Luke W. Guddat

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.

Structure, function, and regulation of tartrate-resistant acid phosphatase

The tartrate-resistant acid phosphatases (TRAPs) are a class of metalloenzymes that catalyze the hydrolysis of various phosphate esters and anhydrides under acidic reaction conditions. Because the bound metal ions confer an intense color on these enzymes they are also known as purple acid phosphatases (PAPs). Resistance to inhibition by high concentrations of the competitive inhibitor L 1 tartrate distinguishes TRAP from acid phosphatases of lysosomal or prostatic origin present in many mammalian cells and tissues. TRAP enzymes have been isolated from many mammalian sources, including: bovine and rat spleen; the spleens of patients affected with hairy cell leukemia and Gaucher’s disease; human and rat bone; and human lungs and placenta. The TRAP purified from porcine allantoic fluid, which is also known as uteroferrin, was originally recognized as an abundant basic protein in uterine secretions induced by progesterone. The catalytic mechanism, structure, and properties of the iron center of porcine TRAP have been studied extensively by our group. Mammalian isolated TRAP enzymes all have similar physical properties, including a molecular weight of about 35 kDa, a basic isoelectric point (pI 7.6–9.5), and optimal enzyme activity at an acidic pH. The enzyme can be isolated as a single chain polypeptide, but a dimeric nicked form arises from posttranslational cleavage of the single chain enzyme. Cleavage occurs in an exposed loop that is conserved in all mammalian TRAP enzymes and leads to an increase in Vmax/kcat of the enzyme by an unknown mechanism. Several proteolytic enzymes are able to cleave the exposed loop, but only the cysteine proteinases papain and cathepsin B have been able to cause activation among several tested. Ljusberg et al. put forth the view that TRAP, like several other hydrolases, is synthesized as a relatively inactive proenzyme, and cleavage is the physiological mechanism of proenzyme activation in osteoclasts. Mammalian TRAP enzymes are glycoproteins and, like most lysosomal enzymes, possess the mannose-6-phosphate lysosomal targeting sequence, which must presumably be cleaved or modified to permit secretion. TRAP isolated from allantoic fluid of the pig showed a single, unphosphorylated, high-mannose-type oligosaccharide composed of five or six mannose residues and two N-acetylglucosamine residues. In contrast, recombinant porcine TRAP secreted by Chinese hamster ovary (CHO) cells possessed N-linked, high-mannose oligosaccharide chains that were phosphorylated and could not be dephosphorylated by alkaline phosphatase treatment in vitro. This suggests that the uteroferrin oligosaccharide phosphates were not exposed, perhaps as a result of blocking by an N-acetylglucosamine residue. The glycoprotein structure of human bone TRAP was analyzed by lectin binding and, in agreement with the prior analysis of native uteroferrin, contained only N-linked high-mannose carbohydrates, implying that the native secreted protein is normally dephosphorylated. Analysis of TRAP activity present in electrophoretically separated human serum revealed two isoforms, termed 5a and 5b, with each isoform having a different pH optimum (5a: pH 4.9; 5b: pH 5.5–6.0). The carbohydrate content of the isoforms also differed with only isoform 5a containing sialic acid. TRAP contains two iron atoms at its active site, and the intense purple color of the enzyme results from a tyrosinate Fe(III) charge transfer. Reduction of the active site binuclear center to a mixed valency Fe(III)-Fe(II) form is required for activation and this corresponds to a shift in color from purple to pink. Further reduction or the presence of iron chelators can lead to reversible inactivation and formation of a colorless form of the enzyme. The enzyme is also inhibited noncompetitively by incubation with vanadate or simply following more extended incubation at 37°C. The latter case at least, produced a “yellowish” form of the enzyme. TRAP may become irreversibly inactivated by oxidation in the presence of ascorbate. The recent availability of monoclonal antibodies against TRAP has permitted the identification of an inactivated “yellowish” form of the enzyme as the major form in the circulation.

Crystal structures of reduced and oxidized DsbA: investigation of domain motion and thiolate stabilization.

