Evolution of function in the crotonase superfamily: (3S)-methylglutaconyl-CoA hydratase from Pseudomonas putida

Members of the enoyl-CoA hydratase (crotonase) superfamily catalyze different overall reactions that utilize a common catalytic strategy delivered by a shared structural scaffold; the substrates are usually acyl esters of coenzyme A, and the intermediates are usually thioester enolate anions stabilized by a conserved oxyanion hole. In many bacterial genomes, orthologous members that contain homologues of acid/base catalyst Glu164 but not of Glu144 in rat mitochondrial crotonase are encoded by operons of which the functions have not been assigned. Focusing on the orthologues from Pseudomonas aeruginosa and P. putida, we have determined that these operons encode enzymes in leucine catabolism with the unknown enzyme assigned as (3S)-methylglutaconyl-CoA hydratase (MGCH), which catalyzes the syn-hydration of (E)-3-methylglutaconyl-CoA to (3S)-hydroxymethylglutaryl-CoA. The discovery that bacterial MGCHs catalyze hydration of enoyl-CoAs utilizing a single active-site residue contrasts with the paradigm crotonases as well as with the recently identified mammalian MGCHs that use homologues of both Glu144 and Glu164 in crotonase. Substrate analogues lacking a gamma-carboxylate have been shown to be competitive inhibitors of the enzyme, and installation of a glutamate for the "missing" homologue of Glu144 fails to introduce hydratase activity with the substrate analogues. Thus, bacterial MGCHs may provide an example of opportunistic evolution in which a carboxylate group of the substrate functionally replaces one of the active site glutamate residues in the reactions catalyzed by crotonases and the eukaryotic MGCHs.     

Mutation of Lys242 allows Delta3-Delta2-enoyl-CoA isomerase to acquire enoyl-CoA hydratase activity

We report here a novel example of generating hydratase activity through site-directed mutagenesis of a single residue Lys242 of rat liver mitochondrial Delta3-Delta2-enoyl-CoA isomerase, which is one of the key enzymes involved in fatty acid oxidation and a member of the crotonase superfamily. Lys242 is at the C-terminal of the enzyme, which is far from the active site in the crotonase superfamily and forms a salt bridge with Asp149. A variety of mutant expression plasmids were constructed, and it was observed that mutation of Lys242 to nonbasic residues allowed the mutants to have enoyl-CoA hydratase activity confirmed by HPLC analysis of the incubation mixture. Kinetic studies of these mutants were carried out for both isomerase and hydratase activities. Mutant K242C showed a k(cat) value of 1.0 s(-1) for hydration reaction. This activity constitutes about 10% of the total enzyme activity, and the remaining 90% is its natural isomerase activity. To the best of our knowledge, this is the first report on the generation of functional promiscuity through single amino acid mutation far from the active site. This may be a simple and efficient approach to designing a new enzyme based on an existing template. It could perhaps become a general methodology for facilitating an enzyme to acquire a type enzymatic activity that belongs to another member of the same superfamily, by interrupting a key structural element in order to introduce ambiguity, using site-directed mutagenesis.     

Domain swapping in the low-similarity isomerase/hydratase superfamily: the crystal structure of rat mitochondrial Delta3, Delta2-enoyl-CoA isomerase

Two monofunctional Delta(3), Delta(2)-enoyl-CoA isomerases, one in mitochondria (mECI) and the other in both mitochondria and peroxisomes (pECI), belong to the low-similarity isomerase/hydratase superfamily. Both enzymes catalyze the movement of a double bond from C3 to C2 of an unsaturated acyl-CoA substrate for re-entry into the beta-oxidation pathway. Mutagenesis has shown that Glu165 of rat mECI is involved in catalysis; however, the putative catalytic residue in yeast pECI, Glu158, is not conserved in mECI. To elucidate whether Glu165 of mECI is correctly positioned for catalysis, the crystal structure of rat mECI has been solved. Crystal packing suggests the enzyme is trimeric, in contrast to other members of the superfamily, which appear crystallographically to be dimers of trimers. The polypeptide fold of mECI, like pECI, belongs to a subset of this superfamily in which the C-terminal domain of a given monomer interacts with its own N-terminal domain. This differs from that of crotonase and 1,4-dihydroxy-2-naphtoyl-CoA synthase, whose C-terminal domains are involved in domain swapping with an adjacent monomer. The structure confirms Glu165 as the putative catalytic acid/base, positioned to abstract the pro-R proton from C2 and reprotonate at C4 of the acyl chain. The large tunnel-shaped active site cavity observed in the mECI structure explains the relative substrate promiscuity in acyl-chain length and stereochemistry. Comparison with the crystal structure of pECI suggests the catalytic residues from both enzymes are spatially conserved but not in their primary structures, providing a powerful reminder of how catalytic residues cannot be determined solely by sequence alignments.     

