Identifying groups involved in the binding of prephenate to prephenate dehydrogenase from Escherichia coli

Site-directed mutagenesis was used to investigate the importance of Lys178, Arg286, and Arg294 in the binding of prephenate to the bifunctional enzyme chorismate mutase-prephenate dehydrogenase. From comparison of the kinetic parameters of wild-type enzyme and selected mutants, we conclude that only Arg294 interacts specifically with prephenate. The R294Q substitution reduces the enzyme's affinity for prephenate without affecting V/Et of the dehydrogenase reaction or the kinetic parameters of the mutase reaction. Arg294 likely interacts with the ring carboxylate at C-1 of prephenate since the dissociation constants for a series of inhibitors missing the ring carboxyl group were similar for wild-type and R294Q enzymes. The pH dependencies of log (V/KprephenateEt) and of pKi for hydroxyphenyllactate show that the wild-type dehydrogenase possesses a group with a pK of 8.8 that must be protonated for binding prephenate to the enzyme. None of the three conserved residues is this group since its titration is observed in the V/KprephenateEt profiles for the mutants K178Q, R286A, and R294Q. This group is also seen in the pH-rate profiles of the binding of two substrate analogues, hydroxyphenyllactate and deoxoprephenate. Their only common structural feature at C-1 is the side chain carboxylate, indicating that the protonated residue (pK 8.8) must interact with prephenate's side chain carboxylate. Gdn-HCl-induced denaturation was conducted on wild-type and selected mutant proteins. Unfolding of the wild-type enzyme proceeds through a partially unfolded dimer which dissociates into unfolded monomers. The order of stability is wild-type = R294Q > K178Q > R286A > K178R. The least unstable mutants have reduced mutase and dehydrogenase activities.     

A T4-phage deoxycytidylate deaminase mutant that no longer requires deoxycytidine 5'-triphosphate for activation

A deoxycytidylate (dCMP) deaminase encoded in T4-bacteriophage DNA that is induced on phage infection of Escherichia coli was shown earlier (Maley, G. F., Duceman, B. W., Wang, A. M., Martinez, J. M., and Maley, F. (1990) J. Biol. Chem. 265, 47-51) to be similar in size, properties, and amino acid composition to the T2-phage-induced deaminase. Neither enzyme is active in the absence of dCTP or its natural activator, 5-hydroxymethyl-dCTP. However, on changing the arginine (Arg) at residue 115 of the T4-deaminase to either a glutamate (R115E) or a glutamine (R115Q), the resulting mutant enzymes were active in the absence of dCTP, with each mutant possessing a turnover number or k(cat) that is about 15% that of the wild-type deaminase. When compared on the basis of specific activity, however, the mutants are about 40-50% of the wild-type (WT)-enzyme's specific activity. Molecular weight analysis on the wild-type and mutant deaminases using HPLC size exclusion chromatography revealed that the wild-type deaminase was basically a hexamer, particularly in the presence of dCTP, regardless of the extent of dilution. Under similar conditions, R115E remained a dimer, whereas R115Q and F112A varied from hexamers to dimers particularly at concentrations normally present in the assay solution. Activity measurements appear to support the conclusion that the hexameric form of the enzyme is activated by dCTP, while the dimer is not. Another feature emphasizing the difference between the WT and mutant deaminases was observed on their denaturation-renaturation in EDTA, which revealed the mutants to be restored to 50% of their original activities with the WT deaminase only marginally restored.     

Arg352 is a major determinant of charge selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel

The cystic fibrosis transmembrane conductance regulator forms an anion-selective channel. We previously showed that charge selectivity, the ability to discriminate between anions and cations, occurs near the cytoplasmic end of the channel. The molecular determinants of charge selectivity, however, are unknown. We investigated the role of Arg352, a residue flanking the predicted cytoplasmic end of the M6 segment, in the mechanism of charge selectivity. We determined the Cl- to Na+ permeability ratio (PCl/PNa) from the reversal potential measured in a 10-fold NaCl gradient. For the wild type, PCl/PNa was 36 (range of 28-51). For the R352H mutant, PCl/PNa was dependent on cytoplasmic pH. At pH 5.4, the PCl/PNa was 33 (range of 27-41), similar to that of the wild type, but at pH 7.2, where the histidine should be largely uncharged, PCl/PNa was 3 (range of 2.9-3.1). For the R352C and R352Q mutants, PCl/PNa was 7 (range of 6-8) and 4 (range of 3.5-4.4), respectively. Furthermore, Na+ which does not carry a significant fraction of the current through the wild type is measurably conducted through R352Q. Thus, the charge of the side chain at position 352 is a strong determinant of charge selectivity. In the wild type, the positive charge on Arg352 contributes to an electrostatic potential in the channel that forms a barrier to cation permeation. Mutation of Arg352 did not alter the halide selectivity sequence. Selectivity among halides must involve other residues.     

