Crystal Structure of Diaminopimelate Epimerase from Arabidopsis thaliana, an Amino Acid Racemase Critical for l-Lysine Biosynthesis

Diaminopimelate (DAP) epimerase is a key enzyme for the biosynthesis of lysine in plants. Lysine is an essential dietary nutrient for mammals. In both plants and bacteria, DAP epimerase catalyzes the interconversion of ll-DAP and dl(meso)-DAP. The absence of a mammalian homolog makes DAP epimerase a promising target for the design of novel herbicides and antibacterials. This enzyme requires no cofactors and it functions through an unusual mechanism involving two cysteine residues acting in concert and alternating as a base (thiolate) and as an acid (thiol). The present study reports the crystal structures of two enzyme-inhibitor complexes of DAP epimerase from Arabidopsis thaliana with different isomers of the irreversible inhibitor and substrate mimic, 2-(4-amino-4-carboxybutyl)-aziridine-2-carboxylate, at 1.95 and 2.3 Å resolution. These structures provide the first atomic details of a plant amino acid racemase. Structural analysis reveals that ligand binding to a cleft between the two domains of the enzyme is accompanied by domain closure with two strictly conserved cysteine residues, Cys99 and Cys254, optimally positioned to perform acid/base catalysis via a carbanion stabilization mechanism on the stereogenic α-carbon atom of the amino acid. Stereochemical control in catalysis is achieved by means of a highly symmetric catalytic site that can accommodate both the l and d stereogenic centers of DAP at the proximal site, whereas specific interactions at the distal site require only the l configuration. Structural comparisons of the plant enzyme with its bacterial counterpart from Haemophilus influenzae reveal significant conservation of amino acid residues around the active site that extends to their three-dimensional structures and catalytic mechanism.     

Ordering of C-terminal loop and glutaminase domains of glucosamine-6-phosphate synthase promotes sugar ring opening and formation of the ammonia channel

Glucosamine-6-phosphate synthase (GlmS) channels ammonia from glutamine at the glutaminase site to fructose 6-phosphate (Fru6P) at the synthase site. Escherichia coli GlmS is composed of two C-terminal synthase domains that form the dimer interface and two N-terminal glutaminase domains at its periphery. We report the crystal structures of GlmS alone and in complex with the glucosamine-6-phosphate product at 2.95 Å and 2.9 Å resolution, respectively. Surprisingly, although the whole protein is present in this crystal form, no electron density for the glutaminase domain was observed, indicating its mobility. Comparison of the two structures with that of the previously reported GlmS-Fru6P complex shows that, upon sugar binding, the C-terminal loop, which forms the major part of the channel walls, becomes ordered and covers the synthase site. The ordering of the glutaminase domains likely follows Fru6P binding by the anchoring of Trp74, which acts as the gate of the channel, on the closed C-terminal loop. This is accompanied by a major conformational change of the side chain of Lys503^# of the neighboring synthase domain that strengthens the interactions of the synthase domain with the C-terminal loop and completely shields the synthase site. The concomitant conformational change of the Lys503^#-Gly505^# tripeptide places catalytic His504^# in the proper position to open the sugar and buries the linear sugar, which is now in the vicinity of the catalytic groups involved in the sugar isomerization reaction. Together with the previously reported structures of GlmS in complex with Fru6P or glucose 6-phosphate and a glutamine analogue, the new structures reveal the structural changes occurring during the whole catalytic cycle.     

Induced-fit upon ligand binding revealed by crystal structures of the hot-dog fold thioesterase in dynemicin biosynthesis

Dynemicins are structurally related 10-membered enediyne natural products isolated from Micromonospora chernisa with potent antitumor and antibiotic activity. The early biosynthetic steps of the enediyne moiety of dynemicins are catalyzed by an iterative polyketide synthase (DynE8) and a thioesterase (DynE7). Recent studies indicate that the function of DynE7 is to off-load the linear biosynthetic intermediate assembled on DynE8. Here, we report crystal structures of DynE7 in its free form at 2.7 Å resolution and of DynE7 in complex with the DynE8-produced all-trans pentadecen-2-one at 2.1 Å resolution. These crystal structures reveal that upon ligand binding, significant conformational changes throughout the substrate-binding tunnel result in an expanded tunnel that traverses an entire monomer of the tetrameric DynE7 protein. The enlarged inner segment of the channel binds the carbonyl-conjugated polyene mainly through hydrophobic interactions, whereas the putative catalytic residues are located in the outer segment of the channel. The crystallographic information reinforces an unusual catalytic mechanism that involves a strictly conserved arginine residue for this subfamily of hot-dog fold thioesterases, distinct from the typical mechanism for hot-dog fold thioesterases that utilizes an acidic residue for catalysis.     

