Evolution of enzymatic activity in the enolase superfamily: structural studies of the promiscuous o-succinylbenzoate synthase from Amycolatopsis

Divergent evolution of enzyme function is commonly explained by a gene duplication event followed by mutational changes that allow the protein encoded by the copy to acquire a new function. An alternate hypothesis is that this process is facilitated when the progenitor enzyme acquires a second function while maintaining the original activity. This phenomenon has been suggested to occur in the o-succinylbenzoate synthase (OSBS) from a species of Amycolatopsis that catalyzes not only the physiological syn-dehydration reaction of 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate but also an accidental racemization of N-acylamino acids [Palmer, D. R., Garrett, J. B., Sharma, V., Meganathan, R., Babbitt, P. C., and Gerlt, J. A. (1999) Biochemistry 38, 4252-4258]. To understand the molecular basis of this promiscuity, three-dimensional structures of liganded complexes of this enzyme have been determined, including the product of the OSBS reaction and three N-acylamino acid substrates for the N-acylamino acid racemase (NAAAR) reaction, N-acetylmethionine, N-succinylmethionine, and N-succinylphenylglycine, to 2.2, 2.3, 2.1, and 1.9 A resolution, respectively. These structures show how the active-site cavity can accommodate both the hydrophobic substrate for the OSBS reaction and the substrates for the accidental NAAAR reaction. As expected, the N-acylamino acid is sandwiched between lysines 163 and 263, which function as the catalytic bases for the abstraction of the alpha-proton in the (R)- and (S)-racemization reactions, respectively [Taylor Ringia, E. A., Garrett, J. B, Thoden, J. B., Holden, H. M., Rayment, I., and Gerlt, J. A. (2004) Biochemistry 42, 224-229]. Importantly, the protein forms specific favorable interactions with the hydrophobic amino acid side chain, alpha-carbon, carboxylate, and the polar components of the N-acyl linkage. Accommodation of the components of the N-acyl linkage appears to be the reason that this enzyme is capable of a racemization reaction on these substrates, whereas the orthologous OSBS from Escherichia coli lacks this functionality.     

Evolution of structure and function in the o-succinylbenzoate synthase/N-acylamino acid racemase family of the enolase superfamily

Understanding how proteins evolve to provide both exquisite specificity and proficient activity is a fundamental problem in biology that has implications for protein function prediction and protein engineering. To study this problem, we analyzed the evolution of structure and function in the o-succinylbenzoate synthase/N-acylamino acid racemase (OSBS/NAAAR) family, part of the mechanistically diverse enolase superfamily. Although all characterized members of the family catalyze the OSBS reaction, this family is extraordinarily divergent, with some members sharing <15% identity. In addition, a member of this family, Amycolatopsis OSBS/NAAAR, is promiscuous, catalyzing both dehydration and racemization. Although the OSBS/NAAAR family appears to have a single evolutionary origin, no sequence or structural motifs unique to this family could be identified; all residues conserved in the family are also found in enolase superfamily members that have different functions. Based on their species distribution, several uncharacterized proteins similar to Amycolatopsis OSBS/NAAAR appear to have been transmitted by lateral gene transfer. Like Amycolatopsis OSBS/NAAAR, these might have additional or alternative functions to OSBS because many are from organisms lacking the pathway in which OSBS is an intermediate. In addition to functional differences, the OSBS/NAAAR family exhibits surprising structural variations, including large differences in orientation between the two domains. These results offer several insights into protein evolution. First, orthologous proteins can exhibit significant structural variation, and specificity can be maintained with little conservation of ligand-contacting residues. Second, the discovery of a set of proteins similar to Amycolatopsis OSBS/NAAAR supports the hypothesis that new protein functions evolve through promiscuous intermediates. Finally, a combination of evolutionary, structural, and sequence analyses identified characteristics that might prime proteins, such as Amycolatopsis OSBS/NAAAR, for the evolution of new activities.     