Essential role of conserved arginine 160 in intramolecular electron transfer in human sulfite oxidase

Arginine 160 in human sulfite oxidase (SO) is conserved in all SO species sequenced to date. Previous steady-state kinetic studies of the R160Q human SO mutant showed a remarkable decrease in k(cat)/K(m)(sulfite) of nearly 1000-fold, which suggests that Arg 160 in human SO makes an important contribution to the binding of sulfite near the molybdenum cofactor [Garrett, R. M., Johnson, J. L., Graf, T. N., Feigenbaum, A., Rajagopalan, K. V. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 6394-6398]. In the crystal structure of chicken SO, Arg 138, the equivalent of Arg 160 in human SO, is involved in the formation of a positively charged sulfite binding site [Kisker, C., Schindelin, H., Pacheco, A., Wehbi, W., Garnett, R. M., Rajagopalan, K. V., Enemark, J. H., Rees, D. C. (1997) Cell 91, 973-983]. To further assess the role of Arg 160 in human SO, intramolecular electron transfer (IET) rates between the reduced heme [Fe(II)] and oxidized molybdenum [Mo(VI)] centers in the wild type, R160Q, and R160K human SO forms were investigated by laser flash photolysis. In the R160Q mutant, the IET rate constant at pH 6.0 was decreased by nearly 3 orders of magnitude relative to wild type, which indicates that the positive charge of Arg 160 is essential for efficient IET in human SO. Furthermore, the IET rate constant for the R160K mutant is about one-fourth that of the wild type enzyme, which strongly indicates that it is the loss of charge of Arg 160, and not its precise location, that is responsible for the much larger decrease in IET rates in the R160Q mutant. Steady-state kinetic measurements indicate that IET is rate-limiting in the catalytic cycle of the R160Q mutant. Thus, the large decrease in the IET rate constant rationalizes the fatal impact of this mutation in patients with this genetic disorder.     

Defining the role of arginine 96 in green fluorescent protein fluorophore biosynthesis

Aequoria victoria green fluorescent protein (GFP) is a revolutionary molecular biology tool because of its spontaneous peptide backbone cyclization and chromophore formation from residues Ser65, Tyr66, and Gly67. Here we use structure-based design, comprehensive targeted mutagenesis, and high-resolution crystallography to probe the significant functional role of conserved Arg96 (R96) in chromophore maturation. The R96M GFP variant, in which the R96M side chain is similar in volume but lacks the R96 positive charge, exhibits dramatically slower chromophore maturation kinetics (from hours to months). Comparison of the precyclized conformation of the chromophore-forming residues with the mature R96M chromophore reveals a similar Y66 conformer, contrary to the large Y66 conformational change previously defined in the slowly maturing R96A variant [Barondeau, D. P., Putnam, C. D., Kassmann, C. J., Tainer, J. A., and Getzoff, E. D. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 12111-12116]. Comprehensive R96 mutagenesis and fluorescent colony screening indicate that only the R96K substitution restores wild-type maturation kinetics. Further, we show that the slowly maturing R96A variant can be complemented with a Q183R second-site mutation designed to restore the missing R96 positive charge and rapid fluorophore biosynthesis. Moreover, comparative structural analysis of R96M, R96K, R96A/Q183R, and wild-type GFP reveals the importance of the presence of positive charge, rather than its exact position. Together, these structural, mutational, and biochemical results establish a pivotal role for the R96 positive charge in accelerating the GFP post-translational modification, with implications for peptide backbone cyclization in GFP, its homologues, and related biological systems.     

