Crystal structures of substrate complexes of malic enzyme and insights into the catalytic mechanism

Malic enzymes catalyze the oxidative decarboxylation of L-malate to pyruvate and CO(2) with the reduction of the NAD(P)(+) cofactor in the presence of divalent cations. We report the crystal structures at up to 2.1 A resolution of human mitochondrial NAD(P)(+)-dependent malic enzyme in different pentary complexes with the natural substrate malate or pyruvate, the dinucleotide cofactor NAD(+) or NADH, the divalent cation Mn(2+), and the allosteric activator fumarate. Malate is bound deep in the active site, providing two ligands for the cation, and its C4 carboxylate group is out of plane with the C1-C2-C3 atoms, facilitating decarboxylation. The divalent cation is positioned optimally to catalyze the entire reaction. Lys183 is the general base for the oxidation step, extracting the proton from the C2 hydroxyl of malate. Tyr112-Lys183 functions as the general acid-base pair to catalyze the tautomerization of the enolpyruvate product from decarboxylation to pyruvate.     

Crystal structure and substrate binding modeling of the uroporphyrinogen-III decarboxylase from Nicotiana tabacum. Implications for the catalytic mechanism

The enzymatic catalysis of many biological processes of life is supported by the presence of cofactors and prosthetic groups originating from the common tetrapyrrole precursor uroporphyrinogen-III. Uroporphyrinogen-III decarboxylase catalyzes its conversion into coproporphyrinogen-III, leading in plants to chlorophyll and heme biosynthesis. Here we report the first crystal structure of a plant (Nicotiana tabacum) uroporphyrinogen-III decarboxylase, together with the molecular modeling of substrate binding in tobacco and human enzymes. Its structural comparison with the homologous human protein reveals a similar catalytic cleft with six invariant polar residues, Arg(32), Arg(36), Asp(82), Ser(214) (Thr in Escherichia coli), Tyr(159), and His(329) (tobacco numbering). The functional relationships obtained from the structural and modeling analyses of both enzymes allowed the proposal for a refined catalytic mechanism. Asp(82) and Tyr(159) seem to be the catalytic functional groups, whereas the other residues may serve in substrate recognition and binding, with Arg(32) steering its insertion. The crystallographic dimer appears to represent the protein dimer under physiological conditions. The dimeric arrangement offers a plausible mechanism at least for the first two (out of four) decarboxylation steps.     

Crystal structures of creatininase reveal the substrate binding site and provide an insight into the catalytic mechanism

Creatininase from Pseudomonas putida is a member of the urease-related amidohydrolase superfamily. The crystal structure of the Mn-activated enzyme has been solved by the single isomorphous replacement method at 1.8A resolution. The structures of the native creatininase and the Mn-activated creatininase-creatine complex have been determined by a difference Fourier method at 1.85 A and 1.6 A resolution, respectively. We found the disc-shaped hexamer to be roughly 100 A in diameter and 50 A in thickness and arranged as a trimer of dimers with 32 (D3) point group symmetry. The enzyme is a typical Zn2+ enzyme with a binuclear metal center (metal1 and metal2). Atomic absorption spectrometry and X-ray crystallography revealed that Zn2+ at metal1 (Zn1) was easily replaced with Mn2+ (Mn1). In the case of the Mn-activated enzyme, metal1 (Mn1) has a square-pyramidal geometry bound to three protein ligands of Glu34, Asp45, and His120 and two water molecules. Metal2 (Zn2) has a well-ordered tetrahedral geometry bound to the three protein ligands of His36, Asp45, and Glu183 and a water molecule. The crystal structure of the Mn-activated creatininase-creatine complex, which is the first structure as the enzyme-substrate/inhibitor complex of creatininase, reveals that significant conformation changes occur at the flap (between the alpha5 helix and the alpha6 helix) of the active site and the creatine is accommodated in a hydrophobic pocket consisting of Trp174, Trp154, Tyr121, Phe182, Tyr153, and Gly119. The high-resolution crystal structure of the creatininase-creatine complex enables us to identify two water molecules (Wat1 and Wat2) that are possibly essential for the catalytic mechanism of the enzyme. The structure and proposed catalytic mechanism of the creatininase are different from those of urease-related amidohydrolase superfamily enzymes. We propose a new two-step catalytic mechanism possibly common to creatininases in which the Wat1 acts as the attacking nucleophile in the water-adding step and the Wat2 acts as the catalytic acid in the ring-opening step.     

