Structural and kinetic evidence that catalytic reaction of human UDP-glucose 6-dehydrogenase involves covalent thiohemiacetal and thioester enzyme intermediates

Biosynthesis of UDP-glucuronic acid by UDP-glucose 6-dehydrogenase (UGDH) occurs through the four-electron oxidation of the UDP-glucose C6 primary alcohol in two NAD⁺-dependent steps. The catalytic reaction of UGDH is thought to involve a Cys nucleophile that promotes formation of a thiohemiacetal enzyme intermediate in the course of the first oxidation step. The thiohemiacetal undergoes further oxidation into a thioester, and hydrolysis of the thioester completes the catalytic cycle. Herein we present crystallographic and kinetic evidence for the human form of UGDH that clarifies participation of covalent catalysis in the enzymatic mechanism. Substitution of the putative catalytic base for water attack on the thioester (Glu¹⁶¹) by an incompetent analog (Gln¹⁶¹) gave a UGDH variant (E161Q) in which the hydrolysis step had become completely rate-limiting so that a thioester enzyme intermediate accumulated at steady state. By crystallizing E161Q in the presence of 5 mm UDP-glucose and 2 mm NAD⁺, we succeeded in trapping a thiohemiacetal enzyme intermediate and determined its structure at 2.3 Å resolution. Cys²⁷⁶ was covalently modified in the structure, establishing its role as catalytic nucleophile of the reaction. The thiohemiacetal reactive C6 was in a position suitable to become further oxidized by hydride transfer to NAD⁺. The proposed catalytic mechanism of human UGDH involves Lys²²⁰ as general base for UDP-glucose alcohol oxidation and for oxyanion stabilization during formation and breakdown of the thiohemiacetal and thioester enzyme intermediates. Water coordinated to Asp²⁸⁰ deprotonates Cys²⁷⁶ to function as an aldehyde trap and also provides oxyanion stabilization. Glu¹⁶¹ is the Brønsted base catalytically promoting the thioester hydrolysis.     

Structure and Mechanism of Human UDP-xylose Synthase: EVIDENCE FOR A PROMOTING ROLE OF SUGAR RING DISTORTION IN A THREE-STEP CATALYTIC CONVERSION OF UDP-GLUCURONIC ACID

UDP-xylose synthase (UXS) catalyzes decarboxylation of UDP-d-glucuronic acid to UDP-xylose. In mammals, UDP-xylose serves to initiate glycosaminoglycan synthesis on the protein core of extracellular matrix proteoglycans. Lack of UXS activity leads to a defective extracellular matrix, resulting in strong interference with cell signaling pathways. We present comprehensive structural and mechanistic characterization of the human form of UXS. The 1.26-Å crystal structure of the enzyme bound with NAD⁺ and UDP reveals a homodimeric short-chain dehydrogenase/reductase (SDR), belonging to the NDP-sugar epimerases/dehydratases subclass. We show that enzymatic reaction proceeds in three chemical steps via UDP-4-keto-d-glucuronic acid and UDP-4-keto-pentose intermediates. Molecular dynamics simulations reveal that the d-glucuronyl ring accommodated by UXS features a marked ⁴C₁ chair to B_O,3 boat distortion that facilitates catalysis in two different ways. It promotes oxidation at C₄ (step 1) by aligning the enzymatic base Tyr¹⁴⁷ with the reactive substrate hydroxyl and it brings the carboxylate group at C₅ into an almost fully axial position, ideal for decarboxylation of UDP-4-keto-d-glucuronic acid in the second chemical step. The protonated side chain of Tyr¹⁴⁷ stabilizes the enolate of decarboxylated C₄ keto species (²H₁ half-chair) that is then protonated from the Si face at C₅, involving water coordinated by Glu¹²⁰. Arg²⁷⁷, which is positioned by a salt-link interaction with Glu¹²⁰, closes up the catalytic site and prevents release of the UDP-4-keto-pentose and NADH intermediates. Hydrogenation of the C₄ keto group by NADH, assisted by Tyr¹⁴⁷ as catalytic proton donor, yields UDP-xylose adopting the relaxed ⁴C₁ chair conformation (step 3).     