The Na channel voltage sensor associated with inactivation is localized to the external charged residues of domain IV, S4.

Site-3 toxins have been shown to inhibit a component of gating charge (33% of maximum gating charge, Q(max)) in native cardiac Na channels that has been identified with the open-to-inactivated state kinetic transition. To investigate the role of the three outermost arginine amino acid residues in segment 4 domain IV (R1, R2, R3) in gating charge inhibited by site-3 toxins, we recorded ionic and gating currents from human heart Na channels with mutations of the outermost arginines (R1C, R1Q, R2C, and R3C) expressed in fused, mammalian tsA201 cells. All four mutations had ionic currents that activated over the same voltage range with slope factors of their peak conductance-voltage (G-V) relationships similar to those of wild-type channels, although decay of I(Na) was slowest for R1C and R1Q mutant channels and fastest for R3C mutant channels. After Na channel modification by Ap-A toxin, decays of I(Na) were slowed to similar values for all four channel mutants. Toxin modification produced a graded effect on gating charge (Q) of mutant channels, reducing Q(max) by 12% for the R1C and R1Q mutants, by 22% for the R2C mutant, and by 27% for the R3C mutant, only slightly less than the 31% reduction seen for wild-type currents. Consistent with these findings, the relationship of Q(max) to G(max) was significantly shallower for R1 mutants than for R2C and R3C mutant Na channels. These data suggest that site-3 toxins primarily inhibit gating charge associated with movement of the S4 in domain IV, and that the outermost arginine contributes the largest amount to channel gating, with other arginines contributing less.     

Implication by site-directed mutagenesis of Arg314 and Tyr316 in the coenzyme site of pig mitochondrial NADP-dependent isocitrate dehydrogenase

Sequence alignment of pig mitochondrial NADP-dependent isocitrate dehydrogenase with eukaryotic (human, rat, and yeast) and Escherichia coli isocitrate dehydrogenases reveals that Tyr316 is completely conserved and is equivalent to the E. coli Tyr345, which interacts with the 2'-phosphate of NADP in the crystal structure [Hurley et al., Biochemistry 30 (1991) 8671-8678]. Lys321 is also completely conserved in the five isocitrate dehydrogenases. Either an arginine or lysine residue is found among the enzymes from other species at the position corresponding to the pig enzyme Arg314. While Arg323 is not conserved among all species, its proximity to the coenzyme site makes it a good candidate for investigation. The importance of these four amino acids to the function of pig mitochondrial NADP-isocitrate dehydrogenase was studied by site-directed mutagenesis. Mutants (R314Q, Y316F, Y316L, K321Q, and R323Q) were generated by a megaprimer polymerase chain reaction method. Wild-type and mutant enzymes were expressed in E. coli and purified to homogeneity. All mutant and wild-type enzymes exhibited comparable molecular weights indicative of the dimeric enzyme. Mutations do not cause an appreciable change in enzyme secondary structure as revealed by circular dichroism measurements. The kinetic parameters (V(max) and K(M) values) of K321Q and R323Q are similar to those of wild-type, indicating that Lys321 and Arg323 are not involved in enzyme function. R314Q exhibits a 10-fold increase in K(M) for NADP as compared to that of wild-type, while they have comparable V(max) values. These results suggest that Arg314 contributes to the affinity between the enzyme and NADP. The hydroxyl group of Tyr316 is not required for enzyme function since Y316F exhibits similar kinetic parameters to those of wild-type. Y316L shows a 4-fold increase in K(M) for NADP and a decrease in V(max) as compared to wild-type, suggesting that the aromatic ring of the Tyr of isocitrate dehydrogenase contributes to the affinity for coenzyme, as well as to catalysis. The K(i) for NAD of R314Q, Y316F, and Y316L is comparable to that of wild-type, indicating that the Arg314 and Tyr316 may be located near the 2'-phosphate of enzyme-bound NADP.     

