Role of tyrosine 238 in the active site of Rhodotorula gracilis D-amino acid oxidase. A site-directed mutagenesis study.

Y238, one of the very few conserved residues in the active site of d-amino acid oxidases (DAAO), was mutated to phenylalanine and serine in the enzyme from the yeast Rhodotorula gracilis. The mutated proteins are catalytically competent thus eliminating Tyr238 as an active-site acid/base catalyst. Y238F and Y238S mutants exhibit a threefold slower turnover on d-alanine as substrate, which can be attributed to a slower rate of product release relative to the wild-type enzyme (a change of the rate constants for substrate binding was also evident). The Y238 DAAO mutants have spectral properties similar to those of the wild-type enzyme but the degree of stabilization of the flavin semiquinone and the redox properties in the free form of Y238S are different. The binding of the carboxylic acid competitive inhibitors and the substrate d-alanine are changed only slightly, suggesting that the overall substrate binding pocket remains intact. In agreement with data from the pH dependence of ligand binding and with the protein crystal structure, site-directed mutagenesis results emphasize the importance of residue Y238 in controlling access to the active site instead of a role in the substrate/ligand interaction.     

NMR investigation of Tyr105 mutants in TEM-1 beta-lactamase: dynamics are correlated with function

The existence of coupled residue motions on various time scales in enzymes is now well accepted, and their detailed characterization has become an essential element in understanding the role of dynamics in catalysis. To this day, a handful of enzyme systems has been shown to rely on essential residue motions for catalysis, but the generality of such phenomena remains to be elucidated. Using NMR spectroscopy, we investigated the electronic and dynamic effects of several mutations at position 105 in TEM-1 beta-lactamase, an enzyme responsible for antibiotic resistance. Even in absence of substrate, our results show that the number and magnitude of short and long range effects on (1)H-(15)N chemical shifts are correlated with the catalytic efficiencies of the various Y105X mutants investigated. In addition, (15)N relaxation experiments on mutant Y105D show that several active-site residues of TEM-1 display significantly altered motions on both picosecond-nanosecond and microsecond-millisecond time scales despite many being far away from the site of mutation. The altered motions among various active-site residues in mutant Y105D may account for the observed decrease in catalytic efficiency, therefore suggesting that short and long range residue motions could play an important catalytic role in TEM-1 beta-lactamase. These results support previous observations suggesting that internal motions play a role in promoting protein function.     

Substrate binding to histone deacetylases as shown by the crystal structure of the HDAC8-substrate complex

Histone deacetylases (HDACs)-an enzyme family that deacetylates histones and non-histone proteins-are implicated in human diseases such as cancer, and the first-generation of HDAC inhibitors are now in clinical trials. Here, we report the 2.0 A resolution crystal structure of a catalytically inactive HDAC8 active-site mutant, Tyr306Phe, bound to an acetylated peptidic substrate. The structure clarifies the role of active-site residues in the deacetylation reaction and substrate recognition. Notably, the structure shows the unexpected role of a conserved residue at the active-site rim, Asp 101, in positioning the substrate by directly interacting with the peptidic backbone and imposing a constrained cis-conformation. A similar interaction is observed in a new hydroxamate inhibitor-HDAC8 structure that we also solved. The crucial role of Asp 101 in substrate and inhibitor recognition was confirmed by activity and binding assays of wild-type HDAC8 and Asp101Ala, Tyr306Phe and Asp101Ala/Tyr306Phe mutants.     

Site-saturation mutagenesis of Tyr-105 reveals its importance in substrate stabilization and discrimination in TEM-1 beta-lactamase

The conserved Class A beta-lactamase active site residue Tyr-105 was substituted by saturation mutagenesis in TEM-1 beta-lactamase from Escherichia coli in order to clarify its role in enzyme activity and in substrate stabilization and discrimination. Minimum inhibitory concentrations were calculated for E. coli cells harboring each Y105X mutant in the presence of various penicillin and cephalosporin antibiotics. We found that only aromatic residues as well as asparagine replacements conferred high in vivo survival rates for all substrates tested. At position 105, the small residues alanine and glycine provide weak substrate discrimination as evidenced by the difference in benzylpenicillin hydrolysis relative to cephalothin, two typical penicillin and cephalosporin antibiotics. Kinetic analyses of mutants of interest revealed that the Y105X replacements have a greater effect on K(m) than k(cat), highlighting the importance of Tyr-105 in substrate recognition. Finally, by performing a short molecular dynamics study on a restricted set of Y105X mutants of TEM-1, we found that the strong aromatic bias observed at position 105 in Class A beta-lactamases is primarily defined by a structural requirement, selecting planar residues that form a stabilizing wall to the active site. The adopted conformation of residue 105 prevents detrimental steric interactions with the substrate molecule in the active site cavity and provides a rationalization for the strong aromatic bias found in nature at this position among Class A beta-lactamases.     

