Crystal structure of Leishmania major peroxidase and characterization of the compound i tryptophan radical

The parasitic protozoa Leishmania major produces a peroxidase (L. major peroxidase; LmP) that exhibits activities characteristic of both yeast cytochrome c peroxidase (CCP) and plant cytosolic ascorbate peroxidase (APX). One common feature is a key Trp residue, Trp(208) in LmP and Trp(191) in CCP, that is situated adjacent to the proximal His heme ligand in CCP, APX, and LmP. In CCP, Trp(191) forms a stable cationic radical after reaction with H(2)O(2) to form Compound I; in APX, the radical is located on the porphyrin ring. In order to clarify the role of Trp(208) in LmP and to further probe peroxidase structure-function relationships, we have determined the crystal structure of LmP and have studied the role of Trp(208) using electron paramagnetic resonance spectroscopy (EPR), mutagenesis, and enzyme kinetics. Both CCP and LmP have an extended section of β structure near Trp(191) and Trp(208), respectively, which is absent in APX. This region provides stability to the Trp(191) radical in CCP. EPR of LmP Compound I exhibits an intense and stable signal similar to CCP Compound I. In the LmP W208F mutant, this signal disappears, indicating that Trp(208) forms a stable cationic radical. In LmP conversion of the Cys(197) to Thr significantly weakens the Compound I EPR signal and dramatically lowers enzyme activity. These results further support the view that modulation of the local electrostatic environment controls the stability of the Trp radical in peroxidases. Our results also suggest that the biological role of LmP is to function as a cytochrome c peroxidase.     

Structural and biochemical characterization of glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum

We present the first structure of a glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum, both as a product complex with β-D-glucuronic acid (GlcA) and as its trapped covalent 2-fluoroglucuronyl intermediate. This enzyme consists of a catalytic (β/α)(8)-barrel domain and a β-domain with irregular Greek key motifs that is of unknown function. The enzyme showed β-glucuronidase activity and trace levels of β-glucosidase and β-xylosidase activities. In conjunction with mutagenesis studies, these structures identify the catalytic residues as Glu(173) (acid base) and Glu(287) (nucleophile), consistent with the retaining mechanism demonstrated by (1)H NMR analysis. Glu(45), Tyr(243), Tyr(292)-Gly(294), and Tyr(334) form the catalytic pocket and provide substrate discrimination. Consistent with this, the Y292A mutation, which affects the interaction between the main chains of Gln(293) and Gly(294) and the GlcA carboxyl group, resulted in significant loss of β-glucuronidase activity while retaining the side activities at wild-type levels. Likewise, although the β-glucuronidase activity of the Y334F mutant is ~200-fold lower (k(cat)/K(m)) than that of the wild-type enzyme, the β-glucosidase activity is actually 3 times higher and the β-xylosidase activity is only 2.5-fold lower than the equivalent parameters for wild type, consistent with a role for Tyr(334) in recognition of the C6 position of GlcA. The involvement of Glu(45) in discriminating against binding of the O-methyl group at the C4 position of GlcA is revealed in the fact that the E45D mutant hydrolyzes PNP-β-GlcA approximately 300-fold slower (k(cat)/K(m)) than does the wild-type enzyme, whereas 4-O-methyl-GlcA-containing oligosaccharides are hydrolyzed only 7-fold slower.     

Structural and Biochemical Characterization of an Active Arylamine N-Acetyltransferase Possessing a Non-canonical Cys-His-Glu Catalytic Triad

Arylamine N-acetyltransferases (NATs), a class of xenobiotic-metabolizing enzymes, catalyze the acetylation of aromatic amine compounds through a strictly conserved Cys-His-Asp catalytic triad. Each residue is essential for catalysis in both prokaryotic and eukaryotic NATs. Indeed, in (HUMAN)NAT2 variants, mutation of the Asp residue to Asn, Gln, or Glu dramatically impairs enzyme activity. However, a putative atypical NAT harboring a catalytic triad Glu residue was recently identified in Bacillus cereus ((BACCR)NAT3) but has not yet been characterized. We report here the crystal structure and functional characterization of this atypical NAT. The overall fold of (BACCR)NAT3 and the geometry of its Cys-His-Glu catalytic triad are similar to those present in functional NATs. Importantly, the enzyme was found to be active and to acetylate prototypic arylamine NAT substrates. In contrast to (HUMAN) NAT2, the presence of a Glu or Asp in the triad of (BACCR)NAT3 did not significantly affect enzyme structure or function. Computational analysis identified differences in residue packing and steric constraints in the active site of (BACCR)NAT3 that allow it to accommodate a Cys-His-Glu triad. These findings overturn the conventional view, demonstrating that the catalytic triad of this family of acetyltransferases is plastic. Moreover, they highlight the need for further study of the evolutionary history of NATs and the functional significance of the predominant Cys-His-Asp triad in both prokaryotic and eukaryotic forms.     