Role of charged residues at the OmpF porin channel constriction probed by mutagenesis and simulation

The channel constriction of OmpF porin, a pore protein in the bacterial outer membrane, is highly charged due to the presence of three arginines (R42, R82, and R132) and two acidic residues (D113 and E117). The influence of these charges on ion conductance, ion selectivity, and voltage gating has been studied with mutants D113N/E117Q, R42A/R82A/R132A/D113N/E117Q, and V18K/G131K, which were designed to remove or add protein charge at the channel constriction. The crystal structures revealed no or only local changes compared to wild-type OmpF, thus allowing a comparative study. The single-channel conductance of the isosteric D113N/E117Q variant was found to be 2-fold reduced, and that of the pentuple mutant was 70% of the wild-type value, despite a considerably larger pore cross section. Ion selectivity was drastically altered by the mutations with cation/anion permeability ratios ranging from 1 to 12. Ion flow through these and eight other mutants, which have been characterized previously, was simulated by Brownian dynamics based on the detailed crystal structures. The calculated ion selectivity and relative channel conductance values agree well with the experimental data. This demonstrates that ion translocation through porin is mainly governed by pore geometry and charge, the two factors that are properly represented in the simulations.     

Contributions of the substrate-binding arginine residues to maleate-induced closure of the active site of Escherichia coli aspartate aminotransferase.

Crystallography shows that aspartate aminotransferase binds dicarboxylate substrate analogues by bonds to Arg292 and Arg386, respectively [Jager, J, Moser, M. Sauder, U. & Jansonius, J. N. (1994) J. Mol. Biol., 239, 285-305]. The contribution of each interaction to the conformational change that the enzyme undergoes when it binds ligands via these residues, is assessed by probing mutant forms of the enzyme lacking either or both arginines. The probes used are NaH(3)BCN which reduces the cofactor imine, the reactive substrate analogue, cysteine sulfinate and proteolysis by trypsin. The unreactive substrate analogue, maleate, is used to induce closure. Each single mutant reacted only 2.5-fold more slowly with NaH(3)BCN than the wild-type indicating that charge repulsion by the arginines contributes little to maintaining the open conformation. Maleate lowered the rate of reduction of the wild-type enzyme more than 300-fold but had little effect on the reaction of the mutant enzymes indicating that the ability of this dicarboxylate analogue to bridge the arginines precisely makes the major contribution to closure. The R292L mutant reacted 20 times more rapidly with cysteine sulfinate than R386L but 5 x 10(4) times more slowly than the wild-type enzyme, consistent with the proposal that enzyme's catalytic abilities are not developed unless closure is induced by bridging of the arginines. Proteolysis of the mutants with trypsin showed that, in the wild-type enzyme, the bonds most susceptible to trypsin are those contributed by Arg292 and Arg386. Proteolysis of the next most susceptible bond, at Arg25 in the double mutant, was protected by maleate demonstrating the presence of an additional site on the enzyme for binding dicarboxylates.     

Structural, kinetic, and mutational studies of the zinc ion environment in tetrameric cytidine deaminase

The zinc-containing cytidine deaminase (CDA, EC 3.5.4.5) is a pyrimidine salvage enzyme catalyzing the hydrolytic deamination of cytidine and 2'-deoxycytidine forming uridine and 2'-deoxyuridine, respectively. Homodimeric CDA (D-CDA) and homotetrameric CDA (T-CDA) both contain one zinc ion per subunit coordinated to the catalytic water molecule. The zinc ligands in D-CDA are one histidine and two cysteine residues, whereas in T-CDA zinc is coordinated to three cysteines. Two of the zinc coordinating cysteines in T-CDA form hydrogen bonds to the conserved residue Arg56, and this residue together with the dipole moments from two alpha-helices partially neutralizes the additional negative charge in the active site, leading to a catalytic activity similar to D-CDA. Arg56 has been substituted by a glutamine (R56Q), the corresponding residue in D-CDA, an alanine (R56A), and an aspartate (R56D). Moreover, one of the zinc-liganding cysteines has been substituted by histidine to mimic D-CDA, alone (C53H) and in combination with R56Q (C53H/R56Q). R56A, R56Q, and C53H/R56Q contain the same amount of zinc as the wild-type enzyme. The zinc-binding capacity of R56D is reduced. Only R56A, R56Q, and C53H/R56Q yielded measurable CDA activity, R56A and R56Q with similar K(m) but decreased V(max) values compared to wild-type enzyme. Because of dissociation into its inactive subunits, it was impossible to determine the kinetic parameters for C53H/R56Q. R56A and C53H/R56Q display increased apparent pK(a) values compared to the wild-type enzyme and R56Q. On the basis of the structures of R56A, R56Q, and C53H/R56Q an explanation is provided of kinetic results and the apparent instability of C53H/R56Q.     

