The two steps of group II intron self-splicing are mechanistically distinguishable.

The two transesterification reactions catalyzed by self-splicing group II introns take place in either two active sites or two conformations of a single active site involving rearrangements of the positions of the reacting groups. We have investigated the effects on the rates of the chemical steps of the two reactions due to sulfur substitution of nonbridging oxygens at both the 5' and 3' splice sites as well as the deoxyribose substitution of the ribose 2' hydroxyl group at the 5' splice site. The data suggest that the two active sites differ in their interactions with several of these groups. Specifically, sulfur substitution of the pro-Sp nonbridging oxygen at the 5' splice site reduces the chemical rate of the step one branching reaction by at least 250-fold, whereas substitution of the pro-Sp oxygen at the 3' splice site has only a 4.5-fold effect on the chemical rate of step two. Previous work demonstrated that the Rp phosphorothioate substitutions at both the 5' and 3' splice sites reduced the rate of both steps of splicing to an undetectable level. These results suggest that either two distinct active sites catalyze the two steps or that more significant alterations must be made in a single bifunctional active site to accommodate the two different reactions.     

DNA cleavage by EcoRV endonuclease: two metal ions in three metal ion binding sites

Four crystal structures of EcoRV endonuclease mutants K92A and K38A provide new insight into the mechanism of DNA bending and the structural basis for metal-dependent phosphodiester bond cleavage. The removal of a key active site positive charge in the uncleaved K92A-DNA-M(2+) substrate complex results in binding of a sodium ion in the position of the amine nitrogen, suggesting a key role for a positive charge at this position in stabilizing the sharp DNA bend prior to cleavage. By contrast, two structures of K38A cocrystallized with DNA and Mn(2+) ions in different lattice environments reveal cleaved product complexes featuring a common, novel conformation of the scissile phosphate group as compared to all previous EcoRV structures. In these structures, the released 5'-phosphate and 3'-OH groups remain in close juxtaposition with each other and with two Mn(2+) ions that bridge the conserved active site carboxylates. The scissile phosphates are found midway between their positions in the prereactive substrate and postreactive product complexes of the wild-type enzyme. Mn(2+) ions occupy two of the three sites previously described in the prereactive complexes and are plausibly positioned to generate the nucleophilic hydroxide ion, to compensate for the incipient additional negative charge in the transition state, and to ionize a second water for protonation of the 3'-oxyanion. Reconciliation of these findings with earlier X-ray and fluorescence studies suggests a novel mechanism in which a single initially bound metal ion in a third distinct site undergoes a shift in position together with movement of the scissile phosphate deeper into the active site cleft. This reconfigures the local environment to permit binding of the second metal ion followed by movement toward the pentacovalent transition state. The new mechanism suggested here embodies key features of previously proposed two- and three-metal catalytic models, and offers a view of the stereochemical pathway that integrates much of the copious structural and functional data that are available from exhaustive studies in many laboratories.     

Localization of the ATP-binding site in the 23-kDa and 20-kDa regions of the heavy chain of the skeletal muscle myosin head

Three kinds of ATP analogues were synthesized. These ATP analogues can be classified into two conformations, i.e. syn and anti forms with respect to the N-glycosidic bond between adenine and ribose groups of ATP. 3'-O-(N-Methylanthraniloyl)-2-azidoadenosine 5'-triphosphate (MantN2(3)ATP) is recognized as the anti form, as ATP, and the other two, 3'-O-(N-methylanthraniloyl)-8-azidoadenosine 5'-triphosphate (MantN8(3)ATP) and 1,N6-etheno-8-azidoadenosine 5'-triphosphate (epsilon N8(3)ATP) are both syn forms. Mant and etheno groups are both fluorescent which allows detection of their binding to proteins. The photochemical binding of azido groups in ATP analogues to the myosin active site, examined in the presence and absence of ATP, showed that all the analogues bound to the site of myosin ATPase. These analogues also acted as substrates of the ATPase and were hydrolyzed in the active site, as judged by competitive inhibition of the ATPase and by their ATPase activities. Of these analogues, MantN2(3)ATP is very similar to ATP in divalent-cation dependence of its hydrolysis rate and in its ability to trap ADP in the active site with vanadate, while the other two are different from ATP in these respects. The photochemical binding sites of ATP analogues were localized by gel electrophoresis of trypsinized myosin ATPase with photocross-linked ATP analogues and/or by isolating the modified peptides. MantN2(3)ATP was found in the 23-kDa fragment which has a structure common to ATP-binding proteins, i.e. Gly-Xaa-Xaa-Gly-Xaa-Gly-Lys-Thr. Mant N8(3)ATP was found in a region of the 20-kDa fragment where actin is reported to attach.     