Structural basis for natural lactonase and promiscuous phosphotriesterase activities

Organophosphates are the largest class of known insecticides, several of which are potent nerve agents. Consequently, organophosphate-degrading enzymes are of great scientific interest as bioscavengers and biodecontaminants. Recently, a hyperthermophilic phosphotriesterase (known as SsoPox), from the Archaeon Sulfolobus solfataricus, has been isolated and found to possess a very high lactonase activity. Here, we report the three-dimensional structures of SsoPox in the apo form (2.6 Å resolution) and in complex with a quorum-sensing lactone mimic at 2.0 Å resolution. The structure also reveals an unexpected active site topology, and a unique hydrophobic channel that perfectly accommodates the lactone substrate. Structural and mutagenesis evidence allows us to propose a mechanism for lactone hydrolysis and to refine the catalytic mechanism established for phosphotriesterases. In addition, SsoPox structures permit the correlation of experimental lactonase and phosphotriesterase activities and this strongly suggests lactonase activity as the cognate function of SsoPox. This example demonstrates that promiscuous activities probably constitute a large and efficient reservoir for the creation of novel catalytic activities.     

The crystal structure of l-sorbose reductase from Gluconobacter frateurii complexed with NADPH and l-sorbose

l-Sorbose reductase from Gluconobacter frateurii (SR) is an NADPH-dependent oxidoreductase. SR preferentially catalyzes the reversible reaction between d-sorbitol and l-sorbose with high substrate specificity. To elucidate the structural basis of the catalytic mechanism and the substrate specificity of SR, we have determined the structures of apo-SR, SR in complex with NADPH, and the inactive mutant (His116Leu) of SR in complex with NADPH and l-sorbose at 2.83 Å, 1.90 Å, and 1.80 Å resolutions, respectively. Our results show that SR belongs to the short-chain dehydrogenase/reductase (SDR) family and forms a tetrameric structure. Although His116 is not conserved among SDR family enzymes, the structures of SR have revealed that His116 is important for the stabilization of the proton relay system and for active-site conformation as a fourth catalytic residue. In the ternary complex structure, l-sorbose is recognized by 11 hydrogen bonds. Site-directed mutagenesis of residues around the l-sorbose-binding site has shown that the loss of almost full enzymatic activity was caused by not only the substitution of putative catalytic residues but also the substitution of the residue used for the recognition of the C4 hydroxyl groups of l-sorbose (Glu154) and of the residues used for the construction of the substrate-binding pocket (Cys146 and Gly188). The recognition of the C4 hydroxyl group of l-sorbose would be indispensable for the substrate specificity of SR, which recognizes only l-sorbose and d-sorbitol but not other sugars. Our results indicated that these residues were crucial for the substrate recognition and specificity of SR.     

Ultrahigh (0.93 Å) resolution structure of manganese peroxidase from Phanerochaete chrysosporium: Implications for the catalytic mechanism