A key role in catalysis for His89 of adenylosuccinate lyase of Bacillus subtilis

Adenylosuccinate lyase of Bacillus subtilis is a tetrameric enzyme which catalyzes the cleavage of adenylosuccinate to AMP and fumarate. We have mutated His(89), one of three conserved histidines, to Gln, Ala, Glu, and Arg. The enzymes were expressed in Escherichia coli and purified to homogeneity. As compared to a specific activity of 1. 56 micromol of adenylosuccinate converted/min/mg protein for wild-type enzyme, the mutant enzymes exhibit specific activities of 0.0225, 0.0036, 0.0036, and 0.0009 for H89Q, H89A, H89E, and H89R, respectively. Circular dichroism and FPLC gel filtration reveal that mutant enzymes have a similar conformation and oligomeric state to that of wild-type enzyme. In H89Q, the K(M) for adenylosuccinate increases slightly to 2.5-fold that of wild-type, the K(M) for fumarate is elevated 3.3-fold, and the K(M) for AMP is 13 times higher than that observed in wild-type enzyme. The catalytic efficiency of the H89Q enzyme is compromised, with k(cat)/K(M) reduced 174-fold in the direction of AMP formation. These data suggest that His(89) plays a role in both the binding of the AMP portion of the substrate and in correctly orienting the substrate for catalysis. Incubation of H89Q with inactive H141Q enzyme [Lee, T. T., Worby, C., Bao, Z.-Q., Dixon, J. E., and Colman, R. F. (1999) Biochemistry 38, 22-32] leads to a 30-fold increase in activity. This intersubunit complementation indicates that His(89) and His(141) from different subunits participate in the active site and that both are required for catalysis.     

Thermostability of manganese- and iron-superoxide dismutases from Escherichia coli is determined by the characteristic position of a glutamine residue.

The structurally homologous mononuclear iron and manganese superoxide dismutases (FeSOD and MnSOD, respectively) contain a highly conserved glutamine residue in the active site which projects toward the active-site metal centre and participates in an extensive hydrogen bonding network. The position of this residue is different for each SOD isoenzyme (Q69 in FeSOD and Q146 in MnSOD of Escherichia coli). Although site-directed mutant enzymes lacking this glutamine residue (FeSOD[Q69G] and MnSOD[Q146A]) demonstrated a higher degree of selectivity for their respective metal, they showed little or no activity compared with wild types. FeSOD double mutants (FeSOD[Q69G/A141Q]), which mimic the glutamine position in MnSOD, elicited 25% the activity of wild-type FeSOD while the activity of the corresponding MnSOD double mutant (MnSOD[G77Q/Q146A]) increased to 150% (relative to wild-type MnSOD). Both double mutants showed reduced selectivity toward their metal. Differences exhibited in the thermostability of SOD activity was most obvious in the mutants that contained two glutamine residues (FeSOD[A141Q] and MnSOD[G77Q]), where the MnSOD mutant was thermostable and the FeSOD mutant was thermolabile. Significantly, the MnSOD double mutant exhibited a thermal-inactivation profile similar to that of wild-type FeSOD while that of the FeSOD double mutant was similar to wild-type MnSOD. We conclude therefore that the position of this glutamine residue contributes to metal selectivity and is responsible for some of the different physicochemical properties of these SODs, and in particular their characteristic thermostability.     