Conserved catalytic residues of the ALDH1L1 aldehyde dehydrogenase domain control binding and discharging of the coenzyme

The C-terminal domain (C(t)-FDH) of 10-formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1) is an NADP(+)-dependent oxidoreductase and a structural and functional homolog of aldehyde dehydrogenases. Here we report the crystal structures of several C(t)-FDH mutants in which two essential catalytic residues adjacent to the nicotinamide ring of bound NADP(+), Cys-707 and Glu-673, were replaced separately or simultaneously. The replacement of the glutamate with an alanine causes irreversible binding of the coenzyme without any noticeable conformational changes in the vicinity of the nicotinamide ring. Additional replacement of cysteine 707 with an alanine (E673A/C707A double mutant) did not affect this irreversible binding indicating that the lack of the glutamate is solely responsible for the enhanced interaction between the enzyme and the coenzyme. The substitution of the cysteine with an alanine did not affect binding of NADP(+) but resulted in the enzyme lacking the ability to differentiate between the oxidized and reduced coenzyme: unlike the wild-type C(t)-FDH/NADPH complex, in the C707A mutant the position of NADPH is identical to the position of NADP(+) with the nicotinamide ring well ordered within the catalytic center. Thus, whereas the glutamate restricts the affinity for the coenzyme, the cysteine is the sensor of the coenzyme redox state. These conclusions were confirmed by coenzyme binding experiments. Our study further suggests that the binding of the coenzyme is additionally controlled by a long-range communication between the catalytic center and the coenzyme-binding domain and points toward an α-helix involved in the adenine moiety binding as a participant of this communication.     

Crystal Structure of Histamine Dehydrogenase from Nocardioides simplex

Histamine dehydrogenase (HADH) isolated from Nocardioides simplex catalyzes the oxidative deamination of histamine to imidazole acetaldehyde. HADH is highly specific for histamine, and we are interested in understanding the recognition mode of histamine in its active site. We describe the first crystal structure of a recombinant form of HADH (HADH) to 2.7-Å resolution. HADH is a homodimer, where each 76-kDa subunit contains an iron-sulfur cluster ([4Fe-4S]²⁺) and a 6-S-cysteinyl flavin mononucleotide (6-S-Cys-FMN) as redox cofactors. The overall structure of HADH is very similar to that of trimethylamine dehydrogenase (TMADH) from Methylotrophus methylophilus (bacterium W3A1). However, some distinct differences between the structure of HADH and TMADH have been found. Tyr⁶⁰, Trp²⁶⁴, and Trp³⁵⁵ provide the framework for the “aromatic bowl” that serves as a trimethylamine-binding site in TMADH is comprised of Gln⁶⁵, Trp²⁶⁷, and Asp³⁵⁸, respectively, in HADH. The surface Tyr⁴⁴² that is essential in transferring electrons to electron-transfer flavoprotein (ETF) in TMADH is not conserved in HADH. We use this structure to propose the binding mode for histamine in the active site of HADH through molecular modeling and to compare the interactions to those observed for other histamine-binding proteins whose structures are known.     

Molecular cloning and catalytic mechanism of a novel glycosphingolipid-degrading beta-N-acetylgalactosaminidase from Paenibacillus sp. TS12

We report here the molecular cloning, characterization, and catalytic mechanism of a novel glycosphingolipid-degrading β-N-acetylgalactosaminidase (β-NGA) from Paenibacillus sp. TS12 (NgaP). Consisting of 1034 putative amino acid residues, NgaP shares no sequence similarity with known proteins. Recombinant NgaP, expressed in Escherichia coli, cleaved the nonreducing terminal β-GalNAc residues of gangliotriaosylceramide and globotetraosylceramide. The enzyme hydrolyzed para-nitrophenyl-β-N-acetylgalactosaminide ～100 times faster than para-nitrophenyl-β-N-acetylglucosaminide. GalNAc thiazoline, an analog of the oxazolinium intermediate and potent inhibitor for enzymes adopting substrate-assisted catalysis, competitively inhibited the enzyme. The K(i) of the enzyme for GalNAc thiazoline was 1.3 nM, whereas that for GlcNAc thiazoline was 46.8 μM. Comparison of the secondary structure with those of known enzymes exhibiting substrate-assisted catalysis and point mutation analysis indicated that NgaP adopts substrate-assisted catalysis in which Glu-608 and Asp-607 could function as a proton donor and a stabilizer of the 2-acetamide group of the β-GalNAc at the active site, respectively. These results clearly indicate that NgaP is a β-NGA showing substrate-assisted catalysis. This is the first report describing the molecular cloning of a β-NGA adopting substrate-assisted catalysis.     