The active site architecture of Pisum sativum beta-carbonic anhydrase is a mirror image of that of alpha-carbonic anhydrases

We have determined the structure of the beta-carbonic anhydrase from the dicotyledonous plant Pisum sativum at 1.93 A resolution, using a combination of multiple anomalous scattering off the active site zinc ion and non-crystallographic symmetry averaging. The mol- ecule assembles as an octamer with a novel dimer of dimers of dimers arrangement. Two distinct patterns of conservation of active site residues are observed, implying two potentially mechanistically distinct classes of beta-carbonic anhydrases. The active site is located at the interface between two monomers, with Cys160, His220 and Cys223 binding the catalytic zinc ion and residues Asp162 (oriented by Arg164), Gly224, Gln151, Val184, Phe179 and Tyr205 interacting with the substrate analogue, acetic acid. The substrate binding groups have a one to one correspondence with the functional groups in the alpha-carbonic anhydrase active site, with the corresponding residues being closely superimposable by a mirror plane. Therefore, despite differing folds, alpha- and beta-carbonic anhydrase have converged upon a very similar active site design and are likely to share a common mechanism.     

Peroxiredoxin functions as a peroxidase and a regulator and sensor of local peroxides

Peroxiredoxins (Prxs) contain an active site cysteine that is sensitive to oxidation by H(2)O(2). Mammalian cells express six Prx isoforms that are localized to various cellular compartments. The oxidized active site cysteine of Prx can be reduced by a cellular thiol, thus enabling Prx to function as a locally constrained peroxidase. Regulation of Prx via phosphorylation in response to extracellular signals allows the local accumulation of H(2)O(2) and thereby enables its messenger function. The fact that the oxidation state of the active site cysteine of Prx can be transferred to other proteins that are less intrinsically susceptible to H(2)O(2) also allows Prx to function as an H(2)O(2) sensor.     

Study of active site topography of rat liver mitochondrial dicarboxylate transporter using lipophilic substrate derivatives

Earlier it has been demonstrated that the active site (substrate-binding site + active site channel) of rat liver mitochondrial dicarboxylate transporter is characterized by rather complex topography. Probing the active site with 2-monoalkylmalonates revealed the existence of internal and external lipophilic areas separated by a polar region. A two substrate-binding site model of the transporter has been supposed. The correctness of this model has been evaluated by probing the active site with O-acyl-L-malates differing from 2-monoalkylmalonates by 0.23 nm longer distance from the anion groups to the aliphatic chain. Changes in the polar group of the probe did not prevent its binding and showed the same variable lipophilicity pattern for the transporter channel. Probing with alpha,omega-alkylene dimalonates did not reveal the second substrate-binding site at the active site. The substrate-binding site did not show any differences in affinity to O-acyl-derivatives of L-malate and D-malate, except L-malate binds more effectively than D-malate. This suggests involvement of the L-malate hydroxyl group in substrate binding and stereospecific behavior of the transporter substrate-binding site. A modified one substrate-binding site model of the dicarboxylate transporter is discussed.     

Characterization of recombinant rat cathepsin B and nonglycosylated mutants expressed in yeast. New insights into the pH dependence of cathepsin B-catalyzed hydrolyses.

