1.8A X-ray structure of C95M/C1095F double mutant of tethered HIV-1 protease dimer complexed with acetyl pepstatin

Under the selection pressure of drugs, mutations appear in HIV-1 protease even at the sites, which are conserved in the untreated individuals. Cysteine 95 is a highly conserved residue and is believed to be involved in regulation of HIV-1 protease. In some of the virus isolates from patients undergoing heavy treatment with anti-HIV protease drugs, C95F mutation has appeared. The present study reports 1.8A X-ray structure of C95M/C1095F double mutant of tethered HIV-1 protease dimer complexed with acetyl pepstatin. It is found that in this mutant, dimer interface has become more rigid and that the packing at the interface of terminal and core domains is altered. These alterations may be relevant to C95F mutation conferring drug resistance to HIV-1 protease.     

Investigating the effects of double mutation C30A/C75A on onconase structure: Studies at atomic resolution

The structure of onconase C30A/C75A double mutant has been determined at 1.12Å resolution. The structure has high structural homology to other onconase structures. The changes being results of mutation are relatively small, distributed asymmetrically around the two mutated positions, and they are observed not only in the mutation region but expanded to entire molecule. Different conformation of Lys31 side chain that influences the hydrogen bonding network around catalytic triad is probably responsible for lower catalytic efficiency of double mutant. The decrease in thermal stability observed for the onconase variant might be explained by a less dense packing as manifested by the increase of the molecular volume and the solvent accessible surface area. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 454–460, 2014.     

The conserved methionine residue of the metzincins: a site-directed mutagenesis study.

The metalloprotease clan of the metzincins derive their name from the presence of a conserved methionine residue that is located on the C-terminal side of the zinc-binding consensus sequence HEXXHXXGXXH. This methionine residue is located in a rather divergent part of the primary sequence but is structurally very well conserved. It is located under the pyramidal base of the three histidine residues that coordinate the catalytic zinc ion and is not involved in any direct contact with the metal nor the substrate. In order to clarify its role, this methionine residue (M226) of the protease C from Erwinia chrysanthemi has been mutated to various other amino acids. The mutants M226L, M226A, M226I were sufficiently stable to be isolated, while the mutants M226H, M226S and M226N could not be purified. The kinetic properties of these mutants were analysed. All mutants showed decreased activity, whereby increases in K(M) as well as decreases in k(cat) were observed. The M226L mutant and M226C-E189 K double mutant, which has the catalytic glutamic acid substituted as well, could be crystallised. The structure of the M226L mutant was determined to a resolution of 2.0 A and refined to R(free) of 0.20. The structure is isomorphous to the wild-type and does not show large differences, with the exception of a very small movement of the zinc-liganding histidine residues. The M226C-E189 K double mutant crystal structure has been refined to an R(free) of 0.20 at 2.1 A resolution. A small rearrangement of the zinc-liganding histidine residues can be detected, which leads to a slightly different zinc coordination and could explain the decrease in activity.     

Folding mechanism of mutant human lysozyme C77/95A with increased secretion efficiency in yeast.

A mutant human lysozyme C77/95A, in which Cys77 and Cys95 are replaced with alanine, has been characterized by 8-fold greater secretion in yeast (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967) and almost the same three-dimensional structure as wild-type human lysozyme (Inaka, K., Taniyama, Y., Kikuchi, M., Morikawa, K., and Matsushima, M. (1991) J. Biol. Chem. 266, 12599-12603). To clarify the molecular features of C77/95A and the reason for its increased secretion in yeast, the stabilities of the mutant C77/95A and the wild-type proteins were examined by guanidine hydrochloride denaturation, and the unfolding-refolding kinetics were determined from circular dichroism and fluorescence stopped-flow measurements. Equilibrium experiments showed that the delta G of unfolding of C77/95A in water was 5.8 kcal/mol less stable than that of the wild-type protein at pH 4.0 and 10 degrees C. The unfolding rate of C77/95A was 4 orders of magnitude faster than that of the wild-type protein whereas the two proteins shared similar refolding rates. The slowly refolding phase of the wild-type protein disappeared in C77/95A, indicating that the disulfide bond affects this phase. These observations show that the disulfide bond Cys77-Cys95 contributes to the stabilization of the folded form of human lysozyme by suppressing the unfolding rate and that the increase in the unfolding rate, or the disappearance of the slowly refolding phase in vitro, could correlate with the increase in secretion efficiency in vivo.     