Structural characterization of a beta-diketone hydrolase from the cyanobacterium Anabaena sp. PCC 7120 in native and product-bound forms, a coenzyme A-independent member of the crotonase suprafamily

The gene alr4455 from the well-studied cyanobacterium Anabaena sp. PCC 7120 encodes a crotonase orthologue that displays beta-diketone hydrolase activity. Anabaena beta-diketone hydrolase (ABDH), in common with 6-oxocamphor hydrolase (OCH) from Rhodococcus sp. NCIMB 9784, catalyzes the desymmetrization of bicyclo[2.2.2]octane-2,6-dione to yield [(S)-3-oxocyclohexyl]acetic acid, a reaction unusual among the crotonase superfamily as the substrate is not an acyl-CoA thioester. The structure of ABDH has been determined to a resolution of 1.5 A in both native and ligand-bound forms. ABDH forms a hexamer similar to OCH and features one active site per enzyme monomer. The arrangement of side chains in the active site indicates that while the catalytic chemistry may be conserved in OCH orthologues, the structural determinants of substrate specificity are different. In the active site of ligand-bound forms that had been cocrystallized with the bicyclic diketone substrate bicyclo[2.2.2]octane-2,6-dione was found the product of the asymmetric enzymatic retro-Claisen reaction [(S)-3-oxocyclohexyl]acetic acid. The structures of ABDH in both native and ligand-bound forms reveal further details about structural variation and modes of coenzyme A-independent activity within the crotonases and provide further evidence of a wider suprafamily of enzymes that have recruited the crotonase fold for the catalysis of reactions other than those regularly attributed to canonical superfamily members.     

Crystal structure of 3-carboxy-cis,cis-muconate lactonizing enzyme from Pseudomonas putida, a fumarase class II type cycloisomerase: enzyme evolution in parallel pathways

3-Carboxy-cis,cis-muconate lactonizing enzymes (CMLEs), the key enzymes in the protocatechuate branch of the beta-ketoadipate pathway in microorganisms, catalyze the conversion of 3-carboxy-cis,cis-muconate to muconolactones. We have determined the crystal structure of the prokaryotic Pseudomonas putida CMLE (PpCMLE) at 2.6 A resolution. PpCMLE is a homotetramer and belongs to the fumarase class II superfamily. The active site of PpCMLE is formed largely by three regions, which are moderately conserved in the fumarase class II superfamily, from three respective monomers. It has been proposed that residue His141, which is highly conserved in all fumarase class II enzymes and forms a charge relay with residue Glu275 (both His141 and Glu275 are in adenylosuccinate lyase numbering), acts as the general base in most fumarase class II superfamily members. However, this charge relay pair is broken in PpCMLE. The residues corresponding to His141 and Glu275 are Trp153 and Ala289, respectively, in PpCMLE. The structures of prokaryotic MLEs and that of CMLE from the eukaryotic Neurospora crassa are completely different from that of PpCMLE, indicating MLEs and CMLEs, as well as the prokaryotic and eukaryotic CMLEs, evolved from distinct ancestors, although they catalyze similar reactions. The structural differences may be related to recognition by substrates and to differences in the mechanistic pathways by which these enzymes catalyze their respective reactions.     