Adenine Binding Mode Is a Key Factor in Triggering the Early Release of NADH in Coenzyme A-dependent Methylmalonate Semialdehyde Dehydrogenase

Structural dynamics associated with cofactor binding have been shown to play key roles in the catalytic mechanism of hydrolytic NAD(P)-dependent aldehyde dehydrogenases (ALDH). By contrast, no information is available for their CoA-dependent counterparts. We present here the first crystal structure of a CoA-dependent ALDH. The structure of the methylmalonate semialdehyde dehydrogenase (MSDH) from Bacillus subtilis in binary complex with NAD(+) shows that, in contrast to what is observed for hydrolytic ALDHs, the nicotinamide ring is well defined in the electron density due to direct and H(2)O-mediated hydrogen bonds with the carboxamide. The structure also reveals that a conformational isomerization of the NMNH is possible in MSDH, as shown for hydrolytic ALDHs. Finally, the adenine ring is substantially more solvent-exposed, a result that could be explained by the presence of a Val residue at position 229 in helix α(F) that reduces the depth of the binding pocket and the absence of Gly-225 at the N-terminal end of helix α(F). Substitution of glycine for Val-229 and/or insertion of a glycine residue at position 225 resulted in a significant decrease of the rate constant associated with the dissociation of NADH from the NADH/thioacylenzyme complex, thus demonstrating that the weaker stabilization of the adenine ring is a key factor in triggering the early NADH release in the MSDH-catalyzed reaction. This study provides for the first time structural insights into the mechanism whereby the cofactor binding mode is responsible at least in part for the different kinetic behaviors of the hydrolytic and CoA-dependent ALDHs.     

Dynamic mechanism of proton transfer in mannitol 2-dehydrogenase from Pseudomonas fluorescens: mobile GLU292 controls proton relay through a water channel that connects the active site with bulk solvent

The active site of mannitol 2-dehydrogenase from Pseudomonas fluorescens (PfM2DH) is connected with bulk solvent through a narrow protein channel that shows structural resemblance to proton channels utilized by redox-driven proton pumps. A key element of the PfM2DH channel is the "mobile" Glu(292), which was seen crystallographically to adopt distinct positions up and down the channel. It was suggested that the "down → up" conformational change of Glu(292) could play a proton relay function in enzymatic catalysis, through direct proton shuttling by the Glu or because the channel is opened for water molecules forming a chain along which the protons flow. We report evidence from site-directed mutagenesis (Glu(292) → Ala) substantiated by data from molecular dynamics simulations that support a role for Glu(292) as a gate in a water chain (von Grotthuss-type) mechanism of proton translocation. Occupancy of the up and down position of Glu(292) is influenced by the bonding and charge state of the catalytic acid base Lys(295), suggesting that channel opening/closing motions of the Glu are synchronized to the reaction progress. Removal of gatekeeper control in the E292A mutant resulted in a selective, up to 120-fold slowing down of microscopic steps immediately preceding catalytic oxidation of mannitol, consistent with the notion that formation of the productive enzyme-NAD(+)-mannitol complex is promoted by a corresponding position change of Glu(292), which at physiological pH is associated with obligatory deprotonation of Lys(295) to solvent. These results underscore the important role of conformational dynamics in the proton transfer steps of alcohol dehydrogenase catalysis.     