Evolutionary potential of (beta/alpha)8-barrels: in vitro enhancement of a "new" reaction in the enolase superfamily

The repertoire of reactions in the mechanistically diverse enolase superfamily is the result of divergent evolution that conserved enolization of a carboxylate anion substrate but allowed different overall reactions using different substrates. Details of the pathways for the natural evolutionary process are unknown, but the events reasonably involve (1) incremental increases in the level of the "new" reaction that would provide a selective advantage and (2) an accompanying loss of the "old" reaction catalyzed by the progenitor. In an effort to better understand the molecular processes of divergent evolution, the D297G mutant of the l-Ala-d/l-Glu epimerase (AEE) from Escherichia coli was designed so that it could bind the substrate for the o-succinylbenzoate synthase (OSBS) reaction and, as a result, catalyze that reaction [Schmidt, D. M. Z., Mundorff, E. C., Dojka, M., Bermudez, E., Ness, J. E., Govindarajan, S., Babbitt, P. C., Minshull, J., and Gerlt, J. A. (2003) Biochemistry 42, 8387-8393]. The AEE progenitor did not catalyze the OSBS reaction, but the D297G mutant catalyzed a low level of the OSBS reaction (k(cat), 0.013 s(-)(1); K(m), 1.8 mM; k(cat)/K(m), 7.4 M(-)(1) s(-)(1)) that was sufficient to permit anaerobic growth by an OSBS-deficient strain of E. coli; the level of the progenitor's natural AEE reaction was significantly diminished. Using random mutagenesis and an anaerobic metabolic selection, we now have identified the I19F substitution as an additional mutation that enhances both growth of the OSBS-deficient strain and the kinetic constants for the OSBS reaction (k(cat), 0.031 s(-)(1); K(m), 0.34 mM; k(cat)/K(m), 90 M(-)(1) s(-)(1)). Several other substitutions for Ile 19 also enhanced the level of the OSBS reaction. All of the substitutions substantially decreased the level of the AEE reaction from that possessed by the D297G progenitor. The changes in the kinetic constants for both the OSBS and AEE reactions are attributed to a readjustment of substrate specificity so that the substrate for the OSBS reaction is more productively presented to the conserved acid/base catalysts in the active site. These observations support our hypothesis that evolution of "new" functions in the enolase superfamily can occur simply by changes in specificity-determining residues.     

Evolution of enzymatic activities in the enolase superfamily: functional assignment of unknown proteins in Bacillus subtilis and Escherichia coli as L-Ala-D/L-Glu epimerases.

The members of the mechanistically diverse enolase superfamily catalyze different overall reactions by using a common catalytic strategy and structural scaffold. In the muconate lactonizing enzyme (MLE) subgroup of the superfamily, abstraction of a proton adjacent to a carboxylate group initiates reactions, including cycloisomerization (MLE), dehydration [o-succinylbenzoate synthase (OSBS)], and 1,1-proton transfer (catalyzed by an OSBS that also catalyzes a promiscuous N-acylamino acid racemase reaction). The realization that a member of the MLE subgroup could catalyze a 1,1-proton transfer reaction, albeit poorly, led to a search for other enzymes which might catalyze a 1,1-proton transfer as their physiological reaction. YcjG from Escherichia coli and YkfB from Bacillus subtilis, proteins of previously unknown function, were discovered to be L-Ala-D/L-Glu epimerases, although they also catalyze the epimerization of other dipeptides. The values of k(cat)/K(M) for L-Ala-D/L-Glu for both proteins are approximately 10(4) M(-1) s(-1). The genomic context and the substrate specificity of both YcjG and YkfB suggest roles in the metabolism of the murein peptide, of which L-Ala-D-Glu is a component. Homologues possessing L-Ala-D/L-Glu epimerase activity have been identified in at least two other organisms.     

Evolutionary potential of (beta/alpha)8-barrels: stepwise evolution of a "new" reaction in the enolase superfamily

