Factors that affect oxygen activation and coupling of the two redox cycles in the aromatization reaction catalyzed by NikD, an unusual amino acid oxidase.

NikD is a flavoprotein oxidase that catalyzes the oxidation of piperideine-2-carboxylate (P2C) to picolinate in a remarkable aromatization reaction comprising two redox cycles and at least one isomerization step. Tyr258 forms part of an "aromatic cage" that surrounds the ring in picolinate and its precursors. Mutation of Tyr258 to Phe does not perturb the structure of nikD but does affect the coupling of the two redox cycles and causes a 10-fold decrease in turnover rate. Tyr258Phe catalyzes a quantitative two-electron oxidation of P2C, but only 60% of the resulting dihydropicolinate intermediate undergoes a second redox cycle to produce picolinate. The mutation does not affect product yield with an alternate substrate (3,4-dehydro-L-proline) that is aromatized in a single two-electron oxidation step. Wild-type and mutant enzymes exhibit identical rate constants for oxidation of P2C to dihydropicolinate and isomerization of a reduced enzyme.dihydropicolinate complex. The observed rates are 200- and 10-fold faster, respectively, than the mutant turnover rate. Release of picolinate from Tyr258Phe is 100-fold faster than turnover. The presence of a bound substrate or product is a key factor in oxygen activation by wild-type nikD, as judged by the 10-75-fold faster rates observed for complexes of the reduced enzyme with picolinate, benzoate, or 1-cyclohexenoate, a 1-deaza-P2C analogue. The reduced Tyr258Phe x 1-cyclohexenoate complex is 25-fold less reactive with oxygen than the wild-type complex. We postulate that mutation of Tyr258 causes subtle changes in active site dynamics that promote release of the reactive dihydropicolinate intermediate and disrupt the efficient synchronization of oxygen activation observed with wild-type nikD.

NikD, an unusual amino acid oxidase essential for nikkomycin biosynthesis: structures of closed and open forms at 1.15 and 1.90 A resolution.

NikD is an unusual amino-acid-oxidizing enzyme that contains covalently bound FAD, catalyzes a 4-electron oxidation of piperideine-2-carboxylic acid to picolinate, and plays a critical role in the biosynthesis of nikkomycin antibiotics. Crystal structures of closed and open forms of nikD, a two-domain enzyme, have been determined to resolutions of 1.15 and 1.9 A, respectively. The two forms differ by an 11 degrees rotation of the catalytic domain with respect to the FAD-binding domain. The active site is inaccessible to solvent in the closed form; an endogenous ligand, believed to be picolinate, is bound close to and parallel with the flavin ring, an orientation compatible with redox catalysis. The active site is solvent accessible in the open form, but the picolinate ligand is approximately perpendicular to the flavin ring and a tryptophan is stacked above the flavin ring. NikD also contains a mobile cation binding loop.

High resolution crystal structures of free thrombin in the presence of K(+) reveal the molecular basis of monovalent cation selectivity and an inactive slow form.

Structural biology has recently advanced our understanding of the molecular mechanisms of activation and selectivity in monovalent cation activated enzymes. Here we report a 1.9 Angstrom resolution crystal structure of free thrombin, a Na(+) selective enzyme, in the presence of KCl. There are two molecules in the asymmetric unit, one with the cation site bound to K(+) and the other with this site free. The K(+)-bound form shows key differences compared with the Na(+)-bound structure that explain the different kinetics of activation. The cation-free form, on the other hand, assumes a conformation where the monovalent cation binding site is completely disordered, the S1 pocket is inaccessible to substrate and binding to exosite I is compromised by an unprecedented >20 Angstrom shift in the position of the autolysis loop. This form, named S(*), corresponds to the inactive Na(+)-free slow form identified by early kinetic studies. A simple model of thrombin allostery that incorporates the contribution of S(*) is proposed.

Stabilization of the E* form turns thrombin into an anticoagulant.