Three subunits contribute amino acids to the active site of tetrameric adenylosuccinate lyase: Lys268 and Glu275 are required

Tetrameric adenylosuccinate lyase (ASL) of Bacillus subtilis catalyzes the cleavage of adenylosuccinate to form AMP and fumarate. We previously reported that two distinct subunits contribute residues to each active site, including the His68 and His89 from one and His141 from a second subunit [Brosius, J. L., and Colman, R. F. (2000) Biochemistry 39, 13336-13343]. Glu(275) is 2.8 A from His141 in the ASL crystal structure, and Lys268 is also in the active site region; Glu275 and Lys268 come from a third, distinct subunit. Using site-directed mutagenesis, we have replaced Lys268 by Arg, Gln, Glu, and Ala, with specific activities of the purified mutant enzymes being 0.055, 0.00069, 0.00028, and 0.0, respectively, compared to 1.56 units/mg for wild-type (WT) enzyme. Glu275 was substituted by Gln, Asp, Ala, and Arg; none of these homogeneous mutant enzymes has detectable activity. Circular dichroism and light scattering reveal that neither the secondary structure nor the oligomeric state of the Lys268 mutant enzymes has been perturbed. Native gel electrophoresis and circular dichroism indicate that the Glu275 mutant enzymes are tetramers, but their conformation is altered slightly. For K268R, the K(m)s for all substrates are similar to WT enzyme. Binding studies using [2-3H]-adenylosuccinate reveal that none of the Glu275 mutant enzymes, nor inactive K268A, can bind substrate. We propose that Lys268 participates in binding substrate and that Glu275 is essential for catalysis because of its interaction with His141. Incubation of H89Q with K268Q or E275Q leads to restoration of up to 16% WT activity, while incubation of H141Q with K268Q or E275Q results in 6% WT activity. These complementation studies provide the first functional evidence that a third subunit contributes residues to each intersubunit active site of ASL. Thus, adenylosuccinate lyase has four active sites per enzyme tetramer, each of which is formed from regions of three subunits.     

Role of P1 residues Arg336 and Arg562 in the activated-Protein-C-catalysed inactivation of Factor VIIIa

APC (activated Protein C) inactivates human Factor VIIIa following cleavage at residues Arg336 and Arg562 within the A1 and A2 subunits respectively. The role of the P1 arginine in APC-catalysed inactivation of Factor VIIIa was examined by employing recombinant Factor VIIIa molecules where residues 336 and 562 were replaced with alanine and/or glutamine. Stably expressed Factor VIII proteins were activated by thrombin and resultant Factor VIIIa was reacted at high concentration with APC to minimize cofactor inactivation due to A2 subunit dissociation. APC cleaved wild-type Factor VIIIa at the A1 site with a rate approximately 25-fold greater than that for the A2 site. A1 mutants R336A and R336Q were inactivated approximately 9-fold slower than wild-type Factor VIIIa, whereas the A2 mutant R562A was inactivated approximately 2-fold slower. No cleavage at the mutated sites was observed. Taken together, these results suggested that cleavage at the A1 site was the dominant mechanism for Factor VIIIa inactivation catalysed by the proteinase. On the basis of cleavage at Arg336, a K(m) value for wild-type Factor VIIIa of 102 nM was determined, and this value was significantly greater than K(i) values (approximately 9-18 nM) obtained for an R336Q/R562Q Factor VIIIa. Furthermore, evaluation of a series of cluster mutants in the C-terminal region of the A1 subunit revealed a role for acidic residues in segment 341-345 in the APC-catalysed proteolysis of Arg336. Thus, while P1 residues contribute to catalytic efficiency, residues removed from these sites make a primary contribution to the overall binding of APC to Factor VIIIa.     

Protein engineering of CYP105s for their industrial uses

Cytochrome P450 enzymes belonging to the CYP105 family are predominantly found in bacteria belonging to the phylum Actinobacteria and the order Actinomycetales. In this review, we focused on the protein engineering of P450s belonging to the CYP105 family for industrial use. Two Arg substitutions to Ala of CYP105A1 enhanced its vitamin D₃ 25- and 1α-hydroxylation activities by 400 and 100-fold, respectively. The coupling efficiency between product formation and NADPH oxidation was largely improved by the R84A mutation. The quintuple mutant Q87W/T115A/H132L/R194W/G294D of CYP105AB3 showed a 20-fold higher activity than the wild-type enzyme. Amino acids at positions 87 and 191 were located at the substrate entrance channel, and that at position 294 was located close to the heme group. Semi-rational engineering of CYP105A3 selected the best performing mutant, T85F/T119S/V194N/N363Y, for producing pravastatin. The T119S and N363Y mutations synergistically had remarkable effects on the interaction between CYP105A3 and putidaredoxin. Although wild-type CYP105AS1 hydroxylated compactin to 6-epi-pravastatin, the quintuple mutant I95T/Q127R/A180V/L236I/A265N converted almost all compactin to pravastatin. Five amino acid substitutions by two rounds of mutagenesis almost completely changed the stereo-selectivity of CYP105AS1. These results strongly suggest that the protein engineering of CYP105 enzymes greatly increase their industrial utility. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.     