Manganese peroxidase (MnP) is an extracellular heme enzyme produced by the lignin-degrading white-rot fungus Phanerochaete chrysosporium. MnP catalyzes the peroxide-dependent oxidation of Mn^II to Mn^III. The Mn^III is released from the enzyme in complex with oxalate, enabling the oxalate-Mn^III complex to serve as a diffusible redox mediator capable of oxidizing lignin, especially under the mediation of unsaturated fatty acids. One heme propionate and the side chains of Glu35, Glu39 and Asp179 have been identified as Mn^II ligands in our previous crystal structures of native MnP. In our current work, new 0.93 Å and 1.05 Å crystal structures of MnP with and without bound Mn^II, respectively, have been solved. This represents only the sixth structure of a protein of this size at 0.93 Å resolution. In addition, this is the first structure of a heme peroxidase from a eukaryotic organism at sub-Ångstrom resolution. These new structures reveal an ordering/disordering of the C-terminal loop, which is likely required for Mn binding and release. In addition, the catalytic Arg42 residue at the active site, normally thought to function only in the peroxide activation process, also undergoes ordering/disordering that is coupled to a transient H-bond with the Mn ligand, Glu39. Finally, these high-resolution structures also reveal the exact H atoms in several parts of the structure that are relevant to the catalytic mechanism.     

Conformational Flexibility and Peptide Interaction of the Translocation ATPase SecA

The SecA ATPase forms a functional complex with the protein-conducting SecY channel to translocate polypeptides across the bacterial cell membrane. SecA recognizes the translocation substrate and catalyzes its unidirectional movement through the SecY channel. The recent crystal structure of the Thermotoga maritima SecA-SecYEG complex shows the ATPase in a conformation where the nucleotide-binding domains (NBDs) have closed around a bound ADP-BeFx complex and SecA's polypeptide-binding clamp is shut. Here, we present the crystal structure of T. maritima SecA in isolation, determined in its ADP-bound form at 3.1 Å resolution. SecA alone has a drastically different conformation in which the nucleotide-binding pocket between NBD1 and NBD2 is open and the preprotein cross-linking domain has rotated away from both NBDs, thereby opening the polypeptide-binding clamp. To investigate how this clamp binds polypeptide substrates, we also determined a structure of Bacillus subtilis SecA in complex with a peptide at 2.5 Å resolution. This structure shows that the peptide augments the highly conserved β-sheet at the back of the clamp. Taken together, these structures suggest a mechanism by which ATP hydrolysis can lead to polypeptide translocation.     

Mechanistic implications of mutations to the active site lysine of porphobilinogen synthase

Porphobilinogen synthase (PBGS) is a homo-octameric protein that catalyzes the complex asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). The only characterized intermediate in the PBGS-catalyzed reaction is a Schiff base that forms between the first ALA that binds and a conserved lysine, which in Escherichia coli PBGS is Lys-246 and in human PBGS is Lys-252. In this study, E. coli PBGS mutants K246H, K246M, K246W, K246N, and K246G and human PBGS mutant K252G were characterized. Alterations to this lysine result in a disabled but not totally inactive protein suggesting an alternate mechanism in which proximity and orientation are major catalytic devices. (13)C NMR studies of [3,5-(13)C]porphobilinogen bound at the active sites of the E. coli PBGS and the mutants show only minor chemical shift differences, i.e. environmental alterations. Mammalian PBGS is established to have four functional active sites, whereas the crystal structure of E. coli PBGS shows eight spatially distinct and structurally equivalent subunits. Biochemical data for E. coli PBGS have been interpreted to support both four and eight active sites. A unifying hypothesis is that formation of the Schiff base between this lysine and ALA triggers a conformational change that results in asymmetry. Product binding studies with wild-type E. coli PBGS and K246G demonstrate that both bind porphobilinogen at four per octamer although the latter cannot form the Schiff base from substrate. Thus, formation of the lysine to ALA Schiff base is not required to initiate the asymmetry that results in half-site reactivity.     

Crystal structures of protein phosphatase-1 bound to nodularin-R and tautomycin: a novel scaffold for structure-based drug design of serine/threonine phosphatase inhibitors

Protein phosphatase 1 occurs in all tissues and regulates many pathways, ranging from cell-cycle progression to carbohydrate metabolism. Many naturally occurring, molecular toxins modulate PP1 activity, though the exact mechanism of this differential regulation is not understood. A detailed elucidation of these interactions is crucial for understanding the cellular basis of phosphatase function and signaling pathways but, more importantly, they can serve as the basis for highly specific therapeutics, e.g. against cancer. We report the crystal structures of PP1 in complex with nodularin-R at 1.63 Å and tautomycin at 1.70 Å resolution. The PP1:nodularin-R complex was used to demonstrate the utility of our improved PP1 production technique, which produces highly active, soluble PP1. Tautomycin is one of the few toxins that reportedly preferentially binds PP1> PP2A. Therefore, the PP1:tautomycin structure is the first complex structure with a toxin with preferred PP1 specificity. Furthermore, since tautomycin is a linear non-peptide-based toxin, our reported structure will aid the design of lead compounds for novel PP1-specific pharmaceuticals.     