The cysteine proteinase rat cathepsin B was expressed in yeast in an active form and was found to be heterogeneously glycosylated at the consensus sequence for N-linked oligosaccharide substitution. Purified enzyme fractions containing the highest levels of glycosylation were shown to have reduced activity. A glycosylation minus mutant constructed by site-directed mutagenesis (by changing the Ser to Ala in the consensus sequence) was still secreted by the yeast and was shown to be functionally identical with purified rat liver cathepsin B. Recombinant cathepsin B was used to further characterize the pH dependence of cathepsin B-catalyzed hydrolyses using 7-amido-4-methylcoumarin (AMC) and p-nitroaniline (pNA) substrates with arginine as the P1, and either arginine or phenylalanine as the P2 residue. The AMC and pNA groups give insights into the leaving group binding site (P') of cathepsin B. These studies show for the first time that at least seven dissociable groups are involved in substrate binding and hydrolysis in cathepsin B activity. Two of these groups, with pKa values of 6.9 and 7.7 in the recombinant enzyme, are in the leaving group binding site and are most likely His110 and His111. The same groups in rat liver cathepsin B have higher pKa values than in recombinant cathepsin B, but have identical function in the two enzymes. Two other groups are probably the active site Cys29 and His199 with pKa values of 3.6 and 8.6, respectively. A group with a pKa of 5.1 interacts with substrates containing Arg at P2, and the group is most likely Glu245. The remaining two groups, one with a pKa of about 4.9 and the other about 5.3, are most likely carboxyl residues possibly interacting with Arg at P1 in the substrate. The possible candidates on the basis of the x-ray structure are Asp22, Asp69, Glu171, and Glu122, all found within a 13 A radius from the active site thiol of Cys29.     

Metal ions bound at the active site of the junction-resolving enzyme T7 endonuclease I

T7 endonuclease I is a nuclease that is selective for the structure of the four-way DNA junction. The active site is similar to those of a number of restriction enzymes. We have solved the crystal structure of endonuclease I with a wild-type active site. Diffusion of manganese ions into the crystal revealed two peaks of electron density per active site, defining two metal ion-binding sites. Site 1 is fully occupied, and the manganese ion is coordinated by the carboxylate groups of Asp55 and Glu65, and the main chain carbonyl of Thr66. Site 2 is partially occupied, and the metal ion has a single protein ligand, the remaining carboxylate oxygen atom of Asp55. Isothermal titration calorimetry showed the sequential exothermic binding of two manganese ions in solution, with dissociation constants of 0.58 +/- 0.019 and 14 +/- 1.5 mM. These results are consistent with a two metal ion mechanism for the cleavage reaction, in which the hydrolytic water molecule is contained in the first coordination sphere of the site 1-bound metal ion.     

Prostanoids contribute to cutaneous active vasodilation in humans

The specific mechanisms by which skin blood flow increases in response to a rise in core body temperature via cutaneous active vasodilation are poorly understood. The primary purpose of this study was to determine whether the cyclooxygenase (COX) pathway contributes to active vasodilation during whole body heat stress (protocol 1; n = 9). A secondary goal was to verify that the COX pathway does not contribute to the cutaneous hyperemic response during local heating (protocol 2; n = 4). For both protocols, four microdialysis fibers were placed in forearm skin. Sites were randomly assigned and perfused with 1) Ringer solution (control site); 2) ketorolac (KETO), a COX-1/COX-2 pathway inhibitor; 3) NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor; and 4) a combination of KETO and L-NAME. During the first protocol, active vasodilation was induced using whole body heating with water-perfused suits. The second protocol used local heaters to induce a local hyperemic response. Red blood cell flux (RBC flux) was indexed at all sites using laser-Doppler flowmetry, and cutaneous vascular conductance (CVC; RBC flux/mean arterial pressure) was normalized to maximal vasodilation at each site. During whole body heating, CVC values at sites perfused with KETO (43 +/- 9% CVCmax), L-NAME (35 +/- 9% CVCmax), and combined KETO/L-NAME (22 +/- 8% CVCmax) were significantly decreased with respect to the control site (59 +/- 7% CVCmax) (P < 0.05). Additionally, CVC at the combined KETO/L-NAME site was significantly decreased compared with sites infused with KETO or L-NAME alone (P < 0.05). In the second protocol, the hyperemic response to local heating did not differ between the control site and KETO site or between the L-NAME and KETO/L-NAME site. These data suggest that prostanoids contribute to active vasodilation, but do not play a role during local thermal hyperemia.     