Streptomyces griseus trypsin is stabilized against autolysis by the cooperation of a salt bridge and cation-pi interaction

Streptomyces griseus trypsin (SGT) is a bacterial trypsin that lacks the conserved disulphide bond surrounding the autolysis loop. We investigated the molecular mechanism by which SGT is stabilized against autolysis. The autolysis loop connects to another surface loop via a salt bridge (Glu146-Arg222), and the Arg222 residue also forms a cation-pi interaction with Tyr217. Elimination of these bonds by site-directed mutagenesis showed that the surface salt bridge at Glu146-Arg222 is the main force stabilizing the enzyme against autolysis. The effect of the cation-pi interaction at Tyr217-Arg222 is small, however, its presence increases the half-life by about five hours and enhances the protein stability more than three-fold considering the catalytic activity in the presence of the salt bridge. The melting temperature also showed cooperation between the salt bridge and cation-pi interaction. These findings show that S. griseus trypsin is stabilized against autolysis through a cooperative network of a salt bridge and cation-pi interaction, which compensate for the absence of the conserved C136-C201 disulphide bond.     

New reactions in the crotonase superfamily: structure of methylmalonyl CoA decarboxylase from Escherichia coli

The molecular structure of methylmalonyl CoA decarboxylase (MMCD), a newly defined member of the crotonase superfamily encoded by the Escherichia coli genome, has been solved by X-ray crystallographic analyses to a resolution of 1.85 A for the unliganded form and to a resolution of 2.7 A for a complex with an inert thioether analogue of methylmalonyl CoA. Like two other structurally characterized members of the crotonase superfamily (crotonase and dienoyl CoA isomerase), MMCD is a hexamer (dimer of trimers) with each polypeptide chain composed of two structural motifs. The larger N-terminal domain contains the active site while the smaller C-terminal motif is alpha-helical and involved primarily in trimerization. Unlike the other members of the crotonase superfamily, however, the C-terminal motif is folded back onto the N-terminal domain such that each active site is wholly contained within a single subunit. The carboxylate group of the thioether analogue of methylmalonyl CoA is hydrogen bonded to the peptidic NH group of Gly 110 and the imidazole ring of His 66. From modeling studies, it appears that Tyr 140 is positioned within the active site to participate in the decarboxylation reaction by orienting the carboxylate group of methylmalonyl CoA so that it is orthogonal to the plane of the thioester carbonyl group. Surprisingly, while the active site of MMCD contains Glu 113, which is homologous to the general acid/base Glu 144 in the active site of crotonase, its carboxylate side chain is hydrogen bonded to Arg 86, suggesting that it is not directly involved in catalysis. The new constellation of putative functional groups observed in the active site of MMCD underscores the diversity of function in this superfamily.     

The 1.3 A crystal structure of human mitochondrial Delta3-Delta2-enoyl-CoA isomerase shows a novel mode of binding for the fatty acyl group

The crystal structure of Delta3-Delta2-enoyl-CoA isomerase from human mitochondria (hmEci), complexed with the substrate analogue octanoyl-CoA, has been refined at 1.3 A resolution. This enzyme takes part in the beta-oxidation of unsaturated fatty acids by converting both cis-3 and trans-3-enoyl-CoA esters (with variable length of the acyl group) to trans-2-enoyl-CoA. hmEci belongs to the hydratase/isomerase (crotonase) superfamily. Most of the enzymes belonging to this superfamily are hexamers, but hmEci is shown to be a trimer. The mode of binding of the ligand, octanoyl-CoA, shows that the omega-end of the acyl group binds in a hydrophobic tunnel formed by residues of the loop preceding helix H4 as well as by side-chains of the kinked helix H9. From the structure of the complex it can be seen that Glu136 is the only catalytic residue. The importance of Glu136 for catalysis is confirmed by mutagenesis studies. A cavity analysis shows the presence of two large, adjacent empty hydrophobic cavities near the active site, which are shaped by side-chains of helices H1, H2, H3 and H4. The structure comparison of hmEci with structures of other superfamily members, in particular of rat mitochondrial hydratase (crotonase) and yeast peroxisomal enoyl-CoA isomerase, highlights the variable mode of binding of the fatty acid moiety in this superfamily.     