Substrate Binding Mechanism of a Type I Extradiol Dioxygenase

A meta-cleavage pathway for the aerobic degradation of aromatic hydrocarbons is catalyzed by extradiol dioxygenases via a two-step mechanism: catechol substrate binding and dioxygen incorporation. The binding of substrate triggers the release of water, thereby opening a coordination site for molecular oxygen. The crystal structures of AkbC, a type I extradiol dioxygenase, and the enzyme substrate (3-methylcatechol) complex revealed the substrate binding process of extradiol dioxygenase. AkbC is composed of an N-domain and an active C-domain, which contains iron coordinated by a 2-His-1-carboxylate facial triad motif. The C-domain includes a β-hairpin structure and a C-terminal tail. In substrate-bound AkbC, 3-methylcatechol interacts with the iron via a single hydroxyl group, which represents an intermediate stage in the substrate binding process. Structure-based mutagenesis revealed that the C-terminal tail and β-hairpin form part of the substrate binding pocket that is responsible for substrate specificity by blocking substrate entry. Once a substrate enters the active site, these structural elements also play a role in the correct positioning of the substrate. Based on the results presented here, a putative substrate binding mechanism is proposed.     

Two Oxidation Sites for Low Redox Potential Substrates: A DIRECTED MUTAGENESIS,KINETIC, AND CRYSTALLOGRAPHIC STUDY ON PLEUROTUS ERYNGII VERSATILE PEROXIDASE*

Versatile peroxidase shares with manganese peroxidase and lignin peroxidase the ability to oxidize Mn²⁺ and high redox potential aromatic compounds, respectively. Moreover, it is also able to oxidize phenols (and low redox potential dyes) at two catalytic sites, as shown by biphasic kinetics. A high efficiency site (with 2,6-dimethoxyphenol and p-hydroquinone catalytic efficiencies of ∼70 and ∼700 s⁻¹ mm⁻¹, respectively) was localized at the same exposed Trp-164 responsible for high redox potential substrate oxidation (as shown by activity loss in the W164S variant). The second site, characterized by low catalytic efficiency (∼3 and ∼50 s⁻¹ mm⁻¹ for 2,6-dimethoxyphenol and p-hydroquinone, respectively) was localized at the main heme access channel. Steady-state and transient-state kinetics for oxidation of phenols and dyes at the latter site were improved when side chains of residues forming the heme channel edge were removed in single and multiple variants. Among them, the E140G/K176G, E140G/P141G/K176G, and E140G/W164S/K176G variants attained catalytic efficiencies for oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) at the heme channel similar to those of the exposed tryptophan site. The heme channel enlargement shown by x-ray diffraction of the E140G, P141G, K176G, and E140G/K176G variants would allow a better substrate accommodation near the heme, as revealed by the up to 26-fold lower K_m values (compared with native VP). The resulting interactions were shown by the x-ray structure of the E140G-guaiacol complex, which includes two H-bonds of the substrate with Arg-43 and Pro-139 in the distal heme pocket (at the end of the heme channel) and several hydrophobic interactions with other residues and the heme cofactor.     

Retuning Rieske-type oxygenases to expand substrate range

Rieske-type oxygenases are promising biocatalysts for the destruction of persistent pollutants or for the synthesis of fine chemicals. In this work, we explored pathways through which Rieske-type oxygenases evolve to expand their substrate range. BphAE(p4), a variant biphenyl dioxygenase generated from Burkholderia xenovorans LB400 BphAE(LB400) by the double substitution T335A/F336M, and BphAE(RR41), obtained by changing Asn(338), Ile(341), and Leu(409) of BphAE(p4) to Gln(338), Val(341), and Phe(409), metabolize dibenzofuran two and three times faster than BphAE(LB400), respectively. Steady-state kinetic measurements of single- and multiple-substitution mutants of BphAE(LB400) showed that the single T335A and the double N338Q/L409F substitutions contribute significantly to enhanced catalytic activity toward dibenzofuran. Analysis of crystal structures showed that the T335A substitution relieves constraints on a segment lining the catalytic cavity, allowing a significant displacement in response to dibenzofuran binding. The combined N338Q/L409F substitutions alter substrate-induced conformational changes of protein groups involved in subunit assembly and in the chemical steps of the reaction. This suggests a responsive induced fit mechanism that retunes the alignment of protein atoms involved in the chemical steps of the reaction. These enzymes can thus expand their substrate range through mutations that alter the constraints or plasticity of the catalytic cavity to accommodate new substrates or that alter the induced fit mechanism required to achieve proper alignment of reaction-critical atoms or groups.     

