sulfonylurea herbicides to acetohydroxyacid synthase.

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) is the target for the sulfonylurea herbicides, which act as potent inhibitors of the enzyme. Chlorsulfuron (marketed as Glean) and sulfometuron methyl (marketed as Oust) are two commercially important members of this family of herbicides. Here we report crystal structures of yeast AHAS in complex with chlorsulfuron (at a resolution of 2.19 A), sulfometuron methyl (2.34 A), and two other sulfonylureas, metsulfuron methyl (2.29 A) and tribenuron methyl (2.58 A). The structures observed suggest why these inhibitors have different potencies and provide clues about the differential effects of mutations in the active site tunnel on various inhibitors. In all of the structures, the thiamin diphosphate cofactor is fragmented, possibly as the result of inhibitor binding. In addition to thiamin diphosphate, AHAS requires FAD for activity. Recently, it has been reported that reduction of FAD can occur as a minor side reaction due to reaction with the carbanion/enamine of the hydroxyethyl-ThDP intermediate that is formed midway through the catalytic cycle. Here we report that the isoalloxazine ring has a bent conformation that would account for its ability to accept electrons from the hydroxyethyl intermediate. Most sequence and mutation data suggest that yeast AHAS is a high-quality model for the plant enzyme.

Facile crystallization of Escherichia coli ketol-acid reductoisomerase.

Ketol-acid reductoisomerase (EC 1.1.1.86) catalyses the second reaction in the biosynthesis of branched-chain amino acids. The reaction involves an Mg2+ -dependent alkyl migration followed by an NADPH-dependent reduction of the 2-keto group. Here, the crystallization of the Escherichia coli enzyme is reported. A form with a C-terminal hexahistidine tag could be crystallized under 18 different conditions in the absence of NADPH or Mg2+ and a further six crystallization conditions were identified with one or both ligands. With the hexahistidine tag on the N-terminus, 20 crystallization conditions were found, some of which required the presence of NADPH, NADP+, Mg2+ or a combination of ligands. Finally, the selenomethionine-substituted enzyme with the N-terminal tag crystallized under 15 conditions. Thus, the enzyme is remarkably easy to crystallize. Most of the crystals diffract poorly but several data sets were collected at better than 3.2 A resolution; attempts to phase them are currently in progress.

Electron transfer in acetohydroxy acid synthase as a side reaction of catalysis. Implications for the reactivity and partitioning of the carbanion/enamine form of (alpha-hydroxyethyl)thiamin diphosphate in a "nonredox" flavoenzyme.

Acetohydroxy acid synthases (AHAS) are thiamin diphosphate- (ThDP-) and FAD-dependent enzymes that catalyze the first common step of branched-chain amino acid biosynthesis in plants, bacteria, and fungi. Although the flavin cofactor is not chemically involved in the physiological reaction of AHAS, it has been shown to be essential for the structural integrity and activity of the enzyme. Here, we report that the enzyme-bound FAD in AHAS is reduced in the course of catalysis in a side reaction. The reduction of the enzyme-bound flavin during turnover of different substrates under aerobic and anaerobic conditions was characterized by stopped-flow kinetics using the intrinsic FAD absorbance. Reduction of enzyme-bound FAD proceeds with a net rate constant of k' = 0.2 s(-1) in the presence of oxygen and approximately 1 s(-1) under anaerobic conditions. No transient flavin radicals are detectable during the reduction process while time-resolved absorbance spectra are recorded. Reconstitution of the binary enzyme-FAD complex with the chemically synthesized intermediate 2-(hydroxyethyl)-ThDP also results in a reduction of the flavin. These data provide evidence for the first time that the key catalytic intermediate 2-(hydroxyethyl)-ThDP in the carbanionic/enamine form is not only subject to covalent addition of 2-keto acids and an oxygenase side reaction but also transfers electrons to the adjacent FAD in an intramolecular redox reaction yielding 2-acetyl-ThDP and reduced FAD. The detection of the electron transfer supports the idea of a common ancestor of acetohydroxy acid synthase and pyruvate oxidase, a homologous ThDP- and FAD-dependent enzyme that, in contrast to AHASs, catalyzes a reaction that relies on intercofactor electron transfer.