The molecular details of the processes involved in divergent evolution of "new" enzymatic functions are ill-defined. Likely starting points are either a progenitor promiscuous for the new reaction or a progenitor capable of catalyzing the new reaction following a single substitution that results from a single base change. However, the molecular (sequence) pathway by which the selective advantage provided by this protein can be improved and ultimately optimized is unclear. In the mechanistically diverse enolase superfamily, we discovered that a monofunctional progenitor could acquire the ability to catalyze a "new" reaction by a single base change: the D297G mutant of the monofunctional l-Ala-d/l-Glu epimerase (AEE) from Escherichia coli catalyzed a low level of the o-succinylbenzoate synthase (OSBS) reaction as well as a reduced level of the AEE reaction [Schmidt, D. M. Z., Mundorff, E. C., Dojka, M., Bermudez, E., Ness, J. E., Govindarajan, S., Babbitt, P. C., Minshull, J., and Gerlt, J. A. (2003) Biochemistry 42, 8387-8393]. We then discovered that the selective advantage and OSBS activity of the D297G mutant are both enhanced by the I19F substitution [Vick, J. E., Schmidt, D. M. Z., and Gerlt, J. A. (2005) Biochemistry 44, 11722-11729]. Both the D297G and I19F substitutions are positioned to alter the substrate specificity so that the substrate for the OSBS reaction is more productively positioned vis a vis the active site catalytic groups. We now report that both the selective advantage and OSBS activity of the D297G/I19F double mutant are enhanced by the R24C (one base change from the wild type Arg codon), R24W (two base changes from the wild type Arg codon and one base change from the R24C codon), and L277W (one base change from the wild type Leu codon) substitutions. The effects of the R24C and L277W mutants are "additive" in the D297G/I19F/R24C/L277W mutant. The greatest selective advantage and OSBS activity are associated with the D297G/I19F/R24W mutant. These "new" substitutions that enhance both the selective advantage and kinetic constants are positioned in the active site where they can alter the specificity, highlighting that the evolution of the "new" OSBS function can be accomplished by changes in substrate specificity.     

Evolution of enzymatic activity in the enolase superfamily: structure of o-succinylbenzoate synthase from Escherichia coli in complex with Mg2+ and o-succinylbenzoate

The X-ray structures of the ligand free (apo) and the Mg(2+)*o-succinylbenzoate (OSB) product complex of o-succinylbenzoate synthase (OSBS) from Escherichia coli have been solved to 1.65 and 1.77 A resolution, respectively. The structure of apo OSBS was solved by multiple isomorphous replacement in space group P2(1)2(1)2(1); the structure of the complex with Mg(2+)*OSB was solved by molecular replacement in space group P2(1)2(1)2. The two domain fold found for OSBS is similar to those found for other members of the enolase superfamily: a mixed alpha/beta capping domain formed from segments at the N- and C-termini of the polypeptide and a larger (beta/alpha)(7)beta barrel domain. Two regions of disorder were found in the structure of apo OSBS: (i) the loop between the first two beta-strands in the alpha/beta domain; and (ii) the first sheet-helix pair in the barrel domain. These regions are ordered in the product complex with Mg(2+)*OSB. As expected, the Mg(2+)*OSB pair is bound at the C-terminal end of the barrel domain. The electron density for the phenyl succinate component of the product is well-defined; however, the 1-carboxylate appears to adopt multiple conformations. The metal is octahedrally coordinated by Asp(161), Glu(190), and Asp(213), two water molecules, and one oxygen of the benzoate carboxylate group of OSB. The loop between the first two beta-strands in the alpha/beta motif interacts with the aromatic ring of OSB. Lys(133) and Lys(235) are positioned to function as acid/base catalysts in the dehydration reaction. Few hydrogen bonding or electrostatic interactions are involved in the binding of OSB to the active site; instead, most of the interactions between OSB and the protein are either indirect via water molecules or via hydrophobic interactions. As a result, evolution of both the shape and the volume of the active site should be subject to few structural constraints. This would provide a structural strategy for the evolution of new catalytic activities in homologues of OSBS and a likely explanation for how the OSBS from Amycolaptosis also can catalyze the racemization of N-acylamino acids [Palmer, D. R., Garrett, J. B., Sharma, V., Meganathan, R., Babbitt, P. C., and Gerlt, J. A. (1999) Biochemistry 38, 4252-4258].     