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such “off-pathway” species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2′- and 3′-deoxy (–H) and −amino (–NH₂) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3′-OH making a nonproductive interaction with an active site metal ion termed M_A and with the adjacent 2′-OH making no interaction. Upon S binding, a rearrangement occurs that allows both –OH groups to contact a different active site metal ion, termed M_C, to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA''s difficulty in specifying a unique conformation and highlighting Nature''s potential to use local transitions of RNA in complex function.     

The role of zinc with special reference to the essential thiol groups in delta-aminolevulinic acid dehydratase of bovine liver.

delta-Aminolevulinic acid dehydratase (5-aminolevulinic acid hydro-lyase (adding 5-aminolevulinic acid and cyclizing), EC 4.2.1.24 purified from bovine liver in the presence of both SH-reducing reagent and zinc during the purification contained one zinc atom and eight SH groups/subunit. This preparation showed the full enzymatic activity even in the absence of thiol activator. It was found that two cysteine residues, one zinc atom and two histidine residues were involved in the active site. The enzyme was fullly active as long as two SH groups in the active site remained in the reduced form even in the absence of zinc. However, the enzymatic activity was completely lost, with a concomitant loss of bound zinc, upon oxidation of the SH groups to a disulfide bond, modification of SH groups with chemical reagents, or mercaptide formation by heavy metals. Thus, it is apparent that the activity depends on the essential SH groups. The zinc is not absolutely essential for the activity but may be required to prevent the essential SH groups from autooxidation by coordination. Binding experiments indicated that there was one binding site of zinc/subunit. Photooxidation of histidine residues diminished both enzymatic activity and bound zinc, suggesting that the histidine residues not only constituted the active site but also served as a possible ligand to zinc.     

Crystal structures of MAP kinase p38 complexed to the docking sites on its nuclear substrate MEF2A and activator MKK3b

The structures of the MAP kinase p38 in complex with docking site peptides containing a phi(A)-X-phi(B) motif, derived from substrate MEF2A and activating enzyme MKK3b, have been solved. The peptides bind to the same site in the C-terminal domain of the kinase, which is both outside the active site and distinct from the "CD" domain previously implicated in docking site interactions. Mutational analysis on the interaction of p38 with the docking sites supports the crystallographic models and has uncovered two novel residues on the docking groove that are critical for binding. The two peptides induce similar large conformational changes local to the peptide binding groove. The peptides also induce unexpected and different conformational changes in the active site, as well as structural disorder in the phosphorylation lip.     

Crystal structure of a murine glutathione S-transferase in complex with a glutathione conjugate of 4-hydroxynon-2-enal in one subunit and glutathione in the other: evidence of signaling across the dimer interface.

mGSTA4-4, a murine glutathione S-transferase (GST) exhibiting high activity in conjugating the lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) with glutathione (GSH), was crystallized in complex with the GSH conjugate of 4-HNE (GS-Hna). The structure has been solved at 2.6 A resolution, which reveals that the active site of one subunit of the dimeric enzyme binds GS-Hna, whereas the other binds GSH. A marked asymmetry between the two subunits is evident. Most noticeable are the differences in the conformation of arginine residues 69 and 15. In all GST structures published previously, the guanidino groups of R69 residues from both subunits stack at the dimer interface and are related by a (pseudo-) 2-fold axis. In the present structure of mGSTA4-4, however, the two R69 side chains point in opposite directions, although their guanidino groups remain in contact. In the subunit with bound GSH, R69 also interacts with R15, and the guanidino group of R15 points away from the active site, whereas in the subunit that binds GS-Hna, R15 pivots into the active site, which breaks its interaction with R69. According to our previous results [Nanduri et al. (1997) Arch. Biochem. Biophys. 335, 305-310], the availability of R15 in the active site assists the conjugation of 4-HNE with GSH. We propose a model for the catalytic mechanism of mGSTA4-4 in conjugating 4-HNE with GSH-i.e., the guanidino group of R15 is available in the active site of only one subunit at any given time and the stacked pair of R69 residues act as a switch that couples the concerted movement of the two R15 side chains. The alternate occupancy of 4-HNE in the two subunits has been confirmed by our kinetic analysis that shows the negative cooperativity of mGSTA4-4 for 4-HNE. Disruption of the signaling between the subunits by mutating the R69 residues released the negative cooperativity with 4-HNE.     