Previous studies have shown that deletion of nine residues in the autolysis loop of thrombin produces a mutant with an anticoagulant propensity of potential clinical relevance, but the molecular origin of the effect has remained unresolved. The x-ray crystal structure of this mutant solved in the free form at 1.55 A resolution reveals an inactive conformation that is practically identical (root mean square deviation of 0.154 A) to the recently identified E* form. The side chain of Trp(215) collapses into the active site by shifting > 10 A from its position in the active E form, and the oxyanion hole is disrupted by a flip of the Glu(192)-Gly(193) peptide bond. This finding confirms the existence of the inactive form E* in essentially the same incarnation as first identified in the structure of the thrombin mutant D102N. In addition, it demonstrates that the anticoagulant profile often caused by a mutation of the thrombin scaffold finds its likely molecular origin in the stabilization of the inactive E* form that is selectively shifted to the active E form upon thrombomodulin and protein C binding.

Engineering protein allostery: 1.05 A resolution structure and enzymatic properties of a Na+-activated trypsin.

Some trypsin-like proteases are endowed with Na(+)-dependent allosteric enhancement of catalytic activity, but this important mechanism has been difficult to engineer in other members of the family. Replacement of 19 amino acids in Streptomyces griseus trypsin targeting the active site and the Na(+)-binding site were found necessary to generate efficient Na(+) activation. Remarkably, this property was linked to the acquisition of a new substrate selectivity profile similar to that of factor Xa, a Na(+)-activated protease involved in blood coagulation. The X-ray crystal structure of the mutant trypsin solved to 1.05 A resolution defines the engineered Na(+) site and active site loops in unprecedented detail. The results demonstrate that trypsin can be engineered into an efficient allosteric protease, and that Na(+) activation is interwoven with substrate selectivity in the trypsin scaffold.

A single methionine residue dictates the kinetic mechanism of interprotein electron transfer from methylamine dehydrogenase to amicyanin.

Amicyanin is a type 1 copper protein that is the natural electron acceptor for the quinoprotein methylamine dehydrogenase (MADH). A P52G amicyanin mutation increased the Kd for complex formation and caused the normally true electron transfer (ET) reaction from O-quinol MADH to amicyanin to become a gated ET reaction (Ma, J. K., Carrell, C. J., Mathews, F. S., and Davidson, V. L. (2006) Biochemistry 45, 8284-8293). One consequence of the P52G mutation was to reposition the side chain of Met51, which is present at the MADH-amicyanin interface. To examine the precise role of Met51 in this interprotein ET reaction, Met51 was converted to Ala, Lys, and Leu. The Kd for complex formation of M51A amicyanin was unchanged but the experimentally determined electronic coupling increased from 12 cm-1 to 142 cm-1, and the reorganization energy increased from 2.3 to 3.1 eV. The rate and salt dependence of the proton transfer-gated ET reaction from N-quinol MADH to amicyanin is also changed by the M51A mutation. These changes in ET parameters and rates for the reactions with M51A amicyanin were similar to those caused by the P52G mutation and indicated that the ET reaction had become gated by a similar process, most likely a conformational rearrangement of the protein ET complex. The results of the M51K and M51L mutations also have consequences on the kinetic mechanism of regulation of the interprotein ET with effects that are intermediate between what is observed for the reaction of the native amicyanin and M51A amicyanin. These data indicate that the loss of the interactions involving Pro52 were primarily responsible for the change in Kd for P52G amicyanin, while the interactions involving the Met51 side chain are entirely responsible for the change in ET parameters and conversion of the true ET reaction of native amicyanin into a conformationally gated ET reaction.

Generation of novel copper sites by mutation of the axial ligand of amicyanin. Atomic resolution structures and spectroscopic properties.