Why is quinidine an inhibitor of cytochrome P450 2D6? The role of key active-site residues in quinidine binding

We have previously shown that Phe(120), Glu(216), and Asp(301) in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolizing enzyme (Paine, M. J., McLaughlin, L. A., Flanagan, J. U., Kemp, C. A., Sutcliffe, M. J., Roberts, G. C., and Wolf, C. R. (2003) J. Biol. Chem. 278, 4021-4027 and Flanagan, J. U., Maréchal, J.-D., Ward, R., Kemp, C. A., McLaughlin, L. A., Sutcliffe, M. J., Roberts, G. C., Paine, M. J., and Wolf, C. R. (2004) Biochem. J. 380, 353-360). We have now examined the effect of mutations of these residues on interactions of the enzyme with the prototypical CYP2D6 inhibitor, quinidine. Abolition of the negative charge at either or both residues 216 and 301 decreased quinidine inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation by at least 100-fold. The apparent dissociation constants (K(d)) for quinidine binding to the wild-type enzyme and the E216D and D301E mutants were 0.25-0.50 microm. The amide substitution of Glu(216) or Asp(301) resulted in 30-64-fold increases in the K(d) for quinidine. The double mutant E216Q/D301Q showed the largest decrease in quinidine affinity, with a K(d) of 65 microm. Alanine substitution of Phe(120), Phe(481),or Phe(483) had only a minor effect on the inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation and on binding. In contrast to the wild-type enzyme, a number of the mutants studied were found to be able to metabolize quinidine. E216F produced O-demethylated quinidine, and F120A and E216Q/D301Q produced both O-demethylated quinidine and 3-hydroxyquinidine metabolites. Homology modeling and molecular docking were used to predict the modes of quinidine binding to the wild-type and mutant enzymes; these were able to rationalize the experimental observations.     

Rescue of mutant alpha-galactosidase A in the endoplasmic reticulum by 1-deoxygalactonojirimycin leads to trafficking to lysosomes

Active-site-specific chaperone therapy for Fabry disease is a genotype-specific therapy using a competitive inhibitor, 1-deoxygalactonojirimycin (DGJ). To elucidate the mechanism of enhancing alpha-galactosidase A (alpha-Gal A) activity by DGJ-treatment, we studied the degradation of a mutant protein and the effect of DGJ in the endoplasmic reticulum (ER). We first established an in vitro translation and translocation system using rabbit reticulocyte lysates and canine pancreas microsomal vesicles for a study on the stability of mutant alpha-Gal A with an amino acid substitution (R301Q) in the ER. R301Q was rapidly degraded, but no degradation of wild-type alpha-Gal A was observed when microsomal vesicles containing wild-type or R301Q alpha-Gal A were isolated and incubated. A pulse-chase experiment on R301Q-expressing TgM/KO mouse fibroblasts showed rapid degradation of R301Q, and its degradation was blocked by the addition of lactacystin, indicating that R301Q was degraded by ER-associated degradation (ERAD). Rapid degradation of R301Q was also observed in TgM/KO mouse fibroblasts treated with brefeldin A, and the amount of R301Q enzyme markedly increased by pretreatment with DGJ starting 12 h prior to addition of brefeldin A. The enhancement of alpha-Gal A activity and its protein level by DGJ-treatment was selectively observed in brefeldin A-treated COS-7 cells expressing R301Q but not in cells expressing the wild-type alpha-Gal A. Observation by immunoelectron microscopy showed that the localization of R301Q in COS-7 cells was in the lysosomes, not the ER. These data suggest that the rescue of R301Q from ERAD is a key step for normalization of intracellular trafficking of R301Q.     