Crystal structures of the complexes of trichosanthin with four substrate analogs and catalytic mechanism of RNA N-glycosidase

Four substrate analogs-nicotinamide adenine dinucleotide, adenylyl (3', 5') guanosine, guanylyl (3',5') adenosine, and adenosine 2', 5'-diphosphate-have been used to prepare the complexes with trichosanthin (TCS), a type I ribosome-inactivating protein that possesses the activity of N-glycosidase. The crystal structures of the complexes have been determined and refined at high resolution. The refined structures show that the N-glycosidic bonds of all the four substrate analogues are hydrolyzed and a common structure is shared by the four complexes, in which only adenine, the product of the enzymatic reaction, is bound in the active center. The structure is compared with those of native trichosanthin and a previously reported trichosanthin-NADPH complex in which the N-glycosidic bond is uncleaved. The structural comparison shows that the conformation of Tyr70 obviously differs from those in the latter two structures, i.e., the side chain of Tyr70 is rotated along its Cbeta-Cgamma bond by approximately 70 degrees. The water molecule found to be preassociated with the N-glycosidic bond in the TCS-NADPH complex structure and proposed to be the water candidate responsible for hydrolyzing the N-glycosidic bond disappears in the trichosanthin-product complex structure. Based on the comparison of the three structures representing the different stages of the enzymatic reaction, the catalytic mechanism of RNA N-glycosidase has been further elucidated. Proteins 2000;39:37-46.     

Crystal structure of the polyketide cyclase AknH with bound substrate and product analogue: implications for catalytic mechanism and product stereoselectivity

AknH is a small polyketide cyclase that catalyses the closure of the fourth carbon ring in aclacinomycin biosynthesis in Streptomyces galilaeus, converting aklanonic acid methyl ester to aklaviketone. The crystal structure analysis of this enzyme, in complex with substrate and product analogue, showed that it is closely related in fold and mechanism to the polyketide cyclase SnoaL that catalyses the corresponding reaction in the biosynthesis of nogalamycin. Similarity is also apparent at a functional level as AknH can convert nogalonic acid methyl ester, the natural substrate of SnoaL, to auraviketone in vitro and in constructs in vivo. Despite the conserved structural and mechanistic features between these enzymes, the reaction products of AknH and SnoaL are stereochemically distinct. Supplied with the same substrate, AknH yields a C9-R product, like most members of this family of polyketide cyclases, whereas the product of SnoaL has the opposite C9-S stereochemistry. A comparison of high-resolution crystal structures of the two enzymes combined with in vitro mutagenesis studies revealed two critical amino acid substitutions in the active sites, which contribute to product stereoselectivity in AknH. Replacement of residues Tyr15 and Asn51 of AknH, located in the vicinity of the main catalytic residue Asp121, by their SnoaL counter-parts phenylalanine and leucine, respectively, results in a complete loss of product stereoselectivity.     

Crystal structures of multidrug-resistant HIV-1 protease in complex with two potent anti-malarial compounds

Two potent inhibitors (compounds 1 and 2) of malarial aspartyl protease, plasmepsin-II, were evaluated against wild type (NL4-3) and multidrug-resistant clinical isolate 769 (MDR) variants of human immunodeficiency virus type-1 (HIV-1) aspartyl protease. Enzyme inhibition assays showed that both 1 and 2 have better potency against NL4-3 than against MDR protease. Crystal structures of MDR protease in complex with 1 and 2 were solved and analyzed. Crystallographic analysis revealed that the MDR protease exhibits a typical wide-open conformation of the flaps (Gly48 to Gly52) causing an overall expansion in the active site cavity, which, in turn caused unstable binding of the inhibitors. Due to the expansion of the active site cavity, both compounds showed loss of direct contacts with the MDR protease compared to the docking models of NL4-3. Multiple water molecules showed a rich network of hydrogen bonds contributing to the stability of the ligand binding in the distorted binding pockets of the MDR protease in both crystal structures. Docking analysis of 1 and 2 showed a decrease in the binding affinity for both compounds against MDR supporting our structure-function studies. Thus, compounds 1 and 2 show promising inhibitory activity against HIV-1 protease variants and hence are good candidates for further development to enhance their potency against NL4-3 as well as MDR HIV-1 protease variants.     