NMR-derived models of amidopyrine and its metabolites in complexes with rabbit cytochrome P450 2B4 reveal a structural mechanism of sequential N-dealkylation

To understand the molecular basis of sequential N-dealkylation by cytochrome P450 2B enzymes, we studied the binding of amidopyrine (AP) as well as the metabolites of this reaction, desmethylamidopyrine (DMAP) and aminoantipyrine (AAP), using the X-ray crystal structure of rabbit P450 2B4 and two nuclear magnetic resonance (NMR) techniques: saturation transfer difference (STD) spectroscopy and longitudinal (T(1)) relaxation NMR. Results of STD NMR of AP and its metabolites bound to P450 2B4 were similar, suggesting that they occupy similar niches within the enzyme's active site. The model-dependent relaxation rates (R(M)) determined from T(1) relaxation NMR of AP and DMAP suggest that the N-linked methyl is closest to the heme. To determine the orientation(s) of AP and its metabolites within the P450 2B4 active site, we used distances calculated from the relaxation rates to constrain the metabolites to the X-ray crystal structure of P450 2B4. Simulated annealing of the complex revealed that the metabolites do indeed occupy similar hydrophobic pockets within the active site, while the N-linked methyls are free to rotate between two binding modes. From these bound structures, a model of N-demethylation in which the N-linked methyl functional groups rotate between catalytic and noncatalytic positions was developed. This study is the first to provide a structural model of a drug and its metabolites complexed to a cytochrome P450 based on NMR and to provide a structural mechanism for how a drug can undergo sequential oxidations without unbinding. The rotation of the amide functional group might represent a common structural mechanism for N-dealkylation reactions for other drugs such as the local anesthetic lidocaine.     

The role of calcium in the regulation of free radical reduction by thioredoxin reductase at the surface of the skin

Membrane associated thioredoxin reductase has been previously shown to reduce free radicals on the outer plasma membranes of human keratinocytes and melanocytes to provide a possible first line of defense against free radical damage at the surface of the skin. Preliminary experiments with cell cultures of human keratinocytes and melanocytes grown in serum-free medium showed that the enzyme activity depends on extracellular calcium concentration in the medium. Thioredoxin reductase activity at the surface of the skin, at the surface of human keratinocytes and melanocytes, and purified thioredoxin reductase from E. coli and adult human keratinocytes all exhibited calcium-dependent allosteric control. Since thioredoxin reductase contains two extremely reactive thiolate groups at the active site with pK values close to neutrality, both of these anions can form covalent complexes with N-ethylmaleimide by nucleophilic attack on the double bond. In our experiments we used spin-labeled maleimide [4-maleimido-tempo] to examine the local environment in the active site of thioredoxin reductase in the presence and absence of calcium. Both spin-labeled thioethers are distinguishable by EPR spectroscopy, with one site being significantly more immobilized than the other. Hence, it has been possible to observe direct evidence for active site closure by calcium. These results suggest that extracellular calcium may play an important role in regulation of thioredoxin reductase activity for the defense mechanism against UV-mediated free radical damage at the surface of human skin.     

X-ray analyses of aspartic proteinases. The three-dimensional structure at 2.1 A resolution of endothiapepsin