Structural and functional evolution of isopropylmalate dehydrogenases in the leucine and glucosinolate pathways of Arabidopsis thaliana

The methionine chain-elongation pathway is required for aliphatic glucosinolate biosynthesis in plants and evolved from leucine biosynthesis. In Arabidopsis thaliana, three 3-isopropylmalate dehydrogenases (AtIPMDHs) play key roles in methionine chain-elongation for the synthesis of aliphatic glucosinolates (e.g. AtIPMDH1) and leucine (e.g. AtIPMDH2 and AtIPMDH3). Here we elucidate the molecular basis underlying the metabolic specialization of these enzymes. The 2.25 Å resolution crystal structure of AtIPMDH2 was solved to provide the first detailed molecular architecture of a plant IPMDH. Modeling of 3-isopropylmalate binding in the AtIPMDH2 active site and sequence comparisons of prokaryotic and eukaryotic IPMDH suggest that substitution of one active site residue may lead to altered substrate specificity and metabolic function. Site-directed mutagenesis of Phe-137 to a leucine in AtIPMDH1 (AtIPMDH1-F137L) reduced activity toward 3-(2′-methylthio)ethylmalate by 200-fold, but enhanced catalytic efficiency with 3-isopropylmalate to levels observed with AtIPMDH2 and AtIPMDH3. Conversely, the AtIPMDH2-L134F and AtIPMDH3-L133F mutants enhanced catalytic efficiency with 3-(2′-methylthio)ethylmalate ∼100-fold and reduced activity for 3-isopropylmalate. Furthermore, the altered in vivo glucosinolate profile of an Arabidopsis ipmdh1 T-DNA knock-out mutant could be restored to wild-type levels by constructs expressing AtIPMDH1, AtIPMDH2-L134F, or AtIPMDH3-L133F, but not by AtIPMDH1-F137L. These results indicate that a single amino acid substitution results in functional divergence of IPMDH in planta to affect substrate specificity and contributes to the evolution of specialized glucosinolate biosynthesis from the ancestral leucine pathway.     

UDP-glucose Dehydrogenase Activity and Optimal Downstream Cellular Function Require Dynamic Reorganization at the Dimer-Dimer Subunit Interfaces

UDP-glucose dehydrogenase (UGDH) provides precursors for steroid elimination, hyaluronan production, and glycosaminoglycan synthesis. The wild-type UGDH enzyme purifies in a hexamer-dimer equilibrium and transiently undergoes dynamic motion that exposes the dimer-dimer interface during catalysis. In the current study we created and characterized point mutations that yielded exclusively dimeric species (obligate dimer, T325D), dimeric species that could be induced to form hexamers in the ternary complex with substrate and cofactor (T325A), and a previously described exclusively hexameric species (UGDHΔ132) to investigate the role of quaternary structure in regulation of the enzyme. Characterization of the purified enzymes revealed a significant decrease in the enzymatic activity of the obligate dimer and hexamer mutants. Kinetic analysis of wild-type UGDH and the inducible hexamer, T325A, showed that upon increasing enzyme concentration, which favors the hexameric species, activity was modestly decreased and exhibited cooperativity. In contrast, cooperative kinetic behavior was not observed in the obligate dimer, T325D. These observations suggest that the regulation of the quaternary assembly of the enzyme is essential for optimal activity and allosteric regulation. Comparison of kinetic and thermal stability parameters revealed structurally dependent properties consistent with a role for controlled assembly and disassembly of the hexamer in the regulation of UGDH. Finally, both T325A and T325D mutants were significantly less efficient in promoting downstream hyaluronan production by HEK293 cells. These data support a model that requires an operational dimer-hexamer equilibrium to function efficiently and preserve regulated activity in the cell.     

Tyrosine latching of a regulatory gate affords allosteric control of aromatic amino acid biosynthesis