The role of Arg165 towards base flipping, base stabilization and catalysis in M.HhaI

Arg165 forms part of a previously identified base flipping motif in the bacterial DNA cytosine methyltransferase, M.HhaI. Replacement of Arg165 with Ala has no detectable effect on either DNA or AdoMet affinity, yet causes the base flipping and restacking transitions to be decreased approximately 16 and 190-fold respectively, thus confirming the importance of this motif. However, these kinetic changes cannot account for the mutant's observed 10(5)-fold decreased catalytic rate. The mutant enzyme/cognate DNA cocrystal structure (2.79 A resolution) shows the target cytosine to be positioned approximately 30 degrees into the major groove, which is consistent with a major groove pathway for nucleotide flipping. The pyrimidine-sugar chi angle is rotated to approximately +171 degrees, from a range of -95 degrees to -120 degrees in B DNA, and -77 degrees in the WT M.HhaI complex. Thus, Arg165 is important for maintaining the cytosine positioned for nucleophilic attack by Cys81. The cytosine sugar pucker is in the C2'-endo-C3'-exo (South conformation), in contrast to the previously reported C3'-endo (North conformation) described for the original 2.70 A resolution cocrystal structure of the WT M.HhaI/DNA complex. We determined a high resolution structure of the WT M.HhaI/DNA complex (1.96 A) to better determine the sugar pucker. This new structure is similar to the original, lower resolution WT M.HhaI complex, but shows that the sugar pucker is O4'-endo (East conformation), intermediate between the South and North conformers. In summary, Arg165 plays significant roles in base flipping, cytosine positioning, and catalysis. Furthermore, the previously proposed M.HhaI-mediated changes in sugar pucker may not be an important contributor to the base flipping mechanism. These results provide insights into the base flipping and catalytic mechanisms for bacterial and eukaryotic DNA methyltransferases.     

Probing domain mobility in a flavocytochrome

The crystal structures of various different members of the family of fumarate reductases and succinate dehydrogenases have allowed the identification of a mobile clamp (or capping) domain [e.g., Taylor, P., Pealing, S. L., Reid, G. A., Chapman, S. K., and Walkinshaw, M. D. (1999) Nat. Struct. Biol. 6, 1108-1112], which has been proposed to be involved in regulating accessibility of the active site to substrate. To investigate this, we have constructed the A251C:S430C double mutant form of the soluble flavocytochrome c(3) fumarate reductase from Shewanella frigidimarina, to introduce an interdomain disulfide bond between the FAD-binding and clamp domains of the enzyme, thus restricting relative mobility between the two. Here, we describe the kinetic and crystallographic analysis of this double mutant enzyme. The 1.6 A resolution crystal structure of the A251C:S430C enzyme under oxidizing conditions reveals the formation of a disulfide bond, while Ellman analysis confirms its presence in the enzyme in solution. Kinetic analyses with the enzyme in both the nonbridged (free thiol) and the disulfide-bridged states indicate a slight decrease in the rate of fumarate reduction when the disulfide bridge is present, while solvent-kinetic-isotope studies indicate that in both wild-type and mutant enzymes the reaction is rate limited by proton and/or hydride transfer during catalysis. The limited effects of the inhibition of clamp domain mobility upon the catalytic reaction would indicate that such mobility is not essential for the regulation of substrate access or product release.     

