Catalytic role of a conserved cysteine residue in the desulfonation reaction by the alkanesulfonate monooxygenase enzyme

Detailed kinetic studies were performed in order to determine the role of the single cysteine residue in the desulfonation reaction catalyzed by SsuD. Mutation of the conserved cysteine at position 54 in SsuD to either serine or alanine had little effect on FMNH₂ binding. The k_cat/K_m value for the C54S SsuD variant increased 3-fold, whereas the k_cat/K_m value for C54A SsuD decreased 6-fold relative to wild-type SsuD. An initial fast phase was observed in kinetic traces obtained for the oxidation of flavin at 370 nm when FMNH₂ was mixed against C54S SsuD (k_obs, 111 s^− 1) in oxygenated buffer that was 10-fold faster than wild-type SsuD (k_obs, 12.9 s^− 1). However, there was no initial fast phase observed in similar kinetic traces obtained for C54A SsuD. This initial fast phase was previously assigned to the formation of the C4a-(hydro)peroxyflavin in studies with wild-type SsuD. There was no evidence for the formation of the C4a-(hydro)peroxyflavin with either SsuD variant when octanesulfonate was included in rapid reaction kinetic studies, even at low octanesulfonate concentrations. The absence of any C4a-(hydro)peroxyflavin accumulation correlates with the increased catalytic activity of C54S SsuD. These results suggest that the conservative serine substitution is able to effectively take the place of cysteine in catalysis. Conversely, decreased accumulation of the C4a-(hydro)peroxyflavin intermediate with the C54A SsuD variant may be due to decreased activity. The data described suggest that Cys54 in SsuD may be either directly or indirectly involved in stabilizing the C4a-(hydro)peroxyflavin intermediate formed during catalysis through hydrogen bonding interactions.     

The critical structural role of a highly conserved histidine residue in group II amino acid decarboxylases

Glutamate decarboxylase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme, belonging to the subset of PLP-dependent decarboxylases classified as group II. Site-directed mutagenesis of Escherichia coli glutamate decarboxylase, combined with analysis of the crystal structure, shows that a histidine residue buried in the protein core is critical for correct folding. This histidine is strictly conserved in the PF00282 PFAM family, which includes the group II decarboxylases. A similar role is proposed for residue Ser269, also highly conserved in this group of enzymes, as it provides one of the interactions stabilising His241.     

Genetic selection reveals the role of a buried, conserved polar residue

The burial of nonpolar surface area is known to enhance markedly the conformational stability of proteins. The contribution from the burial of polar surface area is less clear. Here, we report on the tolerance to substitution of Ser75 of bovine pancreatic ribonuclease (RNase A), a residue that has the unusual attributes of being buried, conserved, and polar. To identify variants that retain biological function, we used a genetic selection based on the intrinsic cytotoxicity of ribonucleolytic activity. Cell growth at 30 degrees C, 37 degrees C, and 44 degrees C correlated with residue size, indicating that the primary attribute of Ser75 is its small size. The side-chain hydroxyl group of Ser75 forms a hydrogen bond with a main-chain nitrogen. The conformational stability of the S75A variant, which lacks this hydrogen bond, was diminished by DeltaDeltaG = 2.5 kcal/mol. Threonine, which can reinstate this hydrogen bond, provided a catalytically active RNase A variant at higher temperatures than did some smaller residues (including aspartate), indicating that a secondary attribute of Ser75 is the ability of its uncharged side chain to accept a hydrogen bond. These results provide insight on the imperatives for the conservation of a buried polar residue.     

The role of a conserved guanosine residue in the hammerhead-type RNA enzyme.

We have designed a hammerhead-type RNA system which consists of three RNA fragments for normal and modified complexes which contain a non-cleavable substrate with 2'-O-methylcytidine and a guanosine-to-inosine replaced enzyme. Examination of the RNA-cleaving activity and conformational properties of the complexes suggests that the 2-amino group of a conserved guanosine residue in the loop region plays an important role for maintaining both the activity and loop conformation.     

Multi-timescale dynamics study of FKBP12 along the rapamycin-mTOR binding coordinate