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.     

Structural Basis for a Bispecific NADP+ and CoA Binding Site in an Archaeal Malonyl-Coenzyme A Reductase

Autotrophic members of the Sulfolobales (crenarchaeota) use the 3-hydroxypropionate/4-hydroxybutyrate cycle to assimilate CO₂ into cell material. The product of the initial acetyl-CoA carboxylation with CO₂, malonyl-CoA, is further reduced to malonic semialdehyde by an NADPH-dependent malonyl-CoA reductase (MCR); the enzyme also catalyzes the reduction of succinyl-CoA to succinic semialdehyde onwards in the cycle. Here, we present the crystal structure of Sulfolobus tokodaii malonyl-CoA reductase in the substrate-free state and in complex with NADP⁺ and CoA. Structural analysis revealed an unexpected reaction cycle in which NADP⁺ and CoA successively occupy identical binding sites. Both coenzymes are pressed into an S-shaped, nearly superimposable structure imposed by a fixed and preformed binding site. The template-governed cofactor shaping implicates the same binding site for the 3′- and 2′-ribose phosphate group of CoA and NADP⁺, respectively, but a different one for the common ADP part: the β-phosphate of CoA aligns with the α-phosphate of NADP⁺. Evolution from an NADP⁺ to a bispecific NADP⁺ and CoA binding site involves many amino acid exchanges within a complex process by which constraints of the CoA structure also influence NADP⁺ binding. Based on the paralogous aspartate-β-semialdehyde dehydrogenase structurally characterized with a covalent Cys-aspartyl adduct, a malonyl/succinyl group can be reliably modeled into MCR and discussed regarding its binding mode, the malonyl/succinyl specificity, and the catalyzed reaction. The modified polypeptide surrounding around the absent ammonium group in malonate/succinate compared with aspartate provides the structural basis for engineering a methylmalonyl-CoA reductase applied for biotechnical polyester building block synthesis.     

Structures of Complexes of a Metal-independent Glycosyltransferase GT6 from Bacteroides ovatus with UDP-N-Acetylgalactosamine (UDP-GalNAc) and Its Hydrolysis Products

Mammalian members of glycosyltransferase family 6 (GT6) of the CAZy database have a GT-A fold containing a conserved Asp-X-Asp (DXD) sequence that binds an essential metal cofactor. Bacteroides ovatus GT6a represents a GT6 clade found in more than 30 Gram-negative bacteria that is similar in sequence to the catalytic domains of mammalian GT6, but has an Asn⁹⁵-Ala-Asn⁹⁷ (NXN) sequence substituted for the DXD motif and metal-independent catalytic activity. Co-crystals of a low activity mutant of BoGT6a (E192Q) with UDP-GalNAc contained protein complexes with intact UDP-GalNAc and two forms with hydrolysis products (UDP plus GalNAc) representing an initial closed complex and later open form primed for product release. Two cationic residues near the C terminus of BoGT6a, Lys²³¹ and Arg²⁴³, interact with the diphosphate moiety of UDP-GalNAc, but only Lys²³¹ interacts with the UDP product and may function in leaving group stabilization. The amide group of Asn⁹⁵, the first Asn of the NXN motif, interacts with the ribose moiety of the substrate. This metal-independent GT6 resembles its metal-dependent homologs in undergoing conformational changes on binding UDP-GalNAc that arise from structuring the C terminus to cover this substrate. It appears that in the GT6 family, the metal cofactor functions specifically in binding the UDP moiety in the donor substrate and transition state, actions that can be efficiently performed by components of the polypeptide chain.     