How essential is the ‘essential’ active-site lysine in dihydrodipicolinate synthase?

Dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52), a validated antibiotic target, catalyses the first committed step in the lysine biosynthetic pathway: the condensation reaction between (S)-aspartate β-semialdehyde [(S)-ASA] and pyruvate via the formation of a Schiff base intermediate between pyruvate and the absolutely conserved active-site lysine. Escherichia coli DHDPS mutants K161A and K161R of the active-site lysine were characterised for the first time. Unexpectedly, the mutant enzymes were still catalytically active, albeit with a significant decrease in activity. The k_cat values for DHDPS-K161A and DHDPS-K161R were 0.06 ± 0.02 s⁻¹ and 0.16 ± 0.06 s⁻¹ respectively, compared to 45 ± 3 s⁻¹ for the wild-type enzyme. Remarkably, the K_M values for pyruvate increased by only 3-fold for DHDPS-K161A and DHDPS-K161R (0.45 ± 0.04 mM and 0.57 ± 0.06 mM, compared to 0.15 ± 0.01 mM for the wild-type DHDPS), while the K_M values for (S)-ASA remained the same for DHDPS-K161R (0.12 ± 0.01 mM) and increased by only 2-fold for DHDPS-K161A (0.23 ± 0.02 mM) and the K_i for lysine was unchanged. The X-ray crystal structures of DHDPS-K161A and DHDPS-K161R were solved at resolutions of 2.0 and 2.1 Å respectively and showed no changes in their secondary or tertiary structures when compared to the wild-type structure. The crystal structure of DHDPS-K161A with pyruvate bound at the active site was solved at a resolution of 2.3 Å and revealed a defined binding pocket for pyruvate that is thus not dependent upon lysine 161. Taken together with ITC and NMR data, it is concluded that although lysine 161 is important in the wild-type DHDPS-catalysed reaction, it is not absolutely essential for catalysis.     

Structural Insight into the Mechanism of c-di-GMP Hydrolysis by EAL Domain Phosphodiesterases

Cyclic diguanylate (or bis-(3′-5′) cyclic dimeric guanosine monophosphate; c-di-GMP) is a ubiquitous second messenger that regulates diverse cellular functions, including motility, biofilm formation, cell cycle progression, and virulence in bacteria. In the cell, degradation of c-di-GMP is catalyzed by highly specific EAL domain phosphodiesterases whose catalytic mechanism is still unclear. Here, we purified 13 EAL domain proteins from various organisms and demonstrated that their catalytic activity is associated with the presence of 10 conserved EAL domain residues. The crystal structure of the TBD1265 EAL domain was determined in free state (1.8 Å) and in complex with c-di-GMP (2.35 Å), and unveiled the role of conserved residues in substrate binding and catalysis. The structure revealed the presence of two metal ions directly coordinated by six conserved residues, two oxygens of c-di-GMP phosphate, and potential catalytic water molecule. Our results support a two-metal-ion catalytic mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases.     

Catalytic mechanism of S-adenosylhomocysteine hydrolase. Site-directed mutagenesis of Asp-130, Lys-185, Asp-189, and Asn-190