Molecular basis for substrate recognition and drug resistance from 1.1 to 1.6 angstroms resolution crystal structures of HIV-1 protease mutants with substrate analogs

HIV-1 protease (PR) and two drug-resistant variants--PR with the V82A mutation (PR(V82A)) and PR with the I84V mutation (PR(I84V))--were studied using reduced peptide analogs of five natural cleavage sites (CA-p2, p2-NC, p6pol-PR, p1-p6 and NC-p1) to understand the structural and kinetic changes. The common drug-resistant mutations V82A and I84V alter residues forming the substrate-binding site. Eight crystal structures were refined at resolutions of 1.10-1.60 A. Differences in the PR-analog interactions depended on the peptide sequence and were consistent with the relative inhibition. Analog p6(pol)-PR formed more hydrogen bonds of P2 Asn with PR and fewer van der Waals contacts at P1' Pro compared with those formed by CA-p2 or p2-NC in PR complexes. The P3 Gly in p1-p6 provided fewer van der Waals contacts and hydrogen bonds at P2-P3 and more water-mediated interactions. PR(I84V) showed reduced van der Waals interactions with inhibitor compared with PR, which was consistent with kinetic data. The structures suggest that the binding affinity for mutants is modulated by the conformational flexibility of the substrate analogs. The complexes of PR(V82A) showed smaller shifts of the main chain atoms of Ala82 relative to PR, but more movement of the peptide analog, compared to complexes with clinical inhibitors. PR(V82A) was able to compensate for the loss of interaction with inhibitor caused by mutation, in agreement with kinetic data, but substrate analogs have more flexibility than the drugs to accommodate the structural changes caused by mutation. Hence, these structures help to explain how HIV can develop drug resistance while retaining the ability of PR to hydrolyze natural substrates.     

Evaluation of the substrate envelope hypothesis for inhibitors of HIV-1 protease

Crystallographic data show that various substrates of HIV protease occupy a remarkably uniform region within the binding site; this region has been termed the substrate envelope. It has been suggested that an inhibitor that fits within the substrate envelope should tend to evade viral resistance because a protease mutation that reduces the affinity of the inhibitor will also tend to reduce the affinity of substrate, and will hence decrease the activity of the enzyme. Accordingly, inhibitors that fit the substrate envelope better should be less susceptible to clinically observed resistant mutations, since these must also allow substrates to bind. The present study describes a quantitative measure of the volume of a bound inhibitor falling outside the substrate envelope, and observes that this quantity correlates with the inhibitor's losses in affinity to clinically relevant mutants. This measure may thus be useful as a penalty function in the design of robust HIV protease inhibitors.     

The evolution of catalytic function in the HIV-1 protease

The evolution of species is a complex phenomenon based on the optimization of a multidimensional function referred to as fitness. At the level of biomolecular evolution, the fitness function can be reduced to include physiochemical properties relevant to the biological function of a particular molecule. In this work, questions involving the physical-chemical mechanisms underlying the evolution of HIV-1 protease are addressed through molecular simulation and subsequent analysis of thermodynamic properties related to the activity of the enzyme. Specifically, the impact of 40 single amino acid mutations on the binding affinity toward the matrix/capsid (MA/CA) substrate and corresponding transition state intermediate has been characterized using a molecular mechanics Poisson-Boltzmann surface area approach. We demonstrate that this approach is capable of extracting statistically significant information relevant to experimentally determined catalytic activity. Further, no correlation was observed between the effect of mutations on substrate and transition state binding, suggesting independent evolutionary pathways toward optimizing substrate specificity and catalytic activity. In addition, a detailed analysis of calculated binding affinity data suggests that ground-state destabilization (reduced binding affinity for the substrate) could be a contributing factor in the evolutionary optimization of HIV-1 protease. A numerical model is developed to demonstrate that ground-state destabilization is a valid mechanism for activity optimization given the high concentrations of substrate experienced by the functional enzyme in vivo.     