Uncoupling metallonuclease metal ion binding sites via nudge mutagenesis

The hydrolysis of phosphodiester bonds by nucleases is critical to nucleic acid processing. Many nucleases utilize metal ion cofactors, and for a number of these enzymes two active-site metal ions have been detected. Testing proposed mechanistic roles for individual bound metal ions has been hampered by the similarity between the sites and cooperative behavior. In the homodimeric PvuII restriction endonuclease, the metal ion dependence of DNA binding is sigmoidal and consistent with two classes of coupled metal ion binding sites. We reasoned that a conservative active-site mutation would perturb the ligand field sufficiently to observe the titration of individual metal ion binding sites without significantly disturbing enzyme function. Indeed, mutation of a Tyr residue 5.5 A from both metal ions in the enzyme-substrate crystal structure (Y94F) renders the metal ion dependence of DNA binding biphasic: two classes of metal ion binding sites become distinct in the presence of DNA. The perturbation in metal ion coordination is supported by 1H-15N heteronuclear single quantum coherence spectra of enzyme-Ca(II) and enzyme-Ca(II)-DNA complexes. Metal ion binding by free Y94F is basically unperturbed: through multiple experiments with different metal ions, the data are consistent with two alkaline earth metal ion binding sites per subunit of low millimolar affinity, behavior which is very similar to that of the wild type. The results presented here indicate a role for the hydroxyl group of Tyr94 in the coupling of metal ion binding sites in the presence of DNA. Its removal causes the affinities for the two metal ion binding sites to be resolved in the presence of substrate. Such tuning of metal ion affinities will be invaluable to efforts to ascertain the contributions of individual bound metal ions to metallonuclease function.     

The C-terminal region of human glutathione transferase A1-1 affects the rate of glutathione binding and the ionization of the active-site Tyr9

In human glutathione transferase (GST) A1-1, the C-terminal region covers the active site and contributes to substrate binding. This region is flexible, but upon binding of an active-site ligand, it is stabilized as an amphipatic alpha-helix. The stabilization has implications for the catalytic activity of the enzyme. In the present study, residue M208 in GST A1-1 has been mutated to Lys and Glu, and residue F220 to Ala and Thr. These mutations are likely to destabilize the C-terminal region due to loss of hydrophobic interactions with the rest of the hydrophobic binding site. The rate constant for binding of glutathione to wild-type GST A1-1 is 450 mM(-)(1) s(-)(1) at 5 degrees C and pH 7.0, which is less than for an association limited by diffusion. However, the M208 and the F220 mutations increase the apparent on-rate constant for glutathione binding to 640-1170 mM(-)(1) s(-)(1). The binding data can be explained by a rapid reversible transition between different enzyme conformations occurring prior to glutathione binding, and restriction of the access to the active site by the C-terminal region. The effect of the mutations appears to be promotion of a less closed conformation, thereby facilitating the association of glutathione and enzyme. Both the M208 and F220 mutants display a lowered pK(a) value ( approximately 0.3 log unit) of the catalytically important Tyr9. Residue 208 does not interact directly with Tyr9 in the active site, and the shift in pK(a) value is therefore ascribed to the proposed dislocation of the C-terminal region caused by the mutation.     

Catalytic mechanism of scytalone dehydratase: site-directed mutagenisis, kinetic isotope effects, and alternate substrates.