Random mutagenesis to enhance the activity of subtilisin in organic solvents: Characterization of Q103R subtilisin E

Q103R subtilisin E was isolated following random mutagenesis and screening for improved activity in the presence of dimethylformamide (DMF). Our goal is to identify the mechanism(s) by which amino acid substitutions can enhance enzyme activity in polar organic solvents. A quantitative framework for comparing substrate binding and catalytic activities of mutant and wild-type enzymes in the presence and absence of DMF is outlined. Kinetic experiments performed at high salt concentration (1M KCl) reveal that the mechanism behind the Q103R variant's enhanced activity toward succinyl-Ala-Ala-Pro-Phe-p-nitroanilide is both electrostatic and nonelectrostatic in origin. Favorable electrostatic interactions between the negatively charged succinyl group of the substrate and the positive charge on Arg 103 are responsible for tighter substrate binding. This conclusion is supported by kinetic experiments performed on the related substrate Ala-Ala-Pro-Phe-p-nitroanilide and the hydrolysis kinetics of the Q103E, Q103K, and Q103S variants constructed by site-directed mutagenesis. These results highlight the importance of the choice of the substrate used to screen for improvements in catalytic activity.     

Identification of residues critical for activity of the wound-induced leucine aminopeptidase (LAP-A) of tomato.

The importance of two putative Zn2+-binding (Asp347, Glu429) and two catalytic (Arg431, Lys354) residues in the tomato leucine aminopeptidase (LAP-A) function was tested. The impact of substitutions at these positions, corresponding to the bovine LAP residues Asp255, Glu334, Arg336, and Lys262, was evaluated in His6-LAP-A fusion proteins expressed in Escherichia coli. Sixty-five percent of the mutant His6-LAP-A proteins were unstable or had complete or partial defects in hexamer assembly or stability. The activity of hexameric His6-LAP-As on Xaa-Leu and Leu-Xaa dipeptides was tested. Most substitutions of Lys354 (a catalytic residue) resulted in His6-LAP-As that cleaved dipeptides at slower rates. The Glu429 mutants (a Zn2+-binding residue) had more diverse phenotypes. Some mutations abolished activity and others retained partial or complete activity. The E429D His6-LAP-A enzyme had Km and kcat values similar to the wild-type His6-LAP-A. One catalytic (Arg431) and one Zn-binding (Asp347) residue were essential for His6-LAP-A activity, as most R431 and D347 mutant His6-LAP-As did not hydrolyze dipeptides. The R431K His6-LAP-A that retained the positive charge had partial activity as reflected in the 4.8-fold decrease in kcat. Surprisingly, while the D347E mutant (that retained a negative charge at position 347) was inactive, the D347R mutant that introduced a positive charge retained partial activity. A model to explain these data is proposed.     

Site-directed mutants of Escherichia coli alpha-ketoglutarate permease (KgtP).

To investigate an active site(s) in the Escherichia coli alpha-ketoglutarate premease, 11 point mutants were made in the corresponding structural gene, kgtP, by oligonucleotide-directed mutagenesis and the polymerase chain reaction. On the basis of sequences conserved in KgtP and related members of a transporter superfamily [Henderson P. J. F., & Maiden, M. C. (1990) Philos. Trans. R. Soc. London B 326, 391], Arg76 was replaced with Ala, Asp, or Lys; Asp88 with Asn or Glu; His90 with Ala; Arg92 with Ala or Lys; and Arg198 with Ala, Asp, or Lys. Mutant proteins expressed using the T7 polymerase system were in each case shown to be membrane-associated. However, they differed in transport activity. Mutants H90A and R198K had activities similar to that of wild type, and R76K and R198A retained 10-60% of the wild-type activity. In all other mutants, alpha-ketoglutarate transport was abolished. The results suggest that Arg92, which is highly conserved among other members of the transporter superfamily, is necessary for activity and also that Asp88 is critical for function, as observed for the tetracycline transporter. These data show further that a positive charge is essential at position 76 and is also important, but not absolutely required, at position 198 for alpha-ketoglutarate transport. Unlike lacY permease which was inactivated by deleting the last helix [McKenna, E., Hardy, D., Pastore, J. C., & Kaback, H. R. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2969], a KgtP truncation mutant missing the last putative membrane-spanning region was relatively stable and also retained 10-50% of the wild-type level of alpha-ketoglutarate transport activity.