Evolutionary potential of (beta/alpha)8-barrels: functional promiscuity produced by single substitutions in the enolase superfamily

The members of the mechanistically diverse, (beta/alpha)(8)-barrel fold-containing enolase superfamily evolved from a common progenitor but catalyze different reactions using a conserved partial reaction. The molecular pathway for natural divergent evolution of function in the superfamily is unknown. We have identified single-site mutants of the (beta/alpha)(8)-barrel domains in both the l-Ala-d/l-Glu epimerase from Escherichia coli (AEE) and the muconate lactonizing enzyme II from Pseudomonas sp. P51 (MLE II) that catalyze the o-succinylbenzoate synthase (OSBS) reaction as well as the wild-type reaction. These enzymes are members of the MLE subgroup of the superfamily, share conserved lysines on opposite sides of their active sites, but catalyze acid- and base-mediated reactions with different mechanisms. A comparison of the structures of AEE and the OSBS from E. coli was used to design the D297G mutant of AEE; the E323G mutant of MLE II was isolated from directed evolution experiments. Although neither wild-type enzyme catalyzes the OSBS reaction, both mutants complement an E. coli OSBS auxotroph and have measurable levels of OSBS activity. The analogous mutations in the D297G mutant of AEE and the E323G mutant of MLE II are each located at the end of the eighth beta-strand of the (beta/alpha)(8)-barrel and alter the ability of AEE and MLE II to bind the substrate of the OSBS reaction. The substitutions relax the substrate specificity, thereby allowing catalysis of the mechanistically diverse OSBS reaction with the assistance of the active site lysines. The generation of functionally promiscuous and mechanistically diverse enzymes via single-amino acid substitutions likely mimics the natural divergent evolution of enzymatic activities and also highlights the utility of the (beta/alpha)(8)-barrel as a scaffold for new function.     

Residues Required for Activity in Escherichia coli o-Succinylbenzoate Synthase (OSBS) Are Not Conserved in All OSBS Enzymes

Understanding how enzyme specificity evolves will provide guiding principles for protein engineering and function prediction. The o-succinylbenzoate synthase (OSBS) family is an excellent model system for elucidating these principles because it has many highly divergent amino acid sequences that are <20% identical, and some members have evolved a second function. The OSBS family belongs to the enolase superfamily, members of which use a set of conserved residues to catalyze a wide variety of reactions. These residues are the only conserved residues in the OSBS family, so they are not sufficient to determine reaction specificity. Some enzymes in the OSBS family catalyze another reaction, N-succinylamino acid racemization (NSAR). NSARs cannot be segregated into a separate family because their sequences are highly similar to those of known OSBSs, and many of them have both OSBS and NSAR activities. To determine how such divergent enzymes can catalyze the same reaction and how NSAR activity evolved, we divided the OSBS family into subfamilies and compared the divergence of their active site residues. Correlating sequence conservation with the effects of mutations in Escherichia coli OSBS identified two nonconserved residues (R159 and G288) at which mutations decrease efficiency ≥200-fold. These residues are not conserved in the subfamily that includes NSAR enzymes. The OSBS/NSAR subfamily binds the substrate in a different orientation, eliminating selective pressure to retain arginine and glycine at these positions. This supports the hypothesis that specificity-determining residues have diverged in the OSBS family and provides insight into the sequence changes required for the evolution of NSAR activity.     

Tyrosine 265 of alanine racemase serves as a base abstracting alpha-hydrogen from L-alanine: the counterpart residue to lysine 39 specific to D-alanine.

Alanine racemase of Bacillus stearothermophilus has been proposed to catalyze alanine racemization by means of two catalytic bases: lysine 39 (K39) abstracting specifically the alpha-hydrogen of D-alanine and tyrosine 265 (Y265) playing the corresponding role for the antipode L-alanine. The role of K39 as indicated has already been verified [Watanabe, A., Kurokawa, Y., Yoshimura, T., Kurihara, T., Soda, K., and Esaki, N. (1999) J. Biol. Chem. 274, 4189-4194]. We here present evidence for the functioning of Y265 as the base catalyst specific to L-alanine. The Y265-->Ala mutant enzyme (Y265A), like Y265S and Y265F, was a poor catalyst for alanine racemization. However, Y265A and Y265S catalyzed transamination with D-alanine much more rapidly than the wild-type enzyme, and the bound coenzyme, pyridoxal 5'-phosphate (PLP), was converted to pyridoxamine 5'-phosphate (PMP). The rate of transamination catalyzed by Y265F was about 9% of that by the wild-type enzyme. However, Y265A, Y265S, and Y265F were similar in that L-alanine was inert as a substrate in transamination. The apo-form of the wild-type enzyme catalyzes the abstraction of tritium non-specifically from both (4'S)- and (4'R)-[4'-(3)H]PMP in the presence of pyruvate. In contrast, apo-Y265A abstracts tritium virtually from only the R-isomer. This indicates that the side-chain of Y265 abstracts the alpha-hydrogen of L-alanine and transfers it supra-facially to the pro-S position at C-4' of PMP. Y265 is the counterpart residue to K39 that transfers the alpha-hydrogen of D-alanine to the pro-R position of PMP.     