Drugs can affect function in proteins by modulating their flexibility. Despite this possibility, there are very few studies on how drug binding affects the dynamics of target macromolecules. FKBP12 (FK506 binding protein 12) is a prolyl cis-trans isomerase and a drug target. The immunosuppressant drug rapamycin exerts its therapeutic effect by serving as an adaptor molecule between FKBP12 and the cell proliferation regulator mTOR (mammalian target of rapamycin). To understand the role of dynamics in rapamycin-based immunosuppression and to gain insight into the role of dynamics in the assembly of supramolecular complexes, we used ¹⁵N, ¹³C, and ²H NMR spin relaxation to characterize FKBP12 along the binding coordinate that leads to cell cycle arrest. We show that sequential addition of rapamycin and mTOR leads to incremental rigidification of the FKBP12 backbone on the picosecond-nanosecond timescale. Both binding events lead to perturbation of main-chain and side-chain dynamics at sites distal to the binding interfaces, suggesting tight coupling interactions dispersed throughout the FKBP12-rapamycin interface. Binding of the first molecule, rapamycin, quenches microsecond-millisecond motions of the FKBP12 80's loop. This loop provides much of the surface buried at the protein-protein interface of the ternary complex, leading us to assert that preorganization upon rapamycin binding facilitates binding of the second molecule, mTOR. Widespread microsecond-millisecond motions of the backbone persist in the drug-bound enzyme, and we provide evidence that these slow motions represent coupled dynamics of the enzyme and isomerization of the bound drug. Finally, the pattern of microsecond-millisecond dynamics reported here in the rapamycin complex is dramatically different from the pattern in the complex with the structurally related drug FK506. This raises the important question of how two complexes that are highly isomorphic based on high-resolution static models have such different flexibilities in solution.     

The strictly conserved Arg-321 residue in the active site of Escherichia coli topoisomerase I plays a critical role in DNA rejoining

The strictly conserved arginine residue proximal to the active site tyrosine of type IA topoisomerases is required for the relaxation of supercoiled DNA and was hypothesized to be required for positioning of the scissile phosphate for DNA cleavage to take place. Mutants of recombinant Yersinia pestis topoisomerase I with hydrophobic substitutions at this position were found in genetic screening to exhibit a dominant lethal phenotype, resulting in drastic loss in Escherichia coli viability when overexpressed. In depth biochemical analysis of E. coli topoisomerase I with the corresponding Arg-321 mutation showed that DNA cleavage can still take place in the absence of this arginine function if Mg(2+) is present to enhance the interaction of the enzyme with the scissile phosphate. However, DNA rejoining is inhibited in the absence of this conserved arginine, resulting in accumulation of the cleaved covalent intermediate and loss of relaxation activity. These new experimental results demonstrate that catalysis of DNA rejoining by type IA topoisomerases has a more stringent requirement than DNA cleavage. In addition to the divalent metal ions, the side chain of this arginine residue is required for the precise positioning of the phosphotyrosine linkage for nucleophilic attack by the 3'-OH end to result in DNA rejoining. Small molecules that can interfere or distort the enzyme-DNA interactions required for DNA rejoining by bacterial type IA topoisomerases could be developed into novel antibacterial drugs.     

The role of a conserved histidine residue in a pyruvate-specific Class II aldolase

Histidine 45 in HpaI was replaced with alanine (H45A) and glutamine (H45Q). In the aldol cleavage reaction, kcat values were lowered by 78- and 2059-fold while Km values were increased by 100- and 42-fold in H45A and H45Q, respectively, compared to the wild-type enzyme. Both mutants displayed higher dissociation constants towards the metal cofactor, pyruvate and the transition state analogue, oxalate. Pyruvate proton exchange rates are consequently reduced in H45A and H45Q. pKa for a catalytic base (6.5) is lost in the mutant enzymes and catalysis is dependent on hydroxide ions. The results show that histidine 45 is important for metal cofactor binding and for facilitating C4-OH proton abstraction of the substrate in the reaction mechanism.     

M42 aminopeptidase catalytic site: the structural and functional role of a strictly conserved aspartate residue

The M42 aminopeptidases are a family of dinuclear aminopeptidases widely distributed in Prokaryotes. They are potentially associated to the proteasome, achieving complete peptide destruction. Their most peculiar characteristic is their quaternary structure, a tetrahedron-shaped particle made of twelve subunits. The catalytic site of M42 aminopeptidases is defined by seven conserved residues. Five of them are involved in metal ion binding which is important to maintain both the activity and the oligomeric state. The sixth conserved residue, a glutamate, is the catalytic base deprotonating the water molecule during peptide bond hydrolysis. The seventh residue is an aspartate whose function remains poorly understood. This aspartate residue, however, must have a critical role as it is strictly conserved in all MH clan enzymes. It forms some kind of catalytic triad with the histidine residue and the metal ion of the M2 binding site. We assess its role in TmPep1050, an M42 aminopeptidase of Thermotoga maritima, through a mutational approach. Asp-62 was substituted with alanine, asparagine, or glutamate residue. The Asp-62 substitutions completely abolished TmPep1050 activity and impeded dodecamer formation. They also interfered with metal ion binding as only one cobalt ion is bound per subunit instead of two. The structure of Asp62Ala variant was solved at 1.5 Å showing how the substitution has an impact on the active site fold. We propose a structural role for Asp-62, helping to stabilize a crucial loop in the active site and to position correctly the catalytic base and a metal ion ligand of the M1 site.     