Glu88 in the non-catalytic domain of acylpeptide hydrolase plays dual roles: charge neutralization for enzymatic activity and formation of salt bridge for thermodynamic stability

Acylpeptide hydrolase of Aeropyrum pernix K1 is composed of a catalytic alpha/beta hydrolase domain and a non-catalytic beta-propeller domain. The Glu88 residue of the propeller domain is highly conserved in the prolyl oligopeptidase family and forms an inter-domain salt bridge with Arg526, a key residue for substrate binding. We have dissected the functions of Glu88 using site-directed mutagenesis, steady-state kinetics analyses, and molecular dynamics simulations. In E88A and E88A/R526K mutants, with a broken inter-domain salt bridge and a positive charge at position 526, catalytic activities for both a peptidase substrate and an esterase substrate were almost abolished. Analysis of the pH dependence of the mutants' reaction kinetics indicates that these mutations lead to changes in the electrostatic environment of the active site, which can be modulated by chloride ions. These findings indicate that the neutralization at position 526 is favorable for the activity of the enzyme, which is also verified by the catalytic behavior of E88A/R526V mutant. All mutants have lower thermodynamic stability than the wild-type. Therefore, Glu88 plays two major roles in the function of the enzyme: neutralizing the positive charge of Arg526, thereby increasing the enzymatic activity, and forming the Glu88-Arg526 salt bridge, thereby stabilizing the protein.     

The 1.3-Angstrom-resolution crystal structure of beta-ketoacyl-acyl carrier protein synthase II from Streptococcus pneumoniae

The beta-ketoacyl-acyl carrier protein synthases are members of the thiolase superfamily and are key regulators of bacterial fatty acid synthesis. As essential components of the bacterial lipid metabolic pathway, they are an attractive target for antibacterial drug discovery. We have determined the 1.3 A resolution crystal structure of the beta-ketoacyl-acyl carrier protein synthase II (FabF) from the pathogenic organism Streptococcus pneumoniae. The protein adopts a duplicated betaalphabetaalphabetaalphabetabeta fold, which is characteristic of the thiolase superfamily. The two-fold pseudosymmetry is broken by the presence of distinct insertions in the two halves of the protein. These insertions have evolved to bind the specific substrates of this particular member of the thiolase superfamily. Docking of the pantetheine moiety of the substrate identifies the loop regions involved in substrate binding and indicates roles for specific, conserved residues in the substrate binding tunnel. The active site triad of this superfamily is present in spFabF as His 303, His 337, and Cys 164. Near the active site is an ion pair, Glu 346 and Lys 332, that is conserved in the condensing enzymes but is unusual in our structure in being stabilized by an Mg(2+) ion which interacts with Glu 346. The active site histidines interact asymmetrically with Lys 332, whose positive charge is closer to His 303, and we propose a specific role for the lysine in polarizing the imidazole ring of this histidine. This asymmetry suggests that the two histidines have unequal roles in catalysis and provides new insights into the catalytic mechanisms of these enzymes.     

Crystal structure of the ECH2 catalytic domain of CurF from Lyngbya majuscula. Insights into a decarboxylase involved in polyketide chain beta-branching

Curacin A is a mixed polyketide/nonribosomal peptide possessing anti-mitotic and anti-proliferative activity. In the biosynthesis of curacin A, the N-terminal domain of the CurF multifunctional protein catalyzes decarboxylation of 3-methylglutaconyl-acyl carrier protein (ACP) to 3-methylcrotonyl-ACP, the postulated precursor of the cyclopropane ring of curacin A. This decarboxylase is encoded within an "HCS cassette" that is used by several other polyketide biosynthetic systems to generate chemical diversity by introduction of a beta-branch functional group to the natural product. The crystal structure of the CurF N-terminal ECH(2) domain establishes that the protein is a crotonase superfamily member. Ala(78) and Gly(118) form an oxyanion hole in the active site that includes only three polar side chains as potential catalytic residues. Site-directed mutagenesis and a biochemical assay established critical functions for His(240) and Lys(86), whereas Tyr(82) was nonessential. A decarboxylation mechanism is proposed in which His(240) serves to stabilize the substrate carboxylate and Lys(86) donates a proton to C-4 of the acyl-ACP enolate intermediate to form the Delta(2) unsaturated isopentenoyl-ACP product. The CurF ECH(2) domain showed a 20-fold selectivity for ACP-over CoA-linked substrates. Specificity for ACP-linked substrates has not been reported for any other crotonase superfamily decarboxylase. Tyr(73) may select against CoA-linked substrates by blocking a contact of Arg(38) with the CoA adenosine 5'-phosphate.     