Mechanism of the reaction catalyzed by mandelate racemase. 1. Chemical and kinetic evidence for a two-base mechanism

The fate of the alpha-hydrogen of mandelate in the reaction catalyzed by mandelate racemase has been investigated by a mass spectroscopic method. The method entails the incubation of (R)- or (S)-[alpha-1H]mandelate in buffered D2O to a low extent of turnover (about 5-8%), esterification of the resulting mixture of mandelates with diazomethane, derivatization of the methyl esters with a chiral derivatizing agent, and quantitation of the isotope content of the alpha-hydrogen of both substrate and product by gas chromatography/mass spectrometric analysis. No significant substrate-derived alpha-protium was found in the product for racemization in either direction. In addition, in the (R) to (S) direction almost no exchange (less than or equal to 0.4%) of the alpha-hydrogen in the remaining (R) substrate pool occurred, but in the (S) to (R) direction 3.5-5.1% exchange of the alpha-hydrogen in the remaining substrate (after 5.1-7.2% net turnover) was found. Qualitatively similar results were obtained in the (S) to (R) direction in H2O when (S)-[alpha-2H]mandelate was used as substrate. In other experiments, an overshoot in the progress curve was observed when the racemization of either enantiomer of [alpha-1H]mandelate in D2O was monitored by following the change in ellipticity of the reaction mixture; the magnitude of the overshoot was greater in the (R) to (S) than in the (S) to (R) direction. All of the available data indicate that the reaction catalyzed by mandelate racemase proceeds by a two-base mechanism, in contrast to earlier proposals.     

A specific enhancing effect of N,N-dimethylsphingosine on epidermal growth factor receptor autophosphorylation. Demonstration of its endogenous occurrence (and the virtual absence of unsubstituted sphingosine) in human epidermoid carcinoma A431 cells

Our previous study suggested that N,N-dimethylsphingosine, but not unsubstituted sphingosine, could be a modulator of protein kinase C in epidermoid carcinoma A431 cells, since N,N-dimethyl-D-erythrosphingenine showed a stronger stereospecific effect on protein kinase C activity in comparison with N,N-dimethyl-L-erythrosphingenine, unsubstituted D- or L-erythrosphingenine, and gangliosides (Igarashi, Y., Hakomori, S., Toyokuni, T., Dean, B., Fujita, S., Sugimoto, M., Ogawa, T., El-Ghendy, K., and Racker, E. (1989) Biochemistry 28, 6796-6800). Other studies also indicated that commercial sphingosine preparation has an enhancing effect on epidermal growth factor (EGF) receptor kinase activity in A431 cells (Davis, R. J., Girones, N., and Faucher, M. F. (1988) J. Biol. Chem. 263, 5373-5379; Faucher, M. F., Girones, N., Hannun, Y. A., Bell, R. M., and Davis, R. J. (1988) J. Biol. Chem. 263, 5319-5327). In the present paper, we report (i) the effect of N,N-dimethylsphingosine as compared with lyso-glycosphingolipids and other sphingolipid breakdown products on EGF receptor autophosphorylation and (ii) demonstration of endogenous N,N-dimethylsphingosine synthesis and the virtual absence of unsubstituted sphingosine in A431 cells. The autophosphorylation of EGF receptor in the absence of detergent was strongly enhanced by N,N-dimethyl-D-erythrosphingenine; this effect was even obvious in the absence of EGF and synergistic in the presence of EGF. Similar enhancing activity was not produced by N,N-dimethyl-L-erythrosphingenine, D- and L-erythrosphingenine, N-monomethyl-D-erythrosphingenine, N-acetyl-D-erythrosphingenine, or the five lyso-glycosphingolipids tested. Labeling of sphingosine in A431 cells by culturing in medium containing [3H]Ser for various durations, followed by extraction and isolation of sphingolipids by standard procedures, resulted in clear bands corresponding to N,N-dimethylsphingosine and ceramide, whereas the band corresponding to sphingosine was virtually absent. The bands corresponding to N,N-dimethylsphingosine and ceramide intensified when cells were treated with metabolic inhibitor for UDP-Glc:Cer beta-Glc transferase (which causes accumulation of ceramide). These results indicate that N,N-dimethylsphingosine acts as a stereospecific enhancer for EGF receptor kinase and is able to produce EGF-like activity in vitro even in the absence of EGF and detergent. Under physiological conditions, N,N-dimethylsphingosine is the major catabolite resulting from ceramide breakdown.     