Amicyanin from Paracoccus denitrificans is a type 1 copper protein with three strong equatorial copper ligands provided by nitrogens of His53 and His95 and the sulfur of Cys92, with an additional weak axial ligand provided by the sulfur of Met98. Met98 was replaced with either Gln or Ala. As isolated, the M98A and M98Q mutant proteins contain zinc in the active site. The zinc is then removed and replaced with copper so that the copper-containing proteins may be studied. Each of the mutant amicyanins exhibits a marked decrease in thermal stability relative to that of native amicyanin, consistent with the weaker affinity for copper. Crystal structures were obtained for the oxidized and reduced forms of M98A and M98Q amicyanins at atomic resolution (

Site-directed mutagenesis of proline 52 to glycine in amicyanin converts a true electron transfer reaction into one that is conformationally gated.

Amicyanin is a type I copper protein that is the natural electron acceptor for the quinoprotein methylamine dehydrogenase (MADH). The conversion of Proline52 of amicyanin to a glycine does not alter the physical and spectroscopic properties of the copper binding site, but it does alter the rate of electron transfer (ET) from MADH. The values of electronic coupling (H(AB)) and reorganization energy (lambda) that are associated with the true ET reaction from the reduced O-quinol tryptophan tryptophylquinone (TTQ) of MADH to oxidized amicyanin are significantly altered as a consequence of the P52G mutation. The experimentally determined H(AB) increases from 12 to 78 cm(-1), and lambda increases from 2.3 to 2.8 eV. The rate and salt-dependence of the proton transfer-gated ET reaction from N-quinol MADH to amicyanin are also changed by the P52G mutation. Kinetic data suggests that a new common reaction step has become rate-limiting for both the true and gated ET reactions that occur from different redox forms of MADH. A comparison of the crystal structures of P52G amicyanin with those of native amicyanin free and in complex with MADH provided clues as to the basis for the change in ET parameters. The mutation results in the loss of three carbons from Pro52 and the movement of the neighboring residue Met51. This reduces the number of hydrophobic interactions with MADH in the complex and perturbs the protein-protein interface. A model is proposed for the ET reaction with P52G amicyanin in which the most stable conformation of the protein-protein complex with MADH is not optimal for ET. A new preceding kinetic step is introduced prior to true ET that requires P52G amicyanin to switch from this redox-inactive stable complex to a redox-active unstable complex. Thus, the ET reaction of P52G amicyanin is no longer a true ET but one that is conformationally gated by the reorientation of the proteins within the ET protein complex. This same reaction step now also gates the ET from N-quinol MADH, which is normally rate-limited by a proton transfer.

Thrombin functions through its RGD sequence in a non-canonical conformation.

Previous studies have suggested that thrombin interacts with integrins in endothelial cells through its RGD (Arg-187, Gly-188, Asp-189) sequence. All existing crystal structures of thrombin show that most of this sequence is buried under the 220-loop and therefore interaction via RGD implies either partial unfolding of the enzyme or its proteolytic digestion. Here, we demonstrate that surface-absorbed thrombin promotes attachment and migration of endothelial cells through interaction with alpha(v)beta(3) and alpha(5)beta(1) integrins. Using site-directed mutants of thrombin we prove that this effect is mediated by the RGD sequence and does not require catalytic activity. The effect is abrogated when residues of the RGD sequence are mutated to Ala and is not observed with proteases like trypsin and tissue-type plasminogen activator, unless the RGD sequence is introduced at position 187-189. The potent inhibitor hirudin does not abrogate the effect, suggesting that thrombin functions through its RGD sequence in a non-canonical conformation. A 1.9-Angstroms resolution crystal structure of free thrombin grown in the presence of high salt (400 mm KCl) shows two molecules in the asymmetric unit, one of which assumes an unprecedented conformation with the autolysis loop shifted 20 Angstroms away from its canonical position, the 220-loop entirely disordered, and the RGD sequence exposed to the solvent.

Structural studies of two mutants of amicyanin from Paracoccus denitrificans that stabilize the reduced state of the copper.