1.9 A x-ray study shows closed flap conformation in crystals of tethered HIV-1 PR

Three-dimensional structure of an asymmetrically mutated (C95M) tethered human immunodeficiency virus type 1 protease enzyme (HIV-1 PR) has been determined in an unliganded form using X-ray diffraction data to 1.9 A resolution. The structure, refined using X-PLOR to an R factor of 19.5%, is unexpectedly similar to the ligand-bound native enzyme, rather than to the ligand-free native enzyme. In particular, the two flaps in the tethered dimer are in a closed configuration. The environments around M95 and C1095 are identical, showing no structural effect of this asymmetric mutation at position 95. Oxidation of Cys1095 has been observed for the first time. There is one well-defined water molecule that hydrogen bonds to both carboxyl groups of the essential aspartic acids in the active site. Proteins 2001;43:57-64.     

Engineering water to act as an active site acid catalyst in a soluble fumarate reductase

The ability of an arginine residue to function as the active site acid catalyst in the fumarate reductase family of enzymes is now well-established. Recently, a dual role for the arginine during fumarate reduction has been proposed [Mowat, C. G., Moysey, R., Miles, C. S., Leys, D., Doherty, M. K., Taylor, P., Walkinshaw, M. D., Reid, G. A., and Chapman, S. K. (2001) Biochemistry 40, 12292-12298] in which it acts both as a Lewis acid in transition-state stabilization and as a Br?nsted acid in proton delivery. This proposal has led to the prediction that, if appropriately positioned, a water molecule would be capable of functioning as the active site Br?nsted acid. In this paper, we describe the construction and kinetic and crystallographic analysis of the Q363F single mutant and Q363F/R402A double mutant forms of flavocytochrome c(3), the soluble fumarate reductase from Shewanella frigidimarina. Although replacement of the active site acid, Arg402, with alanine has been shown to eliminate fumarate reductase activity, this phenomenon is partially reversed by the additional substitution of Gln363 with phenylalanine. This Gln --> Phe substitution in the inactive R402A mutant enzyme was designed to "push" a water molecule close enough to the substrate C3 atom to allow it to act as a Br?nsted acid. The 2.0 A resolution crystal structure of the Q363F/R402A mutant enzyme does indeed reveal the introduction of a water molecule at the correct position in the active site to allow it to act as the catalytic proton donor. The 1.8 A resolution crystal structure of the Q363F mutant enzyme shows a water molecule similarly positioned, which can account for its measured fumarate reductase activity. However, in this mutant enzyme Michaelis complex formation is impaired due to significant and unpredicted structural changes at the active site.     

Role of Tryptophan 95 in substrate specificity and structural stability of Sulfolobus solfataricus alcohol dehydrogenase

A mutant of the thermostable NAD⁺-dependent (S)-stereospecific alcohol dehydrogenase from Sulfolobus solfataricus (SsADH) which has a single substitution, Trp95Leu, located at the substrate binding pocket, was fully characterized to ascertain the role of Trp95 in discriminating between chiral secondary alcohols suggested by the wild-type SsADH crystallographic structure. The Trp95Leu mutant displays no apparent activity with short-chain primary and secondary alcohols and poor activity with aromatic substrates and coenzyme. Moreover, the Trp → Leu substitution affects the structural stability of the archaeal ADH, decreasing its thermal stability without relevant changes in secondary structure. The double mutant Trp95Leu/Asn249Tyr was also purified to assist in crystallographic analysis. This mutant exhibits higher activity but decreased affinity toward aliphatic alcohols, aldehydes as well as NAD⁺ and NADH compared to the wild-type enzyme. The crystal structure of the Trp95Leu/Asn249Tyr mutant apo form, determined at 2.0 Å resolution, reveals a large local rearrangement of the substrate site with dramatic consequences. The Leu95 side-chain conformation points away from the catalytic metal center and the widening of the substrate site is partially counteracted by a concomitant change of Trp117 side chain conformation. Structural changes at the active site are consistent with the reduced activity on substrates and decreased coenzyme binding.     