Enantioselective plasma protein binding of propafenone: mechanism, drug interaction, and species difference

The interaction of propafenone (PPF) enantiomers with human plasma, human serum albumin (HSA), alpha(1)-acid glycoprotein (AGP), as well as with plasma from rat, rabbit, and cow was investigated using indirect chiral high performance liquid chromatography (HPLC) and ultrafiltration techniques. The stronger binding of the S-PPF found in human plasma was due to AGP. Two classes of binding sites in AGP were identified: one with high-affinity and small binding capacity (K(1(S)) = 7.65 x 10(6) M(-1), n(1(S)) = 0.50; K(1(R)) = 2.81 x 10(6) M(-1), n(1(R)) = 0.46), which revealed stereoselectivity; the other with low-affinity and high-binding capacity (n(2(S)) K(2(S)) = 9.95 x 10(3) M(-1); n(2(R)) K(2(R)) = 9.74 x 10(3) M(-1)). The binding to HSA was found to be weak and not enantioselective (nK(S) = 2.08 x 10(3) M(-1), nK(R) = 2.05 x 10(3) M(-1)). The interaction between enantiomers observed in human plasma was confirmed as a competitive type interacting at the high-affinity site in AGP. The binding mode of both enantiomers with AGP was mainly hydrophobic bond. PPF enantiomers had higher-binding affinity for the F-S variant of human AGP. Drug-drug binding interaction studies showed that verapamil, diazepam, nifedipine, furosemide, nitrendipine, and nimodipine did not affect the binding of PPF enantiomers except quinidine and aprindine at the therapeutic concentration. Comparative studies indicated considerable species-dependent binding stereoselectivity between plasma of the four species investigated.     

Direct interaction of coenzyme M with the active-site Fe-S cluster of heterodisulfide reductase

Heterodisulfide reductase (HDR) catalyzes the formation of coenzyme M (CoM-SH) and coenzyme B (CoB-SH) by the reversible reduction of the heterodisulfide, CoM-S-S-CoB. This reaction recycles the two thiol coenzymes involved in the final step of microbial methanogenesis. Electron paramagnetic resonance (EPR) and variable-temperature magnetic circular dichroism spectroscopic experiments on oxidized HDR incubated with CoM-SH revealed a S=1/2 [4Fe-4S]3) cluster, the EPR spectrum of which is broadened in the presence of CoM-33SH [Duin, E.C., Madadi-Kahkesh, S., Hedderich, R., Clay, M.D. and Johnson, M.K. (2002) Heterodisulfide reductase from Methanothermobacter marburgensis contains an active-site [4Fe-4S] cluster that is directly involved in mediating heterodisulfide reduction. FEBS Lett. 512, 263-268; Duin, E.C., Bauer, C., Jaun, B. and Hedderich, R. (2003) Coenzyme M binds to a [4Fe-4S] cluster in the active site of heterodisulfide reductase as deduced from EPR studies with the [33S]coenzyme M-treated enzyme. FEBS Lett. 538, 81-84]. These results provide indirect evidence that the disulfide binds to the iron-sulfur cluster during reduction. We report here direct structural evidence for this interaction from Se X-ray absorption spectroscopic investigation of HDR treated with the selenium analog of coenzyme M (CoM-SeH). Se K edge extended X-ray absorption fine structure confirms a direct interaction of the Se in CoM-SeH-treated HDR with an Fe atom of the Fe-S cluster at an Fe-Se distance of 2.4A.     