On the basis of the X-ray crystal structure of scytalone dehydratase complexed with an active center inhibitor [Lundqvist, T., Rice, J., Hodge, C. N., Basarab, G. S., Pierce, J. and Lindqvist, Y. (1994) Structure (London) 2, 937-944], eight active-site residues were mutated to examine their roles in the catalytic mechanism. All but one residue (Lys73, a potential base in an anti elimination mechanism) were found to be important to catalysis or substrate binding. Steady-state kinetic parameters for the mutants support the native roles for the residues (Asn131, Asp31, His85, His110, Ser129, Tyr30, and Tyr50) within a syn elimination mechanism. Relative substrate specificities for the two physiological substrates, scytalone and veremelone, versus a Ser129 mutant help assign the orientation of the substrates within the active site. His85Asn was the most damaging mutation to catalysis consistent with its native roles as a general base and a general acid in a syn elimination. The additive effect of Tyr30Phe and Tyr50Phe mutations in the double mutant is consistent with their roles in protonating the substrate's carbonyl through a water molecule. Studies on a synthetic substrate, which has an anomeric carbon atom which can better stabilize a carbocation than the physiological substrate (vermelone), suggest that His110Asn prefers this substrate over vermelone in order to balance the mutation-imposed weakness in promoting the elimination of hydroxide from substrates. All mutant enzymes bound a potent active-site inhibitor in near 1:1 stoichiometry, thereby supporting their active-site integrity. An X-ray crystal structure of the Tyr50Phe mutant indicated that both active-site waters were retained, likely accounting for its residual catalytic activity. Steady-state kinetic parameters with deuterated scytalone gave kinetic isotope effects of 2.7 on kcat and 4.2 on kcat/Km, suggesting that steps after dehydration partially limit kcat. Pre-steady-state measurements of a single-enzyme turnover with scytalone gave a rate that was 6-fold larger than kcat. kcat/Km with scytalone has a pKa of 7.9 similar to the pKa value for the ionization of the substrate's C6 phenolic hydroxyl, whereas kcat was unaffected by pH, indicating that the anionic form of scytalone does not bind well to enzyme. With an alternate substrate having a pKa above 11, kcat/Km had a pKa of 9.3 likely due to the ionization of Tyr50. The non-enzyme-catalyzed rate of dehydration of scytalone was nearly a billion-fold slower than the enzyme-catalyzed rate at pH 7.0 and 25 degrees C. The non-enzyme-catalyzed rate of dehydration of scytalone had a deuterium kinetic isotope effect of 1.2 at pH 7.0 and 25 degrees C, and scytalone incorporated deuterium from D2O in the C2 position about 70-fold more rapidly than the dehydration rate. Thus, scytalone dehydrates through an E1cb mechanism off the enzyme.     

Improving the affinity and activity of CYP101D2 for hydrophobic substrates

CYP101D2 is a cytochrome P450 monooxygenase from Novosphingobium aromaticivorans which is closely related to CYP101A1 (P450cam) from Pseudomonas putida. Both enzymes selectively hydroxylate camphor to 5-exo-hydroxycamphor, and the residues that line the active sites of both enzymes are similar including the pre-eminent Tyr96 residue. However, Met98 and Leu253 in CYP101D2 replace Phe98 and Val247 in CYP101A1, and camphor binding only results in a maximal change in the spin state to 40 % high-spin. Substitutions at Tyr96, Met98 and Leu253 in CYP101D2 reduced both the spin state shift on camphor binding and the camphor oxidation activity. The Tyr96Ala mutant increased the affinity of CYP101D2 for hydrocarbon substrates including adamantane, cyclooctane, hexane and 2-methylpentane. The monooxygenase activity of the Tyr96Ala variant towards alkane substrates was also enhanced compared with the wild-type enzyme. The crystal structure of the substrate-free form of this variant shows the enzyme in an open conformation (PDB: 4DXY), similar to that observed with the wild-type enzyme (PDB: 3NV5), with the side chain of Ala96 pointing away from the heme. Despite this, the binding and activity data suggest that this residue plays an important role in substrate binding, evidencing that the enzyme probably undergoes catalysis in a more closed conformation, similar to those observed in the crystal structures of CYP101A1 (PDB: 2CPP) and CYP101D1 (PDB: 3LXI).     

Identification of amino acid substitutions that alter the substrate specificity of TEM-1 beta-lactamase.

TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Recently, TEM beta-lactamase variants with amino acid substitutions in the active-site pocket of the enzyme have been identified in natural isolates with increased resistance to extended-spectrum cephalosporins. To identify other amino acid substitutions that alter the activity of TEM-1 towards extended-spectrum cephalosporins, we probed regions around the active-site pocket by random-replacement mutagenesis. This mutagenesis technique involves randomizing the DNA sequence of three to six codons in the blaTEM-1 gene to form a library containing all or nearly all of the possible substitutions for the region randomized. In total, 20 different residue positions that had been randomized were screened for amino acid substitutions that increased enzyme activity towards the extended-spectrum cephalosporin cefotaxime. Substitutions at positions 104, 168, and 238 in the TEM-1 beta-lactamase that resulted in increased enzyme activity towards extended-spectrum cephalosporins were found. In addition, small deletions in the loop containing residues 166 to 170 drastically altered the substrate specificity of the enzyme by increasing activity towards extended-spectrum cephalosporins while virtually eliminating activity towards ampicillin.     

Crystal structure of nitrated human manganese superoxide dismutase: mechanism of inactivation

A cellular consequence of the reaction of superoxide and nitric oxide is enhanced peroxynitrite levels. Reaction of peroxynitrite with manganese superoxide dismutase (MnSOD) causes nitration of the active-site residue Tyr34 and nearly complete inhibition of catalysis. We report the crystal structures at 2.4 A resolution of human MnSOD nitrated by peroxynitrite and the unmodified MnSOD. A comparison of these structures showed no significant conformational changes of active-site residues or solvent displacement. The side chain of 3-nitrotyrosine 34 had a single conformation that extended toward the manganese with O1 of the nitro group within hydrogen-bonding distance (3.1 A) of Nepsilon2 of the second-shell ligand Gln143. Also, nitration of Tyr34 caused a weakening, as evidenced by the lengthening, of a hydrogen bond between its phenolic OH and Gln143, part of an extensive hydrogen-bond network in the active site. Inhibition of catalysis can be attributed to a steric effect of 3-nitrotyrosine 34 that impedes substrate access and binding, and alteration of the hydrogen-bond network that supports proton transfer in catalysis. It is also possible that an electrostatic effect of the nitro group has altered the finely tuned redox potential necessary for efficient catalysis, although the redox potential of nitrated MnSOD has not been measured.     

Crystal structure of family 5 uracil-DNA glycosylase bound to DNA

Uracil-DNA glycosylase (UDG) removes uracil generated by the deamination of cytosine or misincorporation of deoxyuridine monophosphate. Within the UDG superfamily, a fifth UDG family lacks a polar residue in the active-site motif, which mediates the hydrolysis of the glycosidic bond by activation of a water molecule in UDG families 1-4. We have determined the crystal structure of a novel family 5 UDG from Thermus thermophilus HB8 complexed with DNA containing an abasic site. The active-site structure suggests this enzyme uses both steric force and water activation for its excision reaction. A conserved asparagine residue acts as a ligand to the catalytic water molecule. The structure also implies that another water molecule acts as a barrier during substrate recognition. Based on no significant open-closed conformational change upon binding to DNA, we propose a "slide-in" mechanism for initial damage recognition.     

Crystal structure analysis,covalent docking,and molecular dynamics calculations reveal a conformational switch in PhaZ7 PHB depolymerase

An open and a closed conformation of a surface loop in PhaZ7 extracellular poly(3‐hydroxybutyrate) depolymerase were identified in two high‐resolution crystal structures of a PhaZ7 Y105E mutant. Molecular dynamics (MD) simulations revealed high root mean square fluctuations (RMSF) of the 281–295 loop, in particular at residue Asp289 (RMSF 7.62 Å). Covalent docking between a 3‐hydroxybutyric acid trimer and the catalytic residue Ser136 showed that the binding energy of the substrate is significantly more favorable in the open loop conformation compared to that in the closed loop conformation. MD simulations with the substrate covalently bound depicted 1 Å RMSF higher values for the residues 281–295 in comparison to the apo (substrate‐free) form. In addition, the presence of the substrate in the active site enhanced the ability of the loop to adopt a closed form. Taken together, the analysis suggests that the flexible loop 281–295 of PhaZ7 depolymerase can act as a lid domain to control substrate access to the active site of the enzyme. Proteins 2017; 85:1351–1361. © 2017 Wiley Periodicals, Inc.     