Structural implications of drug-resistant mutants of HIV-1 protease: high-resolution crystal structures of the mutant protease/substrate analogue complexes

Emergence of drug-resistant mutants of HIV-1 protease is an ongoing problem in the fight against AIDS. The mechanisms governing resistance are both complex and varied. We have determined crystal structures of HIV-1 protease mutants, D30N, K45I, N88D, and L90M complexed with peptide inhibitor analogues of CA-p2 and p2-NC cleavage sites in the Gag-pol precursor in order to study the structural mechanisms underlying resistance. The structures were determined at 1.55-1.9-A resolution and compared with the wild-type structure. The conformational disorder seen for most of the hydrophobic side-chains around the inhibitor binding site indicates flexibility of binding. Eight water molecules are conserved in all 9 structures; their location suggests that they are important for catalysis as well as structural stability. Structural differences among the mutants were analyzed in relation to the observed changes in protease activity and stability. Mutant L90M shows steric contacts with the catalytic Asp25 that could destabilize the catalytic loop at the dimer interface, leading to its observed decreased dimer stability and activity. Mutant K45I reduces the mobility of the flap and the inhibitor and contributes to an enhancement in structural stability and activity. The side-chain variations at residue 30 relative to wild-type are the largest in D30N and the changes are consistent with the altered activity observed with peptide substrates. Polar interactions in D30N are maintained, in agreement with the observed urea sensitivity. The side-chains of D30N and N88D are linked through a water molecule suggesting correlated changes at the two sites, as seen with clinical inhibitors. Structural changes seen in N88D are small; however, water molecules that mediate interactions between Asn88 and Thr74/Thr31/Asp30 in other complexes are missing in N88D.     

Crystal structures of a ligand-free and malonate-bound human caspase-1: implications for the mechanism of substrate binding

Caspase-1, a mediator of the posttranslational processing of IL-1beta and IL-18, requires an aspartic acid in the P1 position of its substrates. The mechanisms of caspase-1 activation remain poorly understood despite numerous structures of the enzyme complexed with aspartate-based inhibitors. Here we report a crystal structure of ligand-free caspase-1 that displays dramatic rearrangements of loops defining the active site to generate a closed conformation that is incompatible with substrate binding. A structure of the enzyme complexed with malonate shows the protein in its open (active-site ligand-bound) conformation in which malonate reproduces the hydrogen bonding network observed in structures with covalent inhibitors. These results illustrate the essential function of the obligatory aspartate recognition element that opens the active site of caspase-1 to substrates and may be the determinant responsible for the conformational changes between ligand-free and -bound forms of the enzyme, and suggest a new approach for identifying novel aspartic acid mimetics.     

Curling of flap tips in HIV-1 protease as a mechanism for substrate entry and tolerance of drug resistance

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) protease is an essential viral protein that is a major drug target in the fight against Acquired Immune Deficiency Syndrome (AIDS). Access to the active site of this homodimeric enzyme is gained when two large flaps, one from each monomer, open. The flap movements are therefore central to the function of the enzyme, yet determining how these flaps move at an atomic level has not been experimentally possible. RESULTS: In the present study, we observe the flaps of HIV-1 protease completely opening during a 10 ns solvated molecular dynamics simulation starting from the unliganded crystal structure. This movement is on the time scale observed by Nuclear Magnetic Resonance (NMR) relaxation data. The highly flexible tips of the flaps, with the sequence Gly-Gly-Ile-Gly-Gly, are seen curling back into the protein and thereby burying many hydrophobic residues. CONCLUSIONS: This curled-in conformational change has never been previously described. Previous models of this movement, with the flaps as rigid levers, are not consistent with the experimental data. The residues that participate in this hydrophobic cluster as a result of the conformational change are highly sensitive to mutation and often contribute to drug resistance when they do change. However, several of these residues are not part of the active site cavity, and their essential role in causing drug resistance could possibly be rationalized if this conformational change actually occurs. Trapping HIV-1 protease in this inactive conformation would provide a unique opportunity for future drug design.     

The crystal structures of phosphopantetheine adenylyltransferase with bound substrates reveal the enzyme's catalytic mechanism.

Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme in the coenzyme A pathway that catalyzes the reversible transfer of an adenylyl group from ATP to 4'-phosphopantetheine (Ppant) in the presence of magnesium. To investigate the reaction mechanism, the high-resolution crystal structures of the Escherichia coli PPAT have been determined in the presence of either ATP or Ppant. Structural details of the catalytic center revealed specific roles for individual amino acid residues involved in substrate binding and catalysis. The side-chain of His18 stabilizes the expected pentacovalent intermediate, whereas the side-chains of Thr10 and Lys42 orient the nucleophile for an in-line displacement mechanism. The binding site for the manganese ion that interacts with the phosphate groups of the nucleotide has also been identified. Within the PPAT hexamer, one trimer is in its substrate-free state, whereas the other is in a substrate-bound state.     