Evolution of enzymatic activity in the enolase superfamily: structural and mutagenic studies of the mechanism of the reaction catalyzed by o-succinylbenzoate synthase from Escherichia coli

o-Succinylbenzoate synthase (OSBS) from Escherichia coli, a member of the enolase superfamily, catalyzes an exergonic dehydration reaction in the menaquinone biosynthetic pathway in which 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) is converted to 4-(2'-carboxyphenyl)-4-oxobutyrate (o-succinylbenzoate or OSB). Our previous structural studies of the Mg(2+).OSB complex established that OSBS is a member of the muconate lactonizing enzyme subgroup of the superfamily: the essential Mg(2+) is coordinated to carboxylate ligands at the ends of the third, fourth, and fifth beta-strands of the (beta/alpha)(7)beta-barrel catalytic domain, and the OSB product is located between the Lys 133 at the end of the second beta-strand and the Lys 235 at the end of the sixth beta-strand [Thompson, T. B., Garrett, J. B., Taylor, E. A, Meganathan, R., Gerlt, J. A., and Rayment, I. (2000) Biochemistry 39, 10662-76]. Both Lys 133 and Lys 235 were separately replaced with Ala, Ser, and Arg residues; all six mutants displayed no detectable catalytic activity. The structure of the Mg(2+).SHCHC complex of the K133R mutant has been solved at 1.62 A resolution by molecular replacement starting from the structure of the Mg(2+).OSB complex. This establishes the absolute configuration of SHCHC: the C1-carboxylate and the C6-OH leaving group are in a trans orientation, requiring that the dehydration proceed via a syn stereochemical course. The side chain of Arg 133 is pointed out of the active site so that it cannot function as a general base, whereas in the wild-type enzyme complexed with Mg(2+).OSB, the side chain of Lys 133 is appropriately positioned to function as the only acid/base catalyst in the syn dehydration. The epsilon-ammonium group of Lys 235 forms a cation-pi interaction with the cyclohexadienyl moiety of SHCHC, suggesting that Lys 235 also stabilizes the enediolate anion intermediate in the syn dehydration via a similar interaction.     

Chiral recognition based on enantioselective interactions of propranolol enantiomers with cyclosophoraoses isolated from Rhizobium meliloti

Cyclosophoraoses isolated from Rhizobium meliloti, as an NMR chiral shift agent, were used to discriminate propranolol enantiomers. Continuous variation plot made from the complex of cyclosophoraoses with propranolol showed that the diastereomeric complex had predominantly 1:1 stoichiometry through UV spectroscopic analysis. The chiral recognition of propranolol enantiomers by cyclosophoraoses was investigated through the determination of binding constant based on the (13)C NMR chemical shift changes. The averaged K(obs) values from the plots were 55.7 M(-1) for (R)-(+)-propranolol and 36.6 M(-1) for (S)-(-)-propranolol, respectively. Enantioselectivity (alpha = K(R+)/K(S(-)) of 1.52 was then obtained. Computational calculation also revealed that (R)-(+) propranolol was more tightly bound with cyclosophoraose than (S)-(-)-propranolol due to the enhanced van der Waals interaction.     

Characterization of site-directed mutants in the lac permease of Escherichia coli. 1. Replacement of histidine residues

Wild-type lac permease from Escherichia coli and two site-directed mutant permeases containing Arg in place of His35 and His39 or His322 were purified and reconstituted into proteoliposomes. H35-39R permease is indistinguishable from wild type with regard to all modes of translocation. In contrast, purified, reconstituted permease with Arg in place of His322 is defective in active transport, efflux, equilibrium exchange, and counterflow but catalyzes downhill influx of lactose without concomitant H+ translocation. Although permease with Arg in place of His205 was thought to be devoid of activity [Padan, E., Sarkar, H. K., Viitanen, P. V., Poonian, M. S., & Kaback, H. R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6765], sequencing of lac Y in pH205R reveals the presence of two additional mutations in the 5' end of the gene, and replacement of this portion of lac Y with a restriction fragment from the wild-type gene yields permease with normal activity. Permeases with Asn, Gln, or Lys in place of His322, like H322R permease, catalyze downhill influx of lactose without H+ translocation but are unable to catalyze active transport, equilibrium exchange, or counterflow. Unlike H322R permease, however, the latter mutants catalyze efflux at rates comparable to that of wild-type permease, although the reaction does not occur in symport with H+. Finally, as evidenced by flow dialysis and photoaffinity labeling experiments, replacement of His322 appears to cause a marked decrease in the affinity of the permease for substrate. The results confirm and extend the contention that His322 is the only His residue in the permease involved in lactose/H+ symport and that an imidazole moiety at position 322 is obligatory.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalysis of partial reactions of ATP synthesis by beef heart mitochondrial adenosine triphosphatase