Computational and experimental studies of the catalytic mechanism of Thermobifida fusca cellulase Cel6A (E2)

Mutagenesis experiments suggest that Asp79 in cellulase Cel6A (E2) from Thermobifida fusca has a catalytic role, in spite of the fact that this residue is more than 13 A from the scissile bond in models of the enzyme-substrate complex built upon the crystal structure of the protein. This suggests that there is a substantial conformational shift in the protein upon substrate binding. Molecular mechanics simulations were used to investigate possible alternate conformations of the protein bound to a tetrasaccharide substrate, primarily involving shifts of the loop containing Asp79, and to model the role of water in the active site complex for both the native conformation and alternative low-energy conformations. Several alternative conformations of reasonable energy have been identified, including one in which the overall energy of the enzyme-substrate complex in solution is lower than that of the conformation in the crystal structure. This conformation was found to be stable in molecular dynamics simulations with a cellotetraose substrate and water. In simulations of the substrate complexed with the native protein conformation, the sugar ring in the -1 binding site was observed to make a spontaneous transition from the (4)C(1) conformation to a twist-boat conformer, consistent with generally accepted glycosidase mechanisms. Also, from these simulations Tyr73 and Arg78 were found to have important roles in the active site. Based on the results of these various MD simulations, a new catalytic mechanism is proposed. Using this mechanism, predictions about the effects of changes in Arg78 were made which were confirmed by site-directed mutagenesis.     

Effect of the Met344His mutation on the conformational dynamics of bovine beta-1,4-galactosyltransferase: crystal structure of the Met344His mutant in complex with chitobiose

Beta-1,4-galactosyltransferase (beta4Gal-T1) in the presence of manganese ion transfers galactose from UDP-galactose (UDP-Gal) to N-acetylglucosamine (GlcNAc) that is either free or linked to an oligosaccharide. Crystallographic studies on bovine beta4Gal-T1 have shown that the primary metal binding site is located in the hinge region of a long flexible loop, which upon Mn(2+) and UDP-Gal binding changes from an open to a closed conformation. This conformational change creates an oligosaccharide binding site in the enzyme. Neither UDP nor UDP analogues efficiently induce these conformational changes in the wild-type enzyme, thereby restricting the structural analysis of the acceptor binding site. The binding of Mn(2+) involves an uncommon coordination to the Sdelta atom of Met344; when it is mutated to His, the mutant M344H, in the presence of Mn(2+) and UDP-hexanolamine, readily changes to a closed conformation, facilitating the structural analysis of the enzyme bound with an oligosaccharide acceptor. Although the mutant M344H loses 98% of its Mn(2+)-dependent activity, it exhibits 25% of its activity in the presence of Mg(2+). The crystal structures of M344H-Gal-T1 in complex with either UDP-Gal.Mn(2+) or UDP-Gal.Mg(2+), determined at 2.3 A resolution, show that the mutant enzyme in these complexes is in a closed conformation, and the coordination stereochemistry of Mg(2+) is quite similar to that of Mn(2+). Although either Mn(2+) or Mg(2+), together with UDP-Gal, binds and changes the conformation of the M344H mutant to the closed one, it is the Mg(2+) complex that engages efficiently in catalyses. Thus, this property enabled us to crystallize the M344H mutant for the first time with the acceptor substrate chitobiose in the presence of UDP-hexanolamine and Mn(2+). The crystal structure determined at 2.3 A resolution reveals that the GlcNAc residue at the nonreducing end of chitobiose makes extensive hydrophobic interactions with the highly conserved Tyr286 residue.     

The role of tyrosine 71 in modulating the flap conformations of BACE1

β-Site amyloid precursor protein cleaving enzyme 1 (BACE1) is a potential target for treating Alzheimer's disease. BACE1's binding site is partially covered by a flexible loop on its N-terminal domain, known as the "flap," which has been found in several conformations in crystal structures of BACE1 and other aspartyl proteases. The side chain of the invariant residue Tyr71 on the flap adopts several rotameric orientations, leading to our hypothesis that the orientation of this residue dictates the movement and conformations available to the flap. We investigated this hypothesis by performing 220 ns of molecular dynamics simulations of bound and unbound wild-type BACE1 as well as the unbound Y71A mutant. Our findings indicate that the flap exhibits various degrees of mobility and adopts different conformations depending on the Tyr71 orientation. Surprisingly, the "self-inhibited" form is stable in our simulations, making it a reasonable target for drug design. The alanine mutant, lacking a large side chain at position 71, displays significant differences in flap dynamics from wild type, freely sampling very open and closed conformations. Our simulations show that Tyr71, in addition to its previously determined functions in catalysis and substrate binding, has the important role of modulating flap conformations in BACE1.     