BACKGROUND The redox proteins that incorporate a thioredoxin fold have diverse properties and functions. The bacterial protein-folding factor DsbA is the most oxidizing of the thioredoxin family. DsbA catalyzes disulfide-bond formation during the folding of secreted proteins. The extremely oxidizing nature of DsbA has been proposed to result from either domain motion or stabilizing active-site interactions in the reduced form. In the domain motion model, hinge bending between the two domains of DsbA occurs as a result of redox-related conformational changes. RESULTS We have determined the crystal structures of reduced and oxidized DsbA in the same crystal form and at the same pH (5.6). The crystal structure of a lower pH form of oxidized DsbA has also been determined (pH 5.0). These new crystal structures of DsbA, and the previously determined structure of oxidized DsbA at pH 6.5, provide the foundation for analysis of structural changes that occur upon reduction of the active-site disulfide bond. CONCLUSIONS The structures of reduced and oxidized DsbA reveal that hinge bending motions do occur between the two domains. These motions are independent of redox state, however, and therefore do not contribute to the energetic differences between the two redox states. Instead, the observed domain motion is proposed to be a consequence of substrate binding. Furthermore, DsbAs highly oxidizing nature is a result of hydrogen bond, electrostatic and helix-dipole interactions that favour the thiolate over the disulfide at the active site.

Molecular basis of sulfonylurea herbicide inhibition of acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS) (acetolactate synthase, EC 4.1.3.18) catalyzes the first step in branched-chain amino acid biosynthesis and is the target for sulfonylurea and imidazolinone herbicides. These compounds are potent and selective inhibitors, but their binding site on AHAS has not been elucidated. Here we report the 2.8 Å resolution crystal structure of yeast AHAS in complex with a sulfonylurea herbicide, chlorimuron ethyl. The inhibitor, which has a K i of 3.3 nm, blocks access to the active site and contacts multiple residues where mutation results in herbicide resistance. The structure provides a starting point for the rational design of further herbicidal compounds.

Crystal structure of mammalian purple acid phosphatase

BACKGROUND Mammalian purple acid phosphatases are highly conserved binuclear metal-containing enzymes produced by osteoclasts, the cells that resorb bone. The enzyme is a target for drug design because there is strong evidence that it is involved in bone resorption. RESULTS The 1.55 A resolution structure of pig purple acid phosphatase has been solved by multiple isomorphous replacement. The enzyme comprises two sandwiched beta sheets flanked by alpha-helical segments. The molecule shows internal symmetry, with the metal ions bound at the interface between the two halves. CONCLUSIONS Despite less than 15% sequence identity, the protein fold resembles that of the catalytic domain of plant purple acid phosphatase and some serine/threonine protein phosphatases. The active-site regions of the mammalian and plant purple acid phosphatases differ significantly, however. The internal symmetry suggests that the binuclear centre evolved as a result of the combination of mononuclear ancestors. The structure of the mammalian enzyme provides a basis for antiosteoporotic drug design.

Identification of mammalian-like purple acid phosphatases in a wide range of plants.

Purple acid phosphatases (PAPs) comprise a family of binuclear metal-containing hydrolases, members of which have been isolated from plants, mammals and fungi. Polypeptide chains differ in size (animal approximately 35kDa, plant approximately 55kDa) and exhibit low sequence homology between kingdoms but all residues involved in co-ordination of the metal ions are invariant. A search of genomic databases was undertaken using a sequence pattern which includes the conserved residues. Several novel potential PAP sequences were detected, including the first known examples from bacterial sources. Ten plant ESTs were also identified which, although possessing the conserved sequence pattern, were not homologous throughout their sequences to previously known plant PAPs. Based on these EST sequences, novel cDNAs from sweet potato, soybean, red kidney bean and Arabidopsis thaliana were cloned and sequenced. These sequences are more closely related to mammalian PAP than to previously characterized plant enzymes. Their predicted secondary structure is similar to that of the mammalian enzyme. A model of the sweet potato enzyme was generated based on the coordinates of pig PAP. These observations strongly suggest that the cloned cDNA sequences represent a second group of plant PAPs with properties more similar to the mammalian enzymes than to the high molecular weight plant enzymes.