We have found that when the ATP hydrolysis activity of beef heart mitochondrial adenosine triphosphatase (F1) is eliminated by either cold treatment or chemical modification, the enzyme attains the ability to catalyze the Pi in equilibrium ATP exchange reaction. The ATP hydrolysis activity of isolated F1 was lost upon chemical modification by phenyglyoxal, butanedione, or 7-chloro-4-nitrobenzene-2-oxa-1,3-diazole. The F1 thus chemically modified was able to catalyze an ADP-dependent Pi in equilibrium ATP exchange reaction. In addition F1 that had been cold-treated to eliminate ATP hydrolysis activity, also catalyzed the Pi in equilibrium ATP exchange reaction. The Pi in equilibrium ATP exchange catalyzed by modified F1 was shown to be totally inhibited by the F1-specific antibiotic efrapeptin. We have previously shown that isolated beef heart mitochondrial ATPase will catalyze the formation of a transition state analog of the ATP synthesis reaction (Bossard, M. J., Vik, T. A., and Schuster, S. M. (1980) J. Biol. Chem. 255, 5342-5346). While the F1-catalyzed ATP hydrolysis activity was lost rapidly upon chemical modification or cold treatment, the ability of the enzyme to produce Pi . adenosine 5'-diphosphate (chromium(III) salt) from phosphate and monodentate adenosine 5'-diphosphate (chromium(III) salt) was unimpaired. The implications of these data with regard to the mechanism of ATP synthesis are discussed.     

Characterization of a (2R,3R)-2,3-butanediol dehydrogenase as the Saccharomyces cerevisiae YAL060W gene product. Disruption and induction of the gene

The completion of the Saccharomyces cerevisiae genome project in 1996 showed that almost 60% of the potential open reading frames of the genome had no experimentally determined function. Using a conserved sequence motif present in the zinc-containing medium-chain alcohol dehydrogenases, we found several potential alcohol dehydrogenase genes with no defined function. One of these, YAL060W, was overexpressed using a multicopy inducible vector, and its protein product was purified to homogeneity. The enzyme was found to be a homodimer that, in the presence of NAD(+), but not of NADP, could catalyze the stereospecific oxidation of (2R,3R)-2, 3-butanediol (K(m) = 14 mm, k(cat) = 78,000 min(-)(1)) and meso-butanediol (K(m) = 65 mm, k(cat) = 46,000 min(-)(1)) to (3R)-acetoin and (3S)-acetoin, respectively. It was unable, however, to further oxidize these acetoins to diacetyl. In the presence of NADH, it could catalyze the stereospecific reduction of racemic acetoin ((3R/3S)- acetoin; K(m) = 4.5 mm, k(cat) = 98,000 min(-)(1)) to (2R,3R)-2,3-butanediol and meso-butanediol, respectively. The substrate stereospecificity was determined by analysis of products by gas-liquid chromatography. The YAL060W gene product can therefore be classified as an NAD-dependent (2R,3R)-2,3-butanediol dehydrogenase (BDH). S. cerevisiae could grow on 2,3-butanediol as the sole carbon and energy source. Under these conditions, a 3. 5-fold increase in (2R,3R)-2,3-butanediol dehydrogenase activity was observed in the total cell extracts. The isoelectric focusing pattern of the induced enzyme coincided with that of the pure BDH (pI 6.9). The disruption of the YAL060W gene was not lethal for the yeast under laboratory conditions. The disrupted strain could also grow on 2,3-butanediol, although attaining a lesser cell density than the wild-type strain. Taking into consideration the substrate specificity of the YAL060W gene product, we propose the name of BDH for this gene. The corresponding enzyme is the first eukaryotic (2R, 3R)-2,3-butanediol dehydrogenase characterized of the medium-chain dehydrogenase/reductase family.