S-Adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the hydrolysis of S-adenosylhomocysteine to form adenosine and homocysteine. On the bases of crystal structures of the wild type enzyme and the D244E mutated enzyme complexed with 3'-keto-adenosine (D244E.Ado*), we have identified the important amino acid residues, Asp-130, Lys-185, Asp-189, and Asn-190, for the catalytic reaction and have proposed a catalytic mechanism (Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (2000) J. Biol. Chem. 275, 32147-32156). To confirm the proposed catalytic mechanism, we have made the D130N, K185N, D189N, and N190S mutated enzymes and measured the catalytic activities. The catalytic rates (k(cat)) of D130N, K185N, D189N, and N190S mutated enzymes are reduced to 0.7%, 0.5%, 0.1%, and 0.5%, respectively, in comparison with the wild type enzyme, indicating that Asp-130, Lys-185, Asp-189, and Asn-190 are involved in the catalytic reaction. K(m) values of the mutated enzymes are increased significantly, except for the N190S mutation, suggesting that Asp-130, Lys-185, and Asp-189 participate in the substrate binding. To interpret the kinetic data, the oxidation states of the bound NAD molecules of the wild type and mutated enzymes were measured during the catalytic reaction by monitoring the absorbance at 340 nm. The crystal structures of the WT and D244E.Ado*, containing four subunits in the crystallographic asymmetric unit, were re-refined to have the same subunit structures. A detailed catalytic mechanism of AdoHcyase has been revealed based on the oxidation states of the bound NAD and the re-refined crystal structures of WT and D244E.Ado*. Lys-185 and Asp-130 abstract hydrogen atoms from 3'-OH and 4'-CH, respectively. Asp-189 removes a proton from Lys-185 and produces the neutral N zeta (-NH(2)), and Asn-190 facilitates formation of the neutral Lys-185. His-54 and His-300 hold and polarize a water molecule, which nucleophilically attacks the C5'- of 3'-keto-4',5'-dehydroadenosine to produce 3'-keto-Ado.     

Structure of Isoprene Synthase Illuminates the Chemical Mechanism of Teragram Atmospheric Carbon Emission

The X-ray crystal structure of recombinant PcISPS (isoprene synthase from gray poplar hybrid Populus × canescens) has been determined at 2.7 Å resolution, and the structure of its complex with three Mg²⁺ and the unreactive substrate analogue dimethylallyl-S-thiolodiphosphate has been determined at 2.8 Å resolution. Analysis of these structures suggests that the generation of isoprene from substrate dimethylallyl diphosphate occurs via a syn-periplanar elimination mechanism in which the diphosphate-leaving group serves as a general base. This chemical mechanism is responsible for the annual atmospheric emission of 100 Tg of isoprene by terrestrial plant life. Importantly, the PcISPS structure promises to guide future protein engineering studies, potentially leading to hydrocarbon fuels and products that do not rely on traditional petrochemical sources.     

Crystal structure of Escherichia coli penicillin-binding protein 5 bound to a tripeptide boronic acid inhibitor: a role for Ser-110 in deacylation

Penicillin-binding protein 5 (PBP 5) from Escherichia coli is a well-characterized d-alanine carboxypeptidase that serves as a prototypical enzyme to elucidate the structure, function, and catalytic mechanism of PBPs. A comprehensive understanding of the catalytic mechanism underlying d-alanine carboxypeptidation and antibiotic binding has proven elusive. In this study, we report the crystal structure at 1.6 A resolution of PBP 5 in complex with a substrate-like peptide boronic acid, which was designed to resemble the transition-state intermediate during the deacylation step of the enzyme-catalyzed reaction with peptide substrates. In the structure of the complex, the boron atom is covalently attached to Ser-44, which in turn is within hydrogen-bonding distance to Lys-47. This arrangement further supports the assignment of Lys-47 as the general base that activates Ser-44 during acylation. One of the two hydroxyls in the boronyl center (O2) is held by the oxyanion hole comprising the amides of Ser-44 and His-216, while the other hydroxyl (O3), which is analogous to the nucleophilic water for hydrolysis of the acyl-enzyme intermediate, is solvated by a water molecule that bridges to Ser-110. Lys-47 is not well-positioned to act as the catalytic base in the deacylation reaction. Instead, these data suggest a mechanism of catalysis for deacylation that uses a hydrogen-bonding network, involving Lys-213, Ser-110, and a bridging water molecule, to polarize the hydrolytic water molecule.     