The molecular structure of endothiapepsin (EC 3.4.23.6), the aspartic proteinase from Endothia parasitica, has been refined to a crystallographic R-factor of 0.178 at 2.1 A resolution. The positions of 2389 protein non-hydrogen atoms have been determined and the present model contains 333 solvent molecules. The structure is bilobal, consisting of two predominantly beta-sheet domains that are related by an approximate 2-fold axis. Of approximately 170 residues, 65 are topologically equivalent when one lobe is superimposed on the other. Twenty beta-strands are arranged as five beta-sheets and are connected by regions involving 29 turns and four helices. A central sheet involves three antiparallel strands from each lobe organized around the dyad axis. Each lobe contains a further local dyad that passes through two sheets arranged as a sandwich and relates two equivalent motifs of four antiparallel strands (a, b, c, d) followed by a helix or an irregular helical region. Sheets 1N and 1C, each contain two interpenetrating psi structures contributed by strands c,d,d' and c',d',d, which are related by the intralobe dyad. A further sheet, 2N or 2C, is formed from two extended beta-hairpins from strands b,c and b',c' that fold above the sheets 1N and 1C, respectively, and are hydrogen-bonded around the local intralobe dyad. Asp32 and Asp215 are related by the interlobe dyad and form an intricate hydrogen-bonded network with the neighbouring residues and comprise the most symmetrical part of the structure. The side-chains of the active site aspartate residues are held coplanar and the nearby main chain makes a "fireman's grip" hydrogen-bonding network. Residues 74 to 83 from strands a'N and b'N in the N-terminal lobe form a beta-hairpin loop with high thermal parameters. This "flap" projects over the active site cleft and shields the active site from the solvent region. Shells of water molecules are found on the surface of the protein molecule and large solvent channels are observed within the crystal. There are only three regions of intermolecular contacts and the crystal packing is stabilized by many solvent molecules forming a network of hydrogen bonds. The three-dimensional structure of endothiapepsin is found to be similar to two other fungal aspartic proteinases, penicillopepsin and rhizopuspepsin. Even though sequence identities of endothiapepsin with rhizopuspepsin and penicillopepsin are only 41% and 51%, respectively, a superposition of the three-dimensional structures of these three enzymes shows that 237 residues (72%) are within a root-mean-square distance of 1.0 A.     

Nonidentical alkylation sites in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase.

These studies establish the specificity of 3,3,3-trifluorobromoacetone for reaction with the active site cysteines of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase and suggest the potential use of trifluoroacetonyl groups as 19F nuclear magnetic resonance probes for study of symmetry relations between the four protomers of the enzyme. The alkylation of the holoenzyme follows biphasic kinetics and indicates either preexistent or induced nonequivalence among the sites; these effects are not predisposed by a low coenzyme/enzyme ratio. Two additional alkylation sites not at the active centers are created by acylation with beta-(2-furyl)acryloyl phosphate: it is concluded that pseudosubstrates cause an intramolecular rearrangement which exposes two sulfhydryl functions besides those of the active site (Cys-149).     

Cofactor assisted gating mechanism in the active site of NADH oxidase from Thermus thermophilus

NADH oxidase (NOX) from Thermus thermophilus is a member of a structurally homologous flavoprotein family of nitroreductases and flavin reductases. The importance of local conformational dynamics in the active site of NOX has been recently demonstrated. The enzyme activity was increased by 250% in the presence of 1 M urea with no apparent perturbation of the native structure of the protein. The present in silico results correlate with the in vitro data and suggest the possible explanation about the effect of urea on NOX activity at the molecular level. Both, X-ray structure and molecular dynamics (MD) simulations, show open conformation of the active site represented by approximately 0.9 nm distance between the indole ring of Trp47 and the isoalloxazine ring of FMN412. In this conformation, the substrate molecule can bind in the active site without sterical restraints. MD simulations also indicate more stable conformation of the active site called "closed" conformation. In this conformation, Trp47 and the isoalloxazine ring of FMN412 are so close to each other (approximately 0.5 nm) that the substrate molecule is unable to bind between them without perturbing this conformation. The open/close transition of the active site between Trp47 and the flavin ring is accompanied by release of the "tightly" bound water molecule from the active site--cofactor assisted gating mechanism. The presence of urea in aqueous solutions of NOX prohibits closing of the active site and even unlocks the closed active site because of the concomitant binding of a urea molecule in the active site cavity. The binding of urea in the active site is stabilized by formation of one/two persistent hydrogen bonds involving the carbonyl group of the urea molecule. Our report represents the first MD study of an enzyme from the novel flavoprotein family of nitroreductases and flavin reductases. The common occurrence of aromatic residues covering the active sites in homologous enzymes suggests the possibility of a general gating mechanism and the importance of local dynamics within this flavoprotein family.