Structural basis for the unusual specificity of Escherichia coli aminopeptidase N

Aminopeptidase N from Escherichia coli is a M1 class aminopeptidase with the active-site region related to that of thermolysin. The enzyme has unusual specificity, cleaving adjacent to the large, nonpolar amino acids Phe and Tyr but also cleaving next to the polar residues Lys and Arg. To try to understand the structural basis for this pattern of hydrolysis, the structure of the enzyme was determined in complex with the amino acids L-arginine, L-lysine, L-phenylalanine, L-tryptophan, and L-tyrosine. These amino acids all bind with their backbone atoms close to the active-site zinc ion and their side chain occupying the S1 subsite. This subsite is in the form of a cylinder, about 10 A in cross-section and 12 A in length. The bottom of the cylinder includes the zinc ion and a number of polar side chains that make multiple hydrogen-bonding and other interactions with the alpha-amino group and the alpha-carboxylate of the bound amino acid. The walls of the S1 cylinder are hydrophobic and accommodate the nonpolar or largely nonpolar side chains of Phe and Tyr. The top of the cylinder is polar in character and includes bound water molecules. The epsilon-amino group of the bound lysine side chain and the guanidinium group of arginine both make multiple hydrogen bonds to this part of the S1 site. At the same time, the hydrocarbon part of the lysine and arginine side chains is accommodated within the nonpolar walls of the S1 cylinder. This combination of hydrophobic and hydrophilic binding surfaces explains the ability of ePepN to cleave Lys, Arg, Phe, and Tyr. Another favored substrate has Ala at the P1 position. The short, nonpolar side chain of this residue can clearly be bound within the hydrophobic part of the S1 cylinder, but the reason for its facile hydrolysis remains uncertain.     

Long-range effects and conformational flexibility of aldolase

The conformational flexibility and long-range interactions in rabbit muscle aldolase induced by active-site ligand binding, cross-linking of the enzyme between Cys72 and Cys338, and removal of the C-terminal tyrosine residue were studied by following the changes in the microenvironments of Cys239 and Cys289 located outside the active site. It was found that substrates induced a conformational change in aldolase, which propagates from the active site to Cys239, which is located close to intersubunit contacts. The response of the enzyme is differential. Ligands having both C-1 and C-6 phosphates or C-1 phosphate only induce the enhancement of Cys239 reactivity, whereas those with C-6 phosphates only decrease Cys239 reactivity. This correlates well with a dramatic difference in kinetic parameters for a cleavage of fructose-1,6-P2 and fructose-1-P. Therefore, these changes can be interpreted as syncatalytic. Cross-linking of the aldolase subunit by an -S-S-bridge between Cys72 and Cys338 inactivates the enzyme, abolishes binding of active-site ligands, and induces a conformational change in the enzyme that can be detected far away (at Cys239 and Cys289) from the site of perturbation. Cys72 and Cys338 are not in the active site. This shows that the region of the active site and the environment of Cys72 and Cys338 are tightly coupled and that residues far away from the active site, through such coupling, can possess properties of active-site residues. Similar, although less dramatic changes are observed upon removal of the C-terminal tyrosine residue. In view of the results obtained in this paper, aldolase seems to be quite a flexible molecule, whose conformation is sensitive to the nature of a substrate bound to the enzyme and is able to transmit the information about a local perturbation over long distances within a molecule.     

Structural Analysis of the Streptomyces avermitilis CYP107W1-Oligomycin A Complex and Role of the Tryptophan 178 Residue

CYP107W1 from Streptomyces avermitilis is a cytochrome P450 enzyme involved in the biosynthesis of macrolide oligomycin A. A previous study reported that CYP107W1 regioselectively hydroxylated C12 of oligomycin C to produce oligomycin A, and the crystal structure of ligand free CYP107W1 was determined. Here, we analyzed the structural properties of the CYP107W1-oligomycin A complex and characterized the functional role of the Trp178 residue in CYP107W1. The crystal structure of the CYP107W1 complex with oligomycin A was determined at a resolution of 2.6 Å. Oligomycin A is bound in the substrate access channel on the upper side of the prosthetic heme mainly by hydrophobic interactions. In particular, the Trp178 residue in the active site intercalates into the large macrolide ring, thereby guiding the substrate into the correct binding orientation for a productive P450 reaction. A Trp178 to Gly mutation resulted in the distortion of binding titration spectra with oligomycin A, whereas binding spectra with azoles were not affected. The Gly178 mutant’s catalytic turnover number for the 12-hydroxylation reaction of oligomycin C was highly reduced. These results indicate that Trp178, located in the open pocket of the active site, may be a critical residue for the productive binding conformation of large macrolide substrates.     