Structure of Novel Enzyme in Mannan Biodegradation Process 4-O-β-d-Mannosyl-d-Glucose Phosphorylase MGP

The crystal structure of a novel component of the mannan biodegradation system, 4-O-β-d-mannosyl-d-glucose phosphorylase (MGP), was determined to a 1.68-Å resolution. The structure of the enzyme revealed a unique homohexameric structure, which was formed by using two helices attached to the N-terminus and C-terminus as a tab for sticking between subunits. The structures of MGP complexes with genuine substrates, 4-O-β-d-mannosyl-d-glucose and phosphate, and the product d-mannose-1-phosphate were also determined. The complex structures revealed that the invariant residue Asp131, which is supposed to be the general acid/base, did not exist close to the glycosidic Glc-O4 atom, which should be protonated in the catalytic reaction. Also, no solvent molecule that might mediate a proton transfer from Asp131 was observed in the substrate complex structure, suggesting that the catalytic mechanism of MGP is different from those of known disaccharide phosphorylases.     

Substrate Binding and Catalysis in Carbamate Kinase Ascertained by Crystallographic and Site-Directed Mutagenesis Studies: Movements and Significance of a Unique Globular Subdomain of This Key Enzyme for Fermentative ATP Production in Bacteria

Carbamate kinase (CK) makes ATP from ADP and carbamoyl phosphate (CP) in the final step of the microbial fermentative catabolism of arginine, agmatine, and oxalurate/allantoin. Two previously reported CK structures failed to clarify CP binding and catalysis and to reveal the significance of the protruding subdomain (PSD) that hangs over the CK active center as an exclusive and characteristic CK feature. We clarify now these three questions by determining two crystal structures of Enterococcus faecalis CK (one at 1.5 Å resolution and containing bound MgADP, and the other at 2.1 Å resolution and having in the active center one sulfate and two fixed water molecules that mimic one bound CP molecule) and by mutating active-center residues, determining the consequences of these mutations on enzyme functionality. Superimposition of the present crystal structures reconstructs the filled active center in the ternary complex, immediately suggesting in-line associative phosphoryl group transfer and a mechanism for enzyme catalysis involving N51, K209, K271, D210, and the PSD residue K128. The large respective increases and decreases in K_m^CP and k_cat triggered by the mutations N51A, K128A, K209A, and D210N corroborate the ternary complex active-site architecture and the catalytic mechanism proposed. The extreme negative effects of K128A demonstrate a key role of the PSD in substrate binding and catalysis. The crystal structures reveal large rigid-body movements of the PSD towards the enzyme body that place K128 next to CP and bury the CP site. A mechanism that connects CP site occupation with the PSD approach, involving V206-I207 in the CP site and P162-S163 in the PSD stem, is identified. The effects of the V206A and V206L mutations support this mechanism. It is concluded that the PSD movement allows CK to select against the abundant CP/carbamate analogues acetylphosphate/acetate and bicarbonate, rendering CK highly selective for CP/carbamate.     

The high-resolution structures of the neutral and the low pH crystals of aminopeptidase from Aeromonas proteolytica

The aminopeptidase from Aeromonas proteolytica (AAP) contains two zinc ions in the active site and catalyzes the degradation of peptides. Herein we report the crystal structures of AAP at 0.95-Å resolution at neutral pH and at 1.24-Å resolution at low pH. The combination of these structures allowed the precise modeling of atomic positions, the identification of the metal bridging oxygen species, and insight into the physical properties of the metal ions. On the basis of these structures, a new putative catalytic mechanism is proposed for AAP that is likely relevant to all binuclear metalloproteases.The coordinates for the 0.95-Å resolution structure and the 1.24-Å structure at pH 4.7 were deposited in the RCSB Protein Data Bank and have PDB ID numbers of 1RTQ and 2DEA, respectively.     

Crystal Structure and Catalytic Mechanism of Leucoanthocyanidin Reductase from Vitis vinifera