The first step of the shikimate pathway for aromatic amino acid biosynthesis is catalyzed by 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS). Thermotoga maritima DAH7PS (TmaDAH7PS) is tetrameric, with monomer units comprised of a core catalytic (β/α)₈ barrel and an N-terminal domain. This enzyme is inhibited strongly by tyrosine and to a lesser extent by the presence of phenylalanine. A truncated mutant of TmaDAH7PS lacking the N-terminal domain was catalytically more active and completely insensitive to tyrosine and phenylalanine, consistent with a role for this domain in allosteric inhibition. The structure of this protein was determined to 2.0 Å. In contrast to the wild-type enzyme, this enzyme is dimeric. Wild-type TmaDAH7PS was co-crystallized with tyrosine, and the structure of this complex was determined to a resolution of 2.35 Å. Tyrosine was found to bind at the interface between two regulatory N-terminal domains, formed from diagonally located monomers of the tetramer, revealing a major reorganization of the regulatory domain with respect to the barrel relative to unliganded enzyme. This significant conformational rearrangement observed in the crystal structures was also clearly evident from small angle X-ray scattering measurements recorded in the presence and absence of tyrosine. The closed conformation adopted by the protein on tyrosine binding impedes substrate entry into the neighboring barrel, revealing an unusual tyrosine-controlled gating mechanism for allosteric control of this enzyme.     

Defining a structural and kinetic rationale for paralogous copies of phenylacetate-CoA ligases from the cystic fibrosis pathogen Burkholderia cenocepacia J2315

The phenylacetic acid (PAA) degradation pathway is the sole aerobic route for phenylacetic acid metabolism in bacteria and facilitates degradation of environmental pollutants such as styrene and ethylbenzene. The PAA pathway also is implicated in promoting Burkholderia cenocepacia infections in cystic fibrosis patients. Intriguingly, the first enzyme in the PAA pathway is present in two copies (paaK1 and paaK2), yet each subsequent enzyme is present in only a single copy. Furthermore, sequence divergence indicates that PaaK1 and PaaK2 form a unique subgroup within the adenylate-forming enzyme (AFE) superfamily. To establish a biochemical rationale for the existence of the PaaK paralogs in B. cenocepacia, we present high resolution x-ray crystal structures of a selenomethionine derivative of PaaK1 in complex with ATP and adenylated phenylacetate intermediate complexes of PaaK1 and PaaK2 in distinct conformations. Structural analysis reveals a novel N-terminal microdomain that may serve to recruit subsequent PAA enzymes, whereas a bifunctional role is proposed for the P-loop in stabilizing the C-terminal domain in conformation 2. The potential for different kinetic profiles was suggested by a structurally divergent extension of the aryl substrate pocket in PaaK1 relative to PaaK2. Functional characterization confirmed this prediction, with PaaK1 possessing a lower K(m) for phenylacetic acid and better able to accommodate 3' and 4' substitutions on the phenyl ring. Collectively, these results offer detailed insight into the reaction mechanism of a novel subgroup of the AFE superfamily and provide a clear biochemical rationale for the presence of paralogous copies of PaaK of B. cenocepacia.     

Vascular Bioactivation of Nitroglycerin by Aldehyde Dehydrogenase-2: REACTION INTERMEDIATES REVEALED BY CRYSTALLOGRAPHY AND MASS SPECTROMETRY*

Aldehyde dehydrogenase-2 (ALDH2) catalyzes the bioactivation of nitroglycerin (glyceryl trinitrate, GTN) in blood vessels, resulting in vasodilation by nitric oxide (NO) or a related species. Because the mechanism of this reaction is still unclear we determined the three-dimensional structures of wild-type (WT) ALDH2 and of a triple mutant of the protein that exhibits low denitration activity (E268Q/C301S/C303S) in complex with GTN. The structure of the triple mutant showed that GTN binds to the active site via polar contacts to the oxyanion hole and to residues 268 and 301 as well as by van der Waals interactions to hydrophobic residues of the catalytic pocket. The structure of the GTN-soaked wild-type protein revealed a thionitrate adduct to Cys-302 as the first reaction intermediate, which was also found by mass spectrometry (MS) experiments. In addition, the MS data identified sulfinic acid as the irreversibly inactivated enzyme species. Assuming that the structures of the triple mutant and wild-type ALDH2 reflect binding of GTN to the catalytic site and the first reaction step, respectively, superposition of the two structures indicates that denitration of GTN is initiated by nucleophilic attack of Cys-302 at one of the terminal nitrate groups, resulting in formation of the observed thionitrate intermediate and release of 1,2-glyceryl dinitrate. Our results shed light on the molecular mechanism of the GTN denitration reaction and provide useful information on the structural requirements for high affinity binding of organic nitrates to the catalytic site of ALDH2.     