The 1.1 å crystal structure of the neuronal acetylcholine receptor antagonist, α-conotoxin PnIA from Conus pennaceus

BACKGROUND alpha-Conotoxins are peptide toxins, isolated from Conus snails, that block the nicotinic acetylcholine receptor (nAChR). The 16-residue peptides PnIA and PnIB from Conus pennaceus incorporate the same disulfide framework as other alpha-conotoxins but differ in function from most alpha-conotoxins by blocking the neuronal nAChR, rather than the skeletal muscle subtype. The crystal structure determination of PnIA was undertaken to identify structural and surface features that might be important for biological activity. RESULTS The 1.1 A crystal structure of synthetic PnIA was determined by direct methods using the Shake-and-Bake program. The three-dimensional structure incorporates a beta turn followed by two alpha-helical turns. The conformation is stabilised by two disulfide bridges that form the interior of the molecule, with all other side chains oriented outwards. CONCLUSIONS The compact architecture of the PnIA toxin provides a rigid framework for presentation of chemical groups that are required for activity. The structure is characterized by distinct hydrophobic and polar surfaces; a 16 A separation of the sole positive and negative charges (these two charged residues being located at opposite ends of the molecule); a hydrophobic region and a protruding tyrosine side chain. These features may be important for the specific interaction of PnIA with neuronal nAChR.

Inhibition of hypoxanthine-guanine phosphoribosyltransferase by acyclic nucleoside phosphonates: a new class of antimalarial therapeutics.

The purine salvage enzyme hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) is essential for purine nucleotide and hence nucleic acid synthesis in the malaria parasite, Plasmodium falciparum. Acyclic nucleoside phosphonates (ANPs) are analogues of the nucleotide product of the reaction, comprising a purine base joined by a linker to a phosphonate moiety. K(i) values for 19 ANPs were determined for Pf HGXPRT and the corresponding human enzyme, HGPRT. Values for Pf HGXPRT were as low as 100 nM, with selectivity for the parasite enzyme of up to 58. Structures of human HGPRT in complex with three ANPs are reported. On binding, a large mobile loop in the free enzyme moves to partly cover the active site. For three ANPs, the IC(50) values for Pf grown in cell culture were 1, 14, and 46 microM, while the cytotoxic concentration for the first compound was 489 microM. These results provide a basis for the design of potent and selective ANP inhibitors of Pf HGXPRT as antimalarial drug leads.

Synthesis of branched 9-[2-(2-phosphonoethoxy)ethyl]purines as a new class of acyclic nucleoside phosphonates which inhibit Plasmodium falciparum hypoxanthine–guanine–xanthine phosphoribosyltransferase

The malarial parasite Plasmodium falciparum (Pf) lacks the de novo pathway and relies on the salvage enzyme, hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT), for the synthesis of the 6-oxopurine nucleoside monophosphates. Specific acyclic nucleoside phosphonates (ANPs) inhibit PfHGXPRT and possess anti-plasmodial activity. Two series of novel branched ANPs derived from 9-[2-(2-phosphonoethoxy)ethyl]purines were synthesized to investigate their inhibition of PfHGXPRT and human HGPRT. The best inhibitor of PfHGXPRT has a K(i) of 1 microM. The data showed that both the position and nature of the hydrophobic substituent change the potency and selectivity of the ANPs.

The Crystal Structures of Klebsiella pneumoniae Acetolactate Synthase with Enzyme-bound Cofactor and with an Unusual Intermediate

Acetohydroxyacid synthase (AHAS) and acetolactate synthase (ALS) are thiamine diphosphate (ThDP)-dependent enzymes that catalyze the decarboxylation of pyruvate to give a cofactor-bound hydroxyethyl group, which is transferred to a second molecule of pyruvate to give 2-acetolactate. AHAS is found in plants, fungi, and bacteria, is involved in the biosynthesis of the branched-chain amino acids, and contains non-catalytic FAD. ALS is found only in some bacteria, is a catabolic enzyme required for the butanediol fermentation, and does not contain FAD. Here we report the 2.3-Å crystal structure of Klebsiella pneumoniae ALS. The overall structure is similar to AHAS except for a groove that accommodates FAD in AHAS, which is filled with amino acid side chains in ALS. The ThDP cofactor has an unusual conformation that is unprecedented among the 26 known three-dimensional structures of nine ThDP-dependent enzymes, including AHAS. This conformation suggests a novel mechanism for ALS. A second structure, at 2.0 Å, is described in which the enzyme is trapped halfway through the catalytic cycle so that it contains the hydroxyethyl intermediate bound to ThDP. The cofactor has a tricyclic structure that has not been observed previously in any ThDP-dependent enzyme, although similar structures are well known for free thiamine. This structure is consistent with our proposed mechanism and probably results from an intramolecular proton transfer within a tricyclic carbanion that is the true reaction intermediate. Modeling of the second molecule of pyruvate into the active site of the enzyme with the bound intermediate is consistent with the stereochemistry and specificity of ALS.