The role of a conserved tyrosine residue in high-potential iron sulfur proteins.

Conserved tyrosine-12 of Ectothiorhodospira halophila high-potential iron sulphur protein (HiPIP) iso-I was substituted with phenylalanine (Y12F), histidine (Y12H), tryptophan (Y12W), isoleucine (Y12I), and alanine (Y12A). Variants Y12A and Y12I were expressed to reasonable levels in cells grown at lower temperatures, but decomposed during purification. Variants Y12F, Y12H, and Y12W were substantially destabilized with respect to the recombinant wild-type HiPIP (rcWT) as determined by differential scanning calorimetry over a pH range of 7.0-11.0. Characterization of the Y12F variant by NMR indicates that the principal structural differences between this variant and the rcWT HiPIP result from the loss of the two hydrogen bonds of the Tyr-12 hydroxyl group with Asn-14 O delta 1 and Lys-59 NH, respectively. The effect of the loss of the latter interaction is propagated through the Lys-59/Val-58 peptide bond, thereby perturbing Gly-46. The delta delta GDapp of Y12F of 2.3 kcal/mol with respect to rcWT HiPIP (25 degrees C, pH 7.0) is entirely consistent with the contribution of these two hydrogen bonds to the stability of the latter. CD measurements show that Tyr-12 influences several electronic transitions within the cluster. The midpoint reduction potentials of variants Y12F, Y12H, and Y12W were 17, 19, and 22 mV (20 mM MOPS, 0.2 M sodium chloride, pH 6.98, 25 degrees C), respectively, higher than that of rcWT HiPIP. The current results indicate that, although conserved Tyr-12 modulates the properties of the cluster, its principle function is to stabilize the HiPIP through hydrogen bonds involving its hydroxyl group and electrostatic interactions involving its aromatic ring.     

Functional role of a conserved arginine residue located on a mobile loop of alkanesulfonate monooxygenase

The structure of the flavin-dependent alkanesulfonate monooxygenase (SsuD) exists as a TIM-barrel structure with an insertion region located over the active site that contains a conserved arginine (Arg297) residue present in all SsuD homologues. Substitution of Arg297 with alanine (R297A SsuD) or lysine (R297K SsuD) was performed to determine the functional role of this conserved residue in SsuD catalysis. While the more conservative R297K SsuD possessed a lower k(cat)/K(m) value (0.04 ± 0.01 μM(-1) min(-1)) relative to wild-type (1.17 ± 0.22 μM(-1) min(-1)), there was no activity observed with the R297A SsuD variant. Each of the arginine variants had similar K(d) values for flavin binding as wild-type SsuD (0.32 ± 0.15 μM), but there was no measurable binding of octanesulfonate. The low levels of activity for the R297A and R297K SsuD variants correlated with the absence of any detectable C4a-(peroxy)flavin formation in stopped-flow kinetic studies. Single-turnover experiments were performed in the presence of SsuE to evaluate both the reductive and oxidative half-reaction. With wild-type SsuD a lag phase is observed following the reductive half-reaction by SsuE that represents flavin transfer or conformational changes associated with the binding of substrates. Evaluation of the Arg297 SsuD variants in the presence of SsuE showed no lag phase following reduction by SsuE, and the flavin was oxidized immediately following the reductive half-reaction. These results corresponded with a lack of detectable changes in the proteolytic susceptibility of R297A and R297K SsuD in the presence of reduced flavin and/or octanesulfonate, signifying the absence of a conformational change in these variants with the substitution of Arg297.     

Aminobacter aminovorans NADH:flavin oxidoreductase His140: a highly conserved residue critical for NADH binding and utilization

Homodimeric FRD(Aa) Class I is an NADH:flavin oxidoreductase from Aminobacter aminovorans. It is unusual because it contains an FMN cofactor but utilizes a sequential-ordered kinetic mechanism. Because little is known about NADH-specific flavin reductases in general and FRD(Aa) in particular, this study aimed to further explore FRD(Aa) by identifying the functionalities of a key residue. A sequence alignment of FRD(Aa) with several known and hypothetical flavoproteins in the same subfamily reveals within the flavin reductase active-site domain a conserved GDH motif, which is believed to be responsible for the enzyme and NADH interaction. Mutation of the His140 in this GDH motif to alanine reduced FRD(Aa) activity to <3%. An ultrafiltration assay and fluorescence quenching demonstrated that H140A FRD(Aa) binds FMN in the same 1:1 stoichiometric ratio as the wild-type enzyme, but with slightly weakened affinity (K(d) = 0.9 microM). Anaerobic stopped-flow studies were carried out using both the native and mutated FRD(Aa). Similar to the native enzyme, H140A FRD(Aa) was also able to reduce the FMN cofactor by NADH although much less efficiently. Kinetic analysis of anaerobic reduction measurements indicated that the His140 residue of FRD(Aa) was essential to NADH binding, as well as important for the reduction of the FMN cofactor. For the native enzyme, the cofactor reduction was followed by at least one slower step in the catalytic pathway.     