Structural Basis for Substrate Recognition and Specificity in Aklavinone-11-Hydroxylase from Rhodomycin Biosynthesis

In the biosynthesis of several anthracyclines, aromatic polyketides produced by many Streptomyces species, the aglycone core is modified by a specific flavin adenine dinucleotide (FAD)- and NAD(P)H-dependent aklavinone-11-hydroxylase. Here, we report the crystal structure of a ternary complex of this enzyme from Streptomyces purpurascens, RdmE, with FAD and the substrate aklavinone. The enzyme is built up of three domains, a FAD-binding domain, a domain involved in substrate binding, and a C-terminal thioredoxin-like domain of unknown function. RdmE exhibits structural similarity to aromatic hydroxylases from the p-hydroxybenzoate hydroxylase family, but unlike most other related enzymes, RdmE is a monomer. The substrate is bound in a hydrophobic pocket in the interior of the enzyme, and access to this pocket is provided through a different route than for the isoalloxazine ring of FAD—the backside of the ligand binding cleft. The architecture of the substrate binding pocket and the observed enzyme-aklavinone interactions provide a structural explanation for the specificity of the enzyme for non-glycosylated substrates with C9-R stereochemistry. The isoalloxazine ring of the flavin cofactor is bound in the “out” conformation but can be modeled in the “in” conformation without invoking large conformational changes of the enzyme. This model places the flavin ring in a position suitable for catalysis, almost perpendicular to the tetracyclic ring system of the substrate and with a distance of the C4a carbon atom of the isoalloxazine ring to the C-11 carbon atom of the substrate of 4.8 Å. The structure suggested that a Tyr224-Arg373 pair might be involved in proton abstraction at the C-6 hydroxyl group, thereby increasing the nucleophilicity of the aromatic ring system and facilitating electrophilic attack by the perhydroxy-flavin intermediate. Replacement of Tyr224 by phenylalanine results in inactive enzyme, whereas mutants at position Arg373 retain catalytic activity close to wild-type level. These data establish an essential role of residue Tyr224 in catalysis, possibly in aligning the substrate in a position suitable for catalysis.     

Human glutathione transferase A4-4 crystal structures and mutagenesis reveal the basis of high catalytic efficiency with toxic lipid peroxidation products.

The oxidation of lipids and cell membranes generates cytotoxic compounds implicated in the etiology of aging, cancer, atherosclerosis, neurodegenerative diseases, and other illnesses. Glutathione transferase (GST) A4-4 is a key component in the defense against the products of this oxidative stress because, unlike other Alpha class GSTs, GST A4-4 shows high catalytic activity with lipid peroxidation products such as 4-hydroxynon-2-enal (HNE). The crystal structure of human apo GST A4-4 unexpectedly possesses an ordered C-terminal alpha-helix, despite the absence of any ligand. The structure of human GST A4-4 in complex with the inhibitor S-(2-iodobenzyl) glutathione reveals key features of the electrophilic substrate-binding pocket which confer specificity toward HNE. Three structural modules form the binding site for electrophilic substrates and thereby govern substrate selectivity: the beta1-alpha1 loop, the end of the alpha4 helix, and the C-terminal alpha9 helix. A few residue changes in GST A4-4 result in alpha9 taking over a predominant role in ligand specificity from the N-terminal loop region important for GST A1-1. Thus, the C-terminal helix alpha9 in GST A4-4 provides pre-existing ligand complementarity rather than acting as a flexible cap as observed in other GST structures. Hydrophobic residues in the alpha9 helix, differing from those in the closely related GST A1-1, delineate a hydrophobic specificity canyon for the binding of lipid peroxidation products. The role of residue Tyr212 as a key catalytic residue, suggested by the crystal structure of the inhibitor complex, is confirmed by mutagenesis results. Tyr212 is positioned to interact with the aldehyde group of the substrate and polarize it for reaction. Tyr212 also coopts part of the binding cleft ordinarily formed by the N-terminal substrate recognition region in the homologous enzyme GST A1-1 to reveal an evolutionary swapping of function between different recognition elements. A structural model of catalysis is presented based on these results.