Mutation of Pro94 to phenylalanine or alanine significantly alters the redox properties of the type I copper center of amicyanin. Each mutation increases the redox midpoint potential (E(m)) value by at least 140 mV and shifts the pK(a) for the pH dependence of the E(m) value to a more acidic value. Atomic resolution (0.99-1.1 A) structures of both the P94F and P94A amicyanin have been determined in the oxidized and reduced states. In each amicyanin mutant, an electron-withdrawing hydrogen bond to the copper-coordinating thiolate sulfur of Cys92 is introduced by movement of the amide nitrogens of Phe94 and Ala94 much closer to the thiolate sulfur than in wild-type amicyanin. This is the likely explanation for the much more positive E(m) values which result from each of these mutations. The observed decrease in the pK(a) value for the pH dependence of the E(m) value that is seen in the mutants seems to be correlated with steric hindrance to the rotation of the His95 copper ligand which results from the mutations. In wild-type amicyanin the His95 side chain undergoes a redox and pH-dependent conformational change which accounts for the pH dependence of the E(m) value of amicyanin. The reduced P94A amicyanin exhibits two alternate conformations with the positions of the copper 1.4 A apart. In one of these conformations, a water molecule appears to have replaced Met98 as a copper ligand. The relevance of these structures to the electron transfer properties of P94F and P94A amicyanin are also discussed.

Crystallographic and NMR investigation of cobalt-substituted amicyanin.

Cobalt(II) amicyanin was prepared by replacing the copper of the type I copper protein amicyanin from Paracoccus denitrificans with cobalt. The structure of the protein and the metal center have been characterized by X-ray crystallography and paramagnetic NMR spectroscopy. The crystal structure indicates that Met98, which provides an axial sulfur ligand in native amicyanin, is no longer bound to the metal in cobalt(II) amicyanin and that a water molecule is recruited from solvent to form the fourth metal ligand. This results in a tetrahedral coordination geometry for the cobalt ion. NMR studies in solution also indicate that the side chain of the methionine residue interacts less strongly with the metal in P. denitrificans amicyanin than in Paracoccus versutus amicyanin. The cobalt(II) amicyanin crystal structure is different from that of cobalt-substituted azurin in which the carbonyl of a glycine residue provides this equivalent ligand. In cobalt(II) amicyanin that residue is a proline, for which the oxygen is structurally inaccessible, so that the water occupies the position held by the glycine carbonyl in cobalt(II) azurin. Such a metal coordination involving water has not previously been reported for a native or metal-substituted type I copper protein.

Molecular dissection of Na+ binding to thrombin.

Na(+) binding near the primary specificity pocket of thrombin promotes the procoagulant, prothrombotic, and signaling functions of the enzyme. The effect is mediated allosterically by a communication between the Na(+) site and regions involved in substrate recognition. Using a panel of 78 Ala mutants of thrombin, we have mapped the allosteric core of residues that are energetically linked to Na(+) binding. These residues are Asp-189, Glu-217, Asp-222, and Tyr-225, all in close proximity to the bound Na(+). Among these residues, Asp-189 shares with Asp-221 the important function of transducing Na(+) binding into enhanced catalytic activity. None of the residues of exosite I, exosite II, or the 60-loop plays a significant role in Na(+) binding and allosteric transduction. X-ray crystal structures of the Na(+)-free (slow) and Na(+)-bound (fast) forms of thrombin, free or bound to the active site inhibitor H-d-Phe-Pro-Arg-chloromethyl-ketone, document the conformational changes induced by Na(+) binding. The slow --> fast transition results in formation of the Arg-187:Asp-222 ion pair, optimal orientation of Asp-189 and Ser-195 for substrate binding, and a significant shift of the side chain of Glu-192 linked to a rearrangement of the network of water molecules that connect the bound Na(+) to Ser-195 in the active site. The changes in the water network and the allosteric core explain the thermodynamic signatures linked to Na(+) binding and the mechanism of thrombin activation by Na(+). The role of the water network uncovered in this study establishes a new paradigm for the allosteric regulation of thrombin and other Na(+)-activated enzymes involved in blood coagulation and the immune response.