Structural mechanism of enoyl-CoA hydratase: three atoms from a single water are added in either an E1cb stepwise or concerted fashion

We have determined the crystal structure of the enzyme enoyl-CoA hydratase (ECH) from rat liver with the bound substrate 4-(N,N-dimethylamino)cinnamoyl-CoA using X-ray diffraction data to a resolution of 2.3 A. In addition to the thiolester substrate, the catalytic water, which is added in the hydration reaction, has been modeled into well-defined electron density in each of the six active sites of the physiological hexamer within the crystallographic asymmetric unit. The catalytic water bridges Glu(144) and Glu(164) of the enzyme and has a lone pair of electrons poised to react with C(3) of the enzyme-bound alpha,beta-unsaturated thiolester. The water molecule, which bridges two glutamate residues, is reminiscent of the enolase active site. However, unlike enolase, which has a lysine available to donate a proton, there are no other sources of protons available from other active site residues in ECH. Furthermore, an analysis of the hydrogen-bonding network of the active site suggests that both Glu(144) and Glu(164) are ionized and carry a negative charge with no reasonable place to have a protonated carboxylate. This lack of hydrogen-bonding acceptors that could accommodate a source of a proton, other than from the water molecule, leads to a hypothesis that the three atoms from a single water molecule are added across the double bond to form the hydrated product. The structural results are discussed in connection with details of the mechanism, which have been elucidated from kinetics, site-directed mutagenesis, and spectroscopy of enzyme-substrate species, in presenting an atomic-resolution mechanism of the reaction. Contrary to the previous interpretation, the structure of the E-S complex together with previously determined kinetic isotope effects is consistent with either a concerted mechanism or an E1cb stepwise mechanism.     

Mutation of Lys242 allows Δ-Δ-enoyl-CoA isomerase to acquire enoyl-CoA hydratase activity

We report here a novel example of generating hydratase activity through site-directed mutagenesis of a single residue Lys242 of rat liver mitochondrial Δ³-Δ²-enoyl-CoA isomerase, which is one of the key enzymes involved in fatty acid oxidation and a member of the crotonase superfamily. Lys242 is at the C-terminal of the enzyme, which is far from the active site in the crotonase superfamily and forms a salt bridge with Asp149. A variety of mutant expression plasmids were constructed, and it was observed that mutation of Lys242 to nonbasic residues allowed the mutants to have enoyl-CoA hydratase activity confirmed by HPLC analysis of the incubation mixture. Kinetic studies of these mutants were carried out for both isomerase and hydratase activities. Mutant K242C showed a k_cat value of 1.0 s^− 1 for hydration reaction. This activity constitutes about 10% of the total enzyme activity, and the remaining 90% is its natural isomerase activity. To the best of our knowledge, this is the first report on the generation of functional promiscuity through single amino acid mutation far from the active site. This may be a simple and efficient approach to designing a new enzyme based on an existing template. It could perhaps become a general methodology for facilitating an enzyme to acquire a type enzymatic activity that belongs to another member of the same superfamily, by interrupting a key structural element in order to introduce ambiguity, using site-directed mutagenesis.     

Constitutive activity of a UV cone opsin

Vertebrate visual pigment proteins contain a conserved carboxylic acid residue in the third transmembrane helix. In rhodopsin, Glu113 serves as a counterion to the positively charged protonated Schiff base formed by 11-cis retinal attached to Lys296. Activation involves breaking of this ion pair. In UV cone pigments, the retinyl Schiff base is unprotonated, and hence such a salt bridge is not present; yet the pigment is inactive in the dark. Mutation of Glu108, which corresponds to rhodopsin's Glu113, to Gln yields a pigment that remains inactive in the dark. The apoproteins of both the wild-type and mutant, however, are constitutively active with the mutant being of significantly higher activity. Thus, one important role for preserving the negatively charged glutamate in the third helix of UV pigments is to maintain a less active opsin in a manner similar to rhodopsin. Ligand binding itself in the absence of a salt bridge is sufficient for deactivation.     