Structural backgrounds for the formation of a catalytically competent complex with NADP(H) during hydride transfer in ferredoxin-NADP(+) reductases

The role of the highly conserved C266 and L268 of pea ferredoxin-NADP(+) reductase (FNR) in formation of the catalytically competent complex of the enzyme with NADP(H) was investigated. Previous studies suggest that the volume of these side-chains, situated facing the side of the C-terminal Y308 catalytic residue not stacking the flavin isoalloxazine ring, may be directly involved in the fine-tuning of the catalytic efficiency of the enzyme. Wild-type pea FNR as well as single and double mutants of C266 and L268 residues were analysed by fast transient-kinetic techniques and their midpoint reduction potentials were determined. For the C266A, C266M and C266A/L268A mutants a significant reduction in the overall hydride transfer (HT) rates was observed along with the absence of charge-transfer complex formation. The HT rate constants for NADPH oxidation were lower than those for NADP(+) reduction, reaching a 30-fold decrease in the double mutant. In agreement, these variants exhibited more negative midpoint potentials with respect to the wild-type enzyme. The three-dimensional structures of C266M and L268V variants were solved. The C266M mutant shows a displacement of E306 away from the relevant residue S90 to accommodate the bulky methionine introduced. The overall findings indicate that in FNR the volume of the residue at position 266 is essential to attain the catalytic architecture between the nicotinamide and isoalloxazine rings at the active site and, therefore, for an efficient HT process. In addition, flexibility of the 268-270 loop appears to be critical for FNR to achieve catalytically competent complexes with NADP(H).     

Rational design, structural and thermodynamic characterization of a hyperstable variant of the villin headpiece helical subdomain

A hyperstable variant of the small independently folded helical subdomain (HP36) derived from the F-actin binding villin headpiece was designed by targeting surface electrostatic interactions and helical propensity. A double mutant N68A, K70M was significantly more stable than wild type. The Tm of wild type in aqueous buffer is 73.0 degrees C, whereas the double mutant did not display a complete unfolding transition. The double mutant could not be completely unfolded even by 10 M urea. In 3 M urea, the Tm of wild type is 54.8 degrees C while that of the N68AK70M double mutant is 73.9 degrees C. Amide H/2H exchange studies show that the pattern of exchange is very similar for wild type and the double mutant. The structures of a K70M single mutant and the double mutant were determined by X-ray crystallography and are identical to that of the wild type. Analytical ultracentrifugation demonstrates that the proteins are monomeric. The hyperstable mutant described here is expected to be useful for folding studies of HP36 because studies of the wild type domain have sometimes been limited by its marginal stability. The results provide direct evidence that naturally occurring miniature protein domains have not been evolutionarily optimized for global stability. The stabilizing effect of this double mutant could not be predicted by sequence analysis because K70 is conserved in the larger intact headpiece for functional reasons.     

Comparison of C40/82A and P27A C40/82A barstar mutants using 19F NMR

Barstar, an inhibitor of the enzyme barnase, contains two phenylalanine residues, three tryptophan residues, and two proline residues. After incorporating either 2-19F-Phe, 4-19F-Phe, or 6-19F-Trp, the structural, dynamic, and folding properties of two mutants (C40/82A, a double mutant, and P27A C40/82A, a triple mutant) were studied by 19F NMR. Experiments were performed as a function of temperature and urea with the two mutants. We show that the consequences of the P27A mutation are extensive. The effect of the mutation is transmitted to distant residues (Phe56 and Trp53) as well as to a residue deeply buried in the hydrophobic core (Phe74). By incorporating 2-19F-Phe, it is shown that Phe56 undergoes a slow ring flipping on the NMR time scale in the triple mutant that is not observed in the double mutant. On the other hand, incorporating 4-19F-Phe shows that the P27A mutation has little effect along the Cbeta-Cgamma axis of Phe56. Labeling with 4-19F-Phe shows, from line broadening, that Phe74 experiences more dynamic motion than does Phe56 in both the double and triple mutant. After incorporating 6-19F-Trp, it is found that, in the triple mutant, Trp53 shows conformational heterogeneity at low temperature while Trp44, which is close to the P27A mutation, does not. At 20 degrees C, residual native-like structure was detected around Trp53 at high concentrations of denaturant. Barstar is cold denatured in the presence of urea. For the double mutant at temperatures below 15 degrees C, and in the presence of 2.5-3.5 M urea, the resonance for Phe74 broadens, and two peaks are observed at 5 degrees C indicative of an exchange process. From line-shape analysis, assuming a two-site conformational exchange, the rate constants as a function of temperature can be extracted. An Eyring plot is linear at 0 M urea but deviates from linearity below 20 degrees C in the presence of 2.5 or 3.5 M urea. The data as a function of urea suggest sequential events in the unfolding process.     