Leucoanthocyanidin reductase (LAR) catalyzes the NADPH-dependent reduction of 2R,3S,4S-flavan-3,4-diols into 2R,3S-flavan-3-ols, a subfamily of flavonoids that is important for plant survival and for human nutrition. LAR1 from Vitis vinifera has been co-crystallized with or without NADPH and one of its natural products, (+)-catechin. Crystals diffract to a resolution between 1.75 and 2.72 Å. The coenzyme and substrate binding pocket is preformed in the apoprotein and not markedly altered upon NADPH binding. The structure of the abortive ternary complex, determined at a resolution of 2.28 Å, indicates the ordering of a short 3₁₀ helix associated with substrate binding and suggests that His122 and Lys140 act as acid-base catalysts. Based on our 3D structures, a two-step catalytic mechanism is proposed, in which a concerted dehydration precedes an NADPH-mediated hydride transfer at C4. The dehydration step involves a Lys-catalyzed deprotonation of the phenolic OH7 through a bridging water molecule and a His-catalyzed protonation of the benzylic hydroxyl at C4. The resulting quinone methide serves as an electrophilic target for hydride transfer at C4. LAR belongs to the short-chain dehydrogenase/reductase superfamily and to the PIP (pinoresinol-lariciresinol reductase, isoflavone reductase, and phenylcoumaran benzylic ether reductase) family. Our data support the concept that all PIP enzymes reduce a quinone methide intermediate and that the major role of the only residue that has been conserved from the short-chain dehydrogenase/reductase catalytic triad (Ser…TyrXXXLys), that is, lysine, is to promote the formation of this intermediate by catalyzing the deprotonation of a phenolic hydroxyl. For some PIP enzymes, this lysine-catalyzed proton abstraction may be sufficient to trigger the extrusion of the leaving group, whereas in LAR, the extrusion of a hydroxide group requires a more sophisticated mechanism of concerted acid-base catalysis that involves histidine and takes advantage of the OH4, OH5, and OH7 substituents of leucoanthocyanidins.     

Structural and mutagenetic analyses of a 1,3–1,4-β-glucanase from Paecilomyces thermophila

The thermostable 1,3–1,4-β-glucanase PtLic16A from the fungus Paecilomyces thermophila catalyzes stringent hydrolysis of barley β-glucan and lichenan with an outstanding efficiency and has great potential for broad industrial applications. Here, we report the crystal structures of PtLic16A and an inactive mutant E113A in ligand-free form and in complex with the ligands cellobiose, cellotetraose and glucotriose at 1.80 Å to 2.25 Å resolution. PtLic16A adopts a typical β-jellyroll fold with a curved surface and the concave face forms an extended ligand binding cleft. These structures suggest that PtLic16A might carry out the hydrolysis via retaining mechanism with E113 and E118 serving as the nucleophile and general acid/base, respectively. Interestingly, in the structure of E113A/1,3–1,4-β-glucotriose complex, the sugar bound to the − 1 subsite adopts an intermediate-like (α-anomeric) configuration. By combining all crystal structures solved here, a comprehensive binding mode for a substrate is proposed. These findings not only help understand the 1,3–1,4-β-glucanase catalytic mechanism but also provide a basis for further enzymatic engineering.     

Crystal structures and mutational analysis of the arginine-, lysine-, histidine-binding protein ArtJ from Geobacillus stearothermophilus. Implications for interactions of ArtJ with its cognate ATP-binding cassette transporter, Art(MP)2

ArtJ is the substrate-binding component (receptor) of the ATP-binding cassette (ABC) transport system ArtJ-(MP)₂ from the thermophilic bacterium Geobacillus stearothermophilus that is specific for arginine, lysine, and histidine. The highest affinity is found for arginine (K_d = 0.039(±0.014) μM), while the affinities for lysine and histidine are about tenfold lower. We have determined the X-ray structures of ArtJ liganded with each of these substrates at resolutions of 1.79 Å (arginine), 1.79 Å (lysine), and 2.35 Å (histidine), respectively. As found for other solute receptors, the polypeptide chain is folded into two distinct domains (lobes) connected by a hinge. The interface between the lobes forms the substrate-binding pocket whose geometry is well preserved in all three ArtJ/amino acid complexes. Structure-derived mutational analyses indicated the crucial role of a region in the carboxy-terminal lobe of ArtJ in contacting the transport pore Art(MP)₂ and revealed the functional importance of Gln132 and Trp68. While variant Gln132Leu exhibited lower binding affinity for arginine but no binding of lysine and histidine, the variant Trp68Leu had lost binding activity for all three substrates. The results are discussed in comparison with known structures of homologous proteins from mesophilic bacteria.