Structure and reaction mechanism of phosphoethanolamine methyltransferase from the malaria parasite Plasmodium falciparum: an antiparasitic drug target

In the malarial parasite Plasmodium falciparum, a multifunctional phosphoethanolamine methyltransferase (PfPMT) catalyzes the methylation of phosphoethanolamine (pEA) to phosphocholine for membrane biogenesis. This pathway is also found in plant and nematodes, but PMT from these organisms use multiple methyltransferase domains for the S-adenosylmethionine (AdoMet) reactions. Because PfPMT is essential for normal growth and survival of Plasmodium and is not found in humans, it is an antiparasitic target. Here we describe the 1.55 Å resolution crystal structure of PfPMT in complex with AdoMet by single-wavelength anomalous dispersion phasing. In addition, 1.19–1.52 Å resolution structures of PfPMT with pEA (substrate), phosphocholine (product), sinefungin (inhibitor), and both pEA and S-adenosylhomocysteine bound were determined. These structures suggest that domain rearrangements occur upon ligand binding and provide insight on active site architecture defining the AdoMet and phosphobase binding sites. Functional characterization of 27 site-directed mutants identifies critical active site residues and suggests that Tyr-19 and His-132 form a catalytic dyad. Kinetic analysis, isothermal titration calorimetry, and protein crystallography of the Y19F and H132A mutants suggest a reaction mechanism for the PMT. Not only are Tyr-19 and His-132 required for phosphobase methylation, but they also form a “catalytic” latch that locks ligands in the active site and orders the site for catalysis. This study provides the first insight on this antiparasitic target enzyme essential for survival of the malaria parasite; however, further studies of the multidomain PMT from plants and nematodes are needed to understand the evolutionary division of metabolic function in the phosphobase pathway of these organisms.     

Binding of NAD+ and l-Threonine Induces Stepwise Structural and Flexibility Changes in Cupriavidus necator l-Threonine Dehydrogenase

Crystal structures of short chain dehydrogenase-like l-threonine dehydrogenase from Cupriavidus necator (CnThrDH) in the apo and holo forms were determined at 2.25 and 2.5 Å, respectively. Structural comparison between the apo and holo forms revealed that four regions of CnThrDH adopted flexible conformations when neither NAD⁺ nor l-Thr were bound: residues 38–59, residues 77–87, residues 180–186, and the catalytic domain. Molecular dynamics simulations performed at the 50-ns time scale revealed that three of these regions remained flexible when NAD⁺ was bound to CnThrDH: residues 80–87, residues 180–186, and the catalytic domain. Molecular dynamics simulations also indicated that the structure of CnThrDH changed from a closed form to an open form upon NAD⁺ binding. The newly formed cleft in the open form may function as a conduit for substrate entry and product exit. These computational results led us to hypothesize that the CnThrDH reaction progresses by switching between the closed and open forms. Enzyme kinetics parameters of the L80G, G184A, and T186N variants also supported this prediction: the k_cat/K_{m, l-Thr} value of the variants was >330-fold lower than that of the wild type; this decrease suggested that the variants mostly adopt the open form when l-Thr is bound to the active site. These results are summarized in a schematic model of the stepwise changes in flexibility and structure that occur in CnThrDH upon binding of NAD⁺ and l-Thr. This demonstrates that the dynamical structural changes of short chain dehydrogenase-like l-threonine dehydrogenase are important for the reactivity and specificity of the enzyme.     

Structural basis for proficient incorporation of dTTP opposite O6-methylguanine by human DNA polymerase iota