The role of water in the catalytic efficiency of triosephosphate isomerase

The structural basis for the effect of the S96P mutation in chicken triosephosphate isomerase (cTIM) has been analyzed using a combination of X-ray crystallography and Fourier transform infrared spectroscopy. The X-ray structure is that of the enzyme complexed with phosphoglycolohydroxamate (PGH), an intermediate analogue, solved at a resolution of 1.9 A. The S96P mutation was identified as a second-site reverent when catalytically crippled mutants, E165D and H95N, were subjected to random mutagenesis. The presence of the second mutation leads to enhanced activity over the single mutation. However, the effect of the S96P mutation alone is to decrease the catalytic efficiency of the enzyme. The crystal structures of the S96P double mutants show that this bulky proline side chain alters the water structure within the active-site cavity (E165D; ref 1) and prevents nonproductive binding conformations of the substrate (H95N; ref 2). Comparison of the S96P single mutant structure with those of the wild-type cTIM, those of the single mutants (E165D and H95N), and those of the double mutants (E165D/S96P and H95N/S96P) begins to address the role of the conserved serine residue at this position. The results indicate that the residue positions the catalytic base E165 optimally for polarization of the substrate carbonyl, thereby aiding in proton abstraction. In addition, this residue is involved in positioning critical water molecules, thereby affecting the way in which water structure influences activity.     

Structural backgrounds for the formation of a catalytically competent complex with NADP(H) during hydride transfer in ferredoxin–NADP+ reductases

The role of the highly conserved C266 and L268 of pea ferredoxin–NADP⁺ reductase (FNR) in formation of the catalytically competent complex of the enzyme with NADP(H) was investigated. Previous studies suggest that the volume of these side-chains, situated facing the side of the C-terminal Y308 catalytic residue not stacking the flavin isoalloxazine ring, may be directly involved in the fine-tuning of the catalytic efficiency of the enzyme. Wild-type pea FNR as well as single and double mutants of C266 and L268 residues were analysed by fast transient-kinetic techniques and their midpoint reduction potentials were determined. For the C266A, C266M and C266A/L268A mutants a significant reduction in the overall hydride transfer (HT) rates was observed along with the absence of charge-transfer complex formation. The HT rate constants for NADPH oxidation were lower than those for NADP⁺ reduction, reaching a 30-fold decrease in the double mutant. In agreement, these variants exhibited more negative midpoint potentials with respect to the wild-type enzyme. The three-dimensional structures of C266M and L268V variants were solved. The C266M mutant shows a displacement of E306 away from the relevant residue S90 to accommodate the bulky methionine introduced. The overall findings indicate that in FNR the volume of the residue at position 266 is essential to attain the catalytic architecture between the nicotinamide and isoalloxazine rings at the active site and, therefore, for an efficient HT process. In addition, flexibility of the 268–270 loop appears to be critical for FNR to achieve catalytically competent complexes with NADP(H).     