Electrostatic guidance of glycosyl cation migration along the reaction coordinate of uracil DNA glycosylase

The DNA repair enzyme uracil DNA glycosylase has been crystallized with a cationic 1-aza-2'-deoxyribose-containing DNA that mimics the ultimate transition state of the reaction in which the water nucleophile attacks the anomeric center of the oxacarbenium ion-uracil anion reaction intermediate. Comparison with substrate and product structures, and the previous structure of the intermediate determined by kinetic isotope effects, reveals an exquisite example of geometric strain, least atomic motion, and electrophile migration in biological catalysis. This structure provides a rare opportunity to reconstruct the detailed structural transformations that occur along an enzymatic reaction coordinate.     

Molecular dynamics study of the metallochaperone Hah1 in its apo and Cu(I)-loaded states: role of the conserved residue M10

Molecular dynamics simulations were performed on both apo and copper forms of the human copper chaperone, Hah1. Wild-type Hah1 and a methionine (M10) to serine mutant were investigated. We have evidenced the central role of residue M10 in stabilizing the hydrophobic core of Hah1 as well as the internal structure of the metal-binding site. When copper(I) is bound, the mobility of Hah1 is reduced whereas mutation of M10 implies a drastic increase of the mobility of apoHah1, stressing the importance of this highly conserved hydrophobic residue for copper sequestration by the apoprotein.     

On the role of a conserved, potentially helix-breaking residue in the tRNA-binding alpha-helix of archaeal CCA-adding enzymes

Archaeal class I CCA-adding enzymes use a ribonucleoprotein template to build and repair the universally conserved 3'-terminal CCA sequence of the acceptor stem of all tRNAs. A wealth of structural and biochemical data indicate that the Archaeoglobus fulgidus CCA-adding enzyme binds primarily to the tRNA acceptor stem through a long, highly conserved alpha-helix that lies nearly parallel to the acceptor stem and makes many contacts with its sugar-phosphate backbone. Although the geometry of this alpha-helix is nearly ideal in all available cocrystal structures, the helix contains a highly conserved, potentially helix-breaking proline or glycine near the N terminus. We performed a mutational analysis to dissect the role of this residue in CCA-addition activity. We found that the phylogenetically permissible P295G mutant and the phylogenetically absent P295T had little effect on CCA addition, whereas P295A and P295S progressively interfered with CCA addition (C74>C75>A76 addition). We also examined the effects of these mutations on tRNA binding and the kinetics of CCA addition, and performed a computational analysis using Rosetta Design to better understand the role of P295 in nucleotide transfer. Our data indicate that CCA-adding activity does not correlate with the stability of the pre-addition cocrystal structures visualized by X-ray crystallography. Rather, the data are consistent with a transient conformational change involving P295 of the tRNA-binding alpha-helix during or between one or more steps in CCA addition.     

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.     

A conserved histidine in vertebrate-type ferredoxins is critical for redox-dependent dynamics

Adrenodoxin (Adx) belongs to the family of Cys(4)Fe(2)S(2) vertebrate-type ferredoxins that shuttle electrons from NAD(P)H-dependent reductases to cytochrome P450 enzymes. The vertebrate-type ferredoxins contain a conserved basic residue, usually a histidine, adjacent to the third cysteine ligand of the Cys(4)Fe(2)S(2) cluster. In bovine Adx the side chain of this residue, His 56, is involved in a hydrogen-bonding network within the domain of Adx that interacts with redox partners. It has been proposed that this network acts as a mechanical link between the metal cluster binding site and the interaction domain, transmitting redox-dependent conformational or dynamical changes from the cluster binding loop to the interaction domain. H/D exchange studies indicate that oxidized Adx (Adx(o)) is more dynamic than reduced Adx (Adx(r)) on the kilosecond time scale in many regions of the protein, including the interaction domain. Dynamical differences on picosecond to nanosecond time scales between the oxidized (Adx(o)) and reduced (Adx(r)) adrenodoxin were probed by measurement of (15)N relaxation parameters. Significant differences between (15)N R(2) rates were observed for all residues that could be measured, with those rates being faster in Adx(o) than in Adx(r). Two mutations of His 56, H56R and H56Q, were also characterized. No systematic redox-dependent differences between (15)N R(2) rates or H/D exchange rates were observed in either mutant, indicating that His 56 is required for the redox-dependent behavior observed in WT Adx. Comparison of chemical shift differences between oxidized and reduced H56Q and H56R Adx confirms that redox-dependent changes are smaller in these mutants than in the wild-type Adx.