Substrate shape determines specificity of recognition for HIV-1 protease: analysis of crystal structures of six substrate complexes

The homodimeric HIV-1 protease is the target of some of the most effective antiviral AIDS therapy, as it facilitates viral maturation by cleaving ten asymmetric and nonhomologous sequences in the Gag and Pol polyproteins. Since the specificity of this enzyme is not easily determined from the sequences of these cleavage sites alone, we solved the crystal structures of complexes of an inactive variant (D25N) of HIV-1 protease with six peptides that correspond to the natural substrate cleavage sites. When the protease binds to its substrate and buries nearly 1000 A2 of surface area, the symmetry of the protease is broken, yet most internal hydrogen bonds and waters are conserved. However, no substrate side chain hydrogen bond is conserved. Specificity of HIV-1 protease appears to be determined by an asymmetric shape rather than a particular amino acid sequence.     

Proteolytic processing of HIV-1 protease precursor, kinetics and mechanism.

Previously it was demonstrated using a model precursor that processing at the N terminus of the HIV-1 protease (PR) precedes processing at its C terminus. We now show the expression, purification, and kinetics of the autoprocessing reaction of a PR precursor linked to 53 amino acids of the native flanking transframe region (DeltaTFP-p6(pol)) of Gag-Pol and containing its two native cleavage sites. The PR contains the two cysteine residues exchanged to alanines, mutations that do not alter the kinetics or the structural stability of the mature PR. DeltaTFP-p6(pol)-PR, which encompasses the known PR inhibitor sequence Glu-Asp-Leu within DeltaTFP, undergoes cleavage at the DeltaTFP/p6(pol) and p6(pol)/PR sites in two consecutive steps to produce the mature PR. Both DeltaTFP-p6(pol)-PR and p6(pol)-PR exhibit low intrinsic enzymatic activity. The appearance of the mature PR is accompanied by a large increase in catalytic activity. It follows first-order kinetics in protein concentration with a rate constant of 0.13 +/- 0.01 min(-1) in 0.1 M acetate at pH 4.8. The pH-rate profile for the observed first-order rate constant is bell-shaped with two ionizable groups of pK(a) 4.9 and 5.1. The rate constant also exhibits approximately 7-fold higher sensitivity to urea denaturation as compared with that of the mature PR, suggesting that the cleavage at the N terminus of the PR domain from the precursor leads to the stabilization of the dimeric structure.     

Crystal structures of homoserine dehydrogenase suggest a novel catalytic mechanism for oxidoreductases

The structure of the antifungal drug target homoserine dehydrogenase (HSD) was determined from Saccharomyces cerevisiae in apo and holo forms, and as a ternary complex with bound products, by X-ray diffraction. The three forms show that the enzyme is a dimer, with each monomer composed of three regions, the nucleotide-binding region, the dimerization region and the catalytic region. The dimerization and catalytic regions have novel folds, whereas the fold of the nucleotide-binding region is a variation on the Rossmann fold. The novel folds impose a novel composition and arrangement of active site residues when compared to all other currently known oxidoreductases. This observation, in conjunction with site-directed mutagenesis of active site residues and steady-state kinetic measurements, suggest that HSD exhibits a new variation on dehydrogenase chemistry.     

Crystal structures of Pasteurella multocida sialyltransferase complexes with acceptor and donor analogues reveal substrate binding sites and catalytic mechanism

Sialyltransferases are key enzymes involved in the biosynthesis of biologically and pathologically important sialic acid-containing molecules in nature. Binary X-ray crystal structures of a multifunctional Pasteurella multocida sialyltransferase (Delta24PmST1) with a donor analogue CMP-3F(a)Neu5Ac or CMP-3F(e)Neu5Ac were determined at 2.0 and 1.9 A resolutions, respectively. Ternary X-ray structures of the protein in complex with CMP or a donor analogue CMP-3F(a)Neu5Ac and an acceptor lactose have been determined at 2.0 and 2.27 A resolutions, respectively. This represents the first sialyltransferase structure and the first GT-B-type glycosyltransferase structure that is bound to both a donor analogue and an acceptor simultaneously. The four structures presented here reveal that binding of the nucleotide-activated donor sugar causes a buried tryptophan to flip out of the protein core to interact with the donor sugar and helps define the acceptor sugar binding site. Additionally, key amino acid residues involved in the catalysis have been identified. Structural and kinetic data support a direct displacement mechanism involving an oxocarbenium ion-like transition state assisted with Asp141 serving as a general base to activate the acceptor hydroxyl group.