O(6)-methylguanine (O(6)-methylG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, even physiological ones (e.g. S-adenosylmethionine). The efficiency of a truncated, catalytic DNA polymerase ι core enzyme was determined for nucleoside triphosphate incorporation opposite O(6)-methylG, using steady-state kinetic analyses. The results presented here corroborate previous work from this laboratory using full-length pol ι, which showed that dTTP incorporation occurs with high efficiency opposite O(6)-methylG. Misincorporation of dTTP opposite O(6)-methylG occurred with ～6-fold higher efficiency than incorporation of dCTP. Crystal structures of the truncated form of pol ι with O(6)-methylG as the template base and incoming dCTP or dTTP were solved and showed that O(6)-methylG is rotated into the syn conformation in the pol ι active site and that dTTP misincorporation by pol ι is the result of Hoogsteen base pairing with the adduct. Both dCTP and dTTP base paired with the Hoogsteen edge of O(6)-methylG. A single, short hydrogen bond formed between the N3 atom of dTTP and the N7 atom of O(6)-methylG. Protonation of the N3 atom of dCTP and bifurcation of the N3 hydrogen between the N7 and O(6) atoms of O(6)-methylG allow base pairing of the lesion with dCTP. We conclude that differences in the Hoogsteen hydrogen bonding between nucleotides is the main factor in the preferential selectivity of dTTP opposite O(6)-methylG by human pol ι, in contrast to the mispairing modes observed previously for O(6)-methylG in the structures of the model DNA polymerases Sulfolobus solfataricus Dpo4 and Bacillus stearothermophilus DNA polymerase I.     

Structural and biochemical elucidation of mechanism for decarboxylative condensation of beta-keto acid by curcumin synthase

The typical reaction catalyzed by type III polyketide synthases (PKSs) is a decarboxylative condensation between acyl-CoA (starter substrate) and malonyl-CoA (extender substrate). In contrast, curcumin synthase 1 (CURS1), which catalyzes curcumin synthesis by condensing feruloyl-CoA with a diketide-CoA, uses a β-keto acid (which is derived from diketide-CoA) as an extender substrate. Here, we determined the crystal structure of CURS1 at 2.32 Å resolution. The overall structure of CURS1 was very similar to the reported structures of type III PKSs and exhibited the αβαβα fold. However, CURS1 had a unique hydrophobic cavity in the CoA-binding tunnel. Replacement of Gly-211 with Phe greatly reduced the enzyme activity. The crystal structure of the G211F mutant (at 2.5 Å resolution) revealed that the side chain of Phe-211 occupied the hydrophobic cavity. Biochemical studies demonstrated that CURS1 catalyzes the decarboxylative condensation of a β-keto acid using a mechanism identical to that for normal decarboxylative condensation of malonyl-CoA by typical type III PKSs. Furthermore, the extender substrate specificity of CURS1 suggested that hydrophobic interaction between CURS1 and a β-keto acid may be important for CURS1 to use an extender substrate lacking the CoA moiety. From these results and a modeling study on substrate binding, we concluded that the hydrophobic cavity is responsible for the hydrophobic interaction between CURS1 and a β-keto acid, and this hydrophobic interaction enables the β-keto acid moiety to access the catalytic center of CURS1 efficiently.     

A twin-track approach has optimized proton and hydride transfer by dynamically coupled tunneling during the evolution of protochlorophyllide oxidoreductase

Protein dynamics are crucial for realizing the catalytic power of enzymes, but how enzymes have evolved to achieve catalysis is unknown. The light-activated enzyme protochlorophyllide oxidoreductase (POR) catalyzes sequential hydride and proton transfers in the photoexcited and ground states, respectively, and is an excellent system for relating the effects of motions to catalysis. Here, we have used the temperature dependence of isotope effects and solvent viscosity measurements to analyze the dynamics coupled to the hydride and proton transfer steps in three cyanobacterial PORs and a related plant enzyme. We have related the dynamic profiles of each enzyme to their evolutionary origin. Motions coupled to light-driven hydride transfer are conserved across all POR enzymes, but those linked to thermally activated proton transfer are variable. Cyanobacterial PORs require complex and solvent-coupled dynamic networks to optimize the proton donor-acceptor distance, but evolutionary pressures appear to have minimized such networks in plant PORs. POR from Gloeobacter violaceus has features of both the cyanobacterial and plant enzymes, suggesting that the dynamic properties have been optimized during the evolution of POR. We infer that the differing trajectories in optimizing a catalytic structure are related to the stringency of the chemistry catalyzed and define a functional adaptation in which active site chemistry is protected from the dynamic effects of distal mutations that might otherwise impact negatively on enzyme catalysis.