Enhancement of 7-methoxyresorufin O-demethylation activity of human cytochrome P450 1A2 by molecular breeding

Alkylresorufins are model substrates for cytochrome P450 (P450) 1A2. The ability of human P450 1A2 to catalyze 7-methoxyresorufin O-demethylation was improved by screening of random mutant libraries (expressed in Escherichia coli) on the basis of 7-methoxyresorufin O-demethylation. After three rounds of mutagenesis and screening, the triple mutant E163K/V193M/K170Q yielded a kcat > five times faster than wild type P450 1A2 in steady-state kinetic analysis using either isolated membrane fractions or purified, reconstituted enzymes. The enhanced catalytic activity was not attributed to changes in substrate affinity. The kinetic hydrogen isotope effect of the triple mutant did not change from wild type enzyme and suggests that C-H bond cleavage is rate-limiting in both enzymes. Homology modeling, based on an X-ray structure of rabbit P450 2C5, suggests that the locations of mutated residues are not close to the substrate binding site and therefore that structural elements outside of this site play roles in changing the catalytic activity. This approach has potential value in understanding P450 1A2 and generating engineered enzymes with enhanced catalytic activity.     

Identification of the residue responsible for catalysing regeneration of activity in the inactive glutamate dehydrogenase mutant D165N

Previously a mutant of clostridial glutamate dehydrogenase with the catalytic Asp-165 replaced by Asn was shown to regain activity through spontaneous, specific deamidation of this residue. A double mutant D165N/K125A has now been constructed, in which the catalytic Lys is also replaced. This was successfully over-expressed and according to several criteria appears to be correctly folded. The double mutant was incubated for 35 days under conditions where D165N reactivates. LC-MS analysis of tryptic digests of timed samples showed no significant deamidation. This confirms that the reactivation of D165N is a consequence of the catalytic chemistry of the enzyme's active site.     

Primary quinone (QA) binding site of bacterial photosynthetic reaction centers: mutations at residue M265 probed by FTIR spectroscopy

In the primary quinone (Q(A)) binding site of Rb. sphaeroides reaction centers (RCs), isoleucine M265 is in extensive van der Waals contact with the ubiquinone headgroup. Substitution of threonine or serine for this residue (mutants M265IT and M265IS), but not valine (mutant M265IV), lowers the redox midpoint potential of Q(A) by about 100 mV (Takahashi et al. (2001) Biochemistry 40, 1020-1028). The unexpectedly large effect of the polar substitutions is not due to reorientation of the methoxy groups as similar redox potential changes are seen for these mutants with either ubiquinone or anthraquinone as Q(A). Using FTIR spectroscopy to compare Q(A)(-)/Q(A) IR difference spectra for wild type and the M265 mutant RCs, we found changes in the polar mutants (M265IT and M265IS) in the quinone C[double bond]O and C[double bond]C stretching region (1600-1660 cm(-1)) and in the semiquinone anion band (1440-1490 cm(-1)), as well as in protein modes. Modeling the mutations into the X-ray structure of the wild-type RC indicates that the hydroxyl group of the mutant polar residues, Thr and Ser, is hydrogen bonded to the peptide C[double bond]O of Thr(M261). It is suggested that the mutational effect is exerted through the extended backbone region that includes Ala(M260), the hydrogen bonding partner to the C1 carbonyl of the quinone headgroup. The resulting structural perturbations are likely to include lengthening of the hydrogen bond between the quinone C1[double bond]O and the peptide NH of Ala(M260). Possible origins of the IR spectroscopic and redox potential effects are discussed.     

Response of dynamic structure to removal of a disulfide bond: normal mode refinement of C77A/C95A mutant of human lysozyme.

In order to investigate the response of dynamic structure to removal of a disulfide bond, the dynamic structure of human lysozyme has been compared to its C77A/C95A mutant. The dynamic structures of the wild type and mutant are determined by normal mode refinement of 1.5-A-resolution X-ray data. The C77A/C95A mutant shows an increase in apparent fluctuations at most residues. However, most of the change originates from an increase in the external fluctuations, reflecting the effect of the mutation on the quality of crystals. The effects of disulfide bond removal on the internal fluctuations are almost exclusively limited to the mutation site at residue 77. No significant change in the correlation of the internal fluctuations is found in either the overall or local dynamics. This indicates that the disulfide bond does not have any substantial role to play in the dynamic structure. A comparison of the wild-type and mutant coordinates suggests that the disulfide bond does not prevent the 2 domains from parting from each other. Instead, the structural changes are characteristic of a cavity-creating mutation, where atoms surrounding the mutation site move cooperatively toward the space created by the smaller alanine side chain. Although this produces tighter packing, more than half of the cavity volume remains unoccupied, thus destabilizing the native state.     