Mutability of an HNH nuclease imidazole general base and exchange of a deprotonation mechanism

Several unique protein folds that catalyze the hydrolysis of phosphodiester bonds have arisen independently in nature, including the PD(D/E)XK superfamily (typified by type II restriction endonucleases and many recombination and repair enzymes) and the HNH superfamily (found in an equally wide array of enzymes, including bacterial colicins and homing endonucleases). Whereas the identity and position of catalytic residues within the PD(D/E)XK superfamily are highly variable, the active sites of HNH nucleases are much more strongly conserved. In this study, the ability of an HNH nuclease to tolerate a mutation of its most conserved catalytic residue (its histidine general base), and the mechanism of the most active enzyme variant, were characterized. Conversion of this residue into several altered chemistries, glutamine, lysine, or glutamate, resulted in measurable activity. The histidine to glutamine mutant displays the highest residual activity and a pH profile similar to that of the wild-type enzyme. This activity is dependent on the presence of a neighboring imidazole ring, which has taken over as a less efficient general base for the reaction. This result implies that mutational pathways to alternative HNH-derived catalytic sites do exist but are not as extensively or successfully diverged or reoptimized in nature as variants of the PD(D/E)XK nuclease superfamily. This is possibly due to multiple steric constraints placed on the compact HNH motif, which is simultaneously involved in protein folding, DNA binding, and catalysis, as well as the use of a planar, aromatic imidazole group as a general base.     

The flavoprotein MrsD catalyzes the oxidative decarboxylation reaction involved in formation of the peptidoglycan biosynthesis inhibitor mersacidin

The lantibiotic mersacidin inhibits peptidoglycan biosynthesis by binding to the peptidoglycan precursor lipid II. Mersacidin contains an unsaturated thioether bridge, which is proposed to be synthesized by posttranslational modifications of threonine residue +15 and the COOH-terminal cysteine residue of the mersacidin precursor peptide MrsA. We show that the flavoprotein MrsD catalyzes the oxidative decarboxylation of the COOH-terminal cysteine residue of MrsA to an aminoenethiol residue. MrsD belongs to the recently described family of homo-oligomeric flavin-containing Cys decarboxylases (i.e., the HFCD protein family). Members of this protein family include the bacterial Dfp proteins (which are involved in coenzyme A biosynthesis), eukaryotic salt tolerance proteins, and further oxidative decarboxylases such as EpiD. In contrast to EpiD and Dfp, MrsD is a FAD and not an FMN-dependent flavoprotein. HFCD enzymes are characterized by a conserved His residue which is part of the active site. Exchange of this His residue for Asn led to inactivation of MrsD. The lantibiotic-synthesizing enzymes EpiD and MrsD have different substrate specificities.     

A revised mechanism for the inactivation of bovine liver enoyl-CoA hydratase by (methylenecyclopropyl)formyl-CoA based on unexpected results with the C114A mutant

The compound (methylenecyclopropyl)formyl-CoA (MCPF-CoA) has been reported earlier as a potent active site-directed inactivator of bovine liver enoyl-CoA hydratase (ECH). It is believed that the mechanism of inactivation involves the attack of Cys114 at C-2' of MCPF-CoA, resulting in ring cleavage and permanent covalent modification of the enzyme. Here, we describe studies with the C114A mutant of bovine liver ECH, which was constructed and purified to determine the role of this residue in the catalytic mechanism of the enzyme. The C114A mutant, which is catalytically competent, shows an unexpected susceptibility to inactivation by MCPF-CoA, indicating that Cys114 is not the primary nucleophile responsible for the inactivation of the enzyme. To determine if catalytic residues Glu115 and Glu135 play a role in the inactivation of the enzyme, the E115Q and E135Q mutants were also constructed and purified. It was determined that these mutants did not react with MCPF-CoA, indicating a possible role for both residues in the inactivation of the wild-type enzyme. Pepsin digestion and subsequent LC-MS/MS analysis of the inactivated wild-type enzyme and C114A mutant revealed that Glu115 was modified in each case, supporting the hypothesis that this residue is the true nucleophile that traps MCPF-CoA and indicating that the covalent modification of Cys114 reported earlier may be a postinactivation artifact. We propose a modified mechanism of inactivation involving Glu115 and Glu135, and suggest that MCPF-CoA may be a mechanism-based inhibitor for bovine liver ECH.     