Asp-99 donates a hydrogen bond not to Tyr-14 but to the steroid directly in the catalytic mechanism of Delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B

Delta 5-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Delta 5-3-ketosteroids at a rate approaching the diffusion limit by an intramolecular transfer of a proton. Despite the extensive studies on the catalytic mechanism, it still remains controversial whether the catalytic residue Asp-99 donates a hydrogen bond to the steroid or to Tyr-14. To clarify the role of Asp-99 in the catalysis, two single mutants of D99E and D99L and three double mutants of Y14F/D99E, Y14F/D99N, and Y14F/D99L have been prepared by site-directed mutagenesis. The D99E mutant whose side chain at position 99 is longer by an additional methylene group exhibits nearly the same kcat as the wild-type while the D99L mutant exhibits ca. 125-fold lower kcat than that of the wild-type. The mutations made at positions 14 and 99 exert synergistic or partially additive effect on kcat in the double mutants, which is inconsistent with the mechanism based on the hydrogen-bonded catalytic dyad, Asp-99 COOH...Tyr-14 OH...C3-O of the steroid. The crystal structure of D99E/D38N complexed with equilenin, an intermediate analogue, at 1.9 A resolution reveals that the distance between Tyr-14 O eta and Glu-99 O epsilon is ca. 4.2 A, which is beyond the range for a hydrogen bond, and that the distance between Glu-99 O epsilon and C3-O of the steroid is maintained to be ca. 2.4 A, short enough for a hydrogen bond to be formed. Taken together, these results strongly support the idea that Asp-99 contributes to the catalysis by donating a hydrogen bond directly to the intermediate.     

S-adenosyl-L-methionine-dependent methyl transfer: observable precatalytic intermediates during DNA cytosine methylation

S-adenosyl-L-methionine- (AdoMet-) dependent methyltransferases are widespread, play critical roles in diverse biological pathways, and are antibiotic and cancer drug targets. Presently missing from our understanding of any AdoMet-dependent methyl-transfer reaction is a high-resolution structure of a precatalytic enzyme/AdoMet/DNA complex. The catalytic mechanism of DNA cytosine methylation was studied by structurally and functionally characterizing several active site mutants of the bacterial enzyme M.HhaI. The 2.64 A resolution protein/DNA/AdoMet structure of the inactive C81A M.HhaI mutant suggests that active site water, an approximately 13 degree tilt of the target base toward the active site nucleophile, and the presence or absence of the cofactor methylsulfonium are coupled via a hydrogen-bonding network involving Tyr167. The active site in the mutant complex is assembled to optimally align the pyrimidine for nucleophilic attack and subsequent methyl transfer, consistent with previous molecular dynamics ab initio and quantum mechanics/molecular mechanics calculations. The mutant/DNA/AdoHcy structure (2.88 A resolution) provides a direct comparison to the postcatalytic complex. A third C81A ternary structure (2.22 A resolution) reveals hydrolysis of AdoMet to adenosine in the active site, further validating the coupling between the methionine portion of AdoMet and ultimately validating the structural observation of a prechemistry/postchemistry water network. Disruption of this hydrogen-bonding network by a Tyr167 to Phe167 mutation does not alter the kinetics of nucleophilic attack or methyl transfer. However, the Y167F mutant shows detectable changes in kcat, caused by the perturbed kinetics of AdoHcy release. These results provide a basis for including an extensive hydrogen-bonding network in controlling the rate-limiting product release steps during cytosine methylation.