Aldehyde dehydrogenase. Maintaining critical active site geometry at motif 8 in the class 3 enzyme.

Alignment of all known, diverse members of the aldehyde dehydrogenase (ALDH) extended family revealed only two strictly conserved, nonglycine residues, a glutamate and a phenylalanine residue. Both occur in one of the highly conserved 'motif' segments and both occupy strategic locations in the tertiary structure at the bottom of the catalytic funnel. In class 3 ALDH, these are Glu333 and Phe335. In addition, Asp247, which is not highly conserved but is characteristic of class 3 ALDHs, hydrogen bonds the main chain between Glu333 and Phe335. These three residues were mutated conservatively. Michaelis constants determined for both NAD/propanal and NADP/benzaldehyde substrate pairs show all three residues to be crucial to effective catalysis, and suggest that the hydrogen bond to Asp247 is a key element in maintaining precise geometry of key elements at the active site.     

Structural and Computational Studies of the Staphylococcus aureus Sortase B-Substrate Complex Reveal a Substrate-stabilized Oxyanion Hole

Sortase cysteine transpeptidases covalently attach proteins to the bacterial cell wall or assemble fiber-like pili that promote bacterial adhesion. Members of this enzyme superfamily are widely distributed in Gram-positive bacteria that frequently utilize multiple sortases to elaborate their peptidoglycan. Sortases catalyze transpeptidation using a conserved active site His-Cys-Arg triad that joins a sorting signal located at the C terminus of their protein substrate to an amino nucleophile located on the cell surface. However, despite extensive study, the catalytic mechanism and molecular basis of substrate recognition remains poorly understood. Here we report the crystal structure of the Staphylococcus aureus sortase B enzyme in a covalent complex with an analog of its NPQTN sorting signal substrate, revealing the structural basis through which it displays the IsdC protein involved in heme-iron scavenging from human hemoglobin. The results of computational modeling, molecular dynamics simulations, and targeted amino acid mutagenesis indicate that the backbone amide of Glu²²⁴ and the side chain of Arg²³³ form an oxyanion hole in sortase B that stabilizes high energy tetrahedral catalytic intermediates. Surprisingly, a highly conserved threonine residue within the bound sorting signal substrate facilitates construction of the oxyanion hole by stabilizing the position of the active site arginine residue via hydrogen bonding. Molecular dynamics simulations and primary sequence conservation suggest that the sorting signal-stabilized oxyanion hole is a universal feature of enzymes within the sortase superfamily.     

X-ray structure of yeast Hal2p, a major target of lithium and sodium toxicity, and identification of framework interactions determining cation sensitivity

The product of the yeast HAL2 gene (Hal2p) is an in vivo target of sodium and lithium toxicity and its overexpression improves salt tolerance in yeast and plants. Hal2p is a metabolic phosphatase which catalyses the hydrolysis of 3'-phosphoadenosine-5'-phosphate (PAP) to AMP. It is, the prototype of an evolutionarily conserved family of PAP phosphatases and the engineering of sodium insensitive enzymes of this group may contribute to the generation of salt-tolerant crops. We have solved the crystal structure of Hal2p in complex with magnesium, lithium and the two products of PAP hydrolysis, AMP and Pi, at 1.6 A resolution. A functional screening of random mutations of the HAL2 gene in growing yeast generated forms of the enzyme with reduced cation sensitivity. Analysis of these mutants defined a salt bridge (Glu238 ellipsis Arg152) and a hydrophobic bond (Va170 ellipsis Trp293) as important framework interactions determining cation sensitivity. Hal2p belongs to a larger superfamily of lithium-sensitive phosphatases which includes inositol monophosphatase. The hydrophobic interaction mutated in Hal2p is conserved in this superfamily and its disruption in human inositol monophosphatase also resulted in reduced cation sensitivity.