Structural and Biochemical Characterization of Free Methionine-R-sulfoxide Reductase from Neisseria meningitidis

A new family of methionine-sulfoxide reductase (Msr) was recently described. The enzyme, named fRMsr, selectively reduces the R isomer at the sulfoxide function of free methionine sulfoxide (Met-R-O). The fRMsrs belong to the GAF fold family. They represent the first GAF domain to show enzymatic activity. Two other Msr families, MsrA and MsrB, were already known. MsrA and MsrB reduce free Met-S-O and Met-R-O, respectively, but exhibit higher catalytic efficiency toward Met-O within a peptide or a protein context. The fold of the three families differs. In the present work, the crystal structure of the fRMsr from Neisseria meningitidis has been determined in complex with S-Met-R-O. Based on biochemical and kinetic data as well as genomic analyses, Cys¹¹⁸ is demonstrated to be the catalytic Cys on which a sulfenic acid is formed. All of the structural factors involved in the stereoselectivity of the l-Met-R-O binding were identified and account for why Met-S-O, DMSO, and a Met-O within a peptide are not substrates. Taking into account the structural, enzymatic, and biochemical information, a scenario of the catalysis for the reductase step is proposed. Based on the thiol content before and after Met-O reduction and the stoichiometry of Met formed per subunit of wild type and Cys-to-Ala mutants, a scenario of the recycling process of the N. meningitidis fRMsr is proposed. All of the biochemical, enzymatic, and structural properties of the N. meningitidis fRMsr are compared with those of MsrA and MsrB and are discussed in terms of the evolution of function of the GAF domain.     

Crystal Structures of β-1,4-Galactosyltransferase 7 Enzyme Reveal Conformational Changes and Substrate Binding

The β-1,4-galactosyltransferase 7 (β4GalT7) enzyme is involved in proteoglycan synthesis. In the presence of a manganese ion, it transfers galactose from UDP-galactose to xylose on a proteoglycan acceptor substrate. We present here the crystal structures of human β4GalT7 in open and closed conformations. A comparison of these crystal structures shows that, upon manganese and UDP or UDP-Gal binding, the enzyme undergoes conformational changes involving a small and a long loop. We also present the crystal structures of Drosophila wild-type β4GalT7 and D211N β4GalT7 mutant enzymes in the closed conformation in the presence of the acceptor substrate xylobiose and the donor substrate UDP-Gal, respectively. To understand the catalytic mechanism, we have crystallized the ternary complex of D211N β4GalT7 mutant enzyme in the presence of manganese with the donor and the acceptor substrates together in the same crystal structure. The galactose moiety of the bound UDP-Gal molecule forms seven hydrogen bonds with the protein molecule. The nonreducing end of the xylose moiety of xylobiose binds to the hydrophobic acceptor sugar binding pocket created by the conformational changes, whereas its extended xylose moiety forms hydrophobic interactions with a Tyr residue. In the ternary complex crystal structure, the nucleophile O4 oxygen atom of the xylose molecule is found in close proximity to the C1 and O5 atoms of the galactose moiety. This is the first time that a Michaelis complex of a glycosyltransferase has been described, and it clearly suggests an S_N2 type catalytic mechanism for the β4GalT7 enzyme.     

Crystal Structure of Reaction Intermediates in Pyruvate Class II Aldolase: SUBSTRATE CLEAVAGE,ENOLATE STABILIZATION,AND SUBSTRATE SPECIFICITY*

Crystal structures of divalent metal-dependent pyruvate aldolase, HpaI, in complex with substrate and cleavage products were determined to 1.8–2.0 Å resolution. The enzyme·substrate complex with 4-hydroxy-2-ketoheptane-1,7-dioate indicates that water molecule W2 bound to the divalent metal ion initiates C3–C4 bond cleavage. The binding mode of the aldehyde donor delineated a solvent-filled capacious binding locus lined with predominantly hydrophobic residues. The absence of direct interactions with the aldehyde aliphatic carbons accounts for the broad specificity and lack of stereospecific control by the enzyme. Enzymatic complex structures formed with keto acceptors, pyruvate, and 2-ketobutyrate revealed bidentate interaction with the divalent metal ion by C1-carboxyl and C2-carbonyl oxygens and water molecule W4 that is within close contact of the C3 carbon. Arg⁷⁰ assumes a multivalent role through its guanidinium moiety interacting with all active site enzymatic species: C2 oxygen in substrate, pyruvate, and ketobutyrate; substrate C4 hydroxyl; aldehyde C1 oxygen; and W4. The multiple interactions made by Arg⁷⁰ stabilize the negatively charged C4 oxygen following proton abstraction, the aldehyde alignment in aldol condensation, and the pyruvate enolate upon aldol cleavage as well as support proton exchange at C3. This role is corroborated by loss of aldol cleavage ability and pyruvate C3 proton exchange activity and by a 730-fold increase in the dissociation constant toward the pyruvate enolate analog oxalate in the R70A mutant. Based on the crystal structures, a mechanism is proposed involving the two enzyme-bound water molecules, W2 and W4, in acid/base catalysis that facilitates reversible aldol cleavage. The same reaction mechanism promotes decarboxylation of oxaloacetate.     

Association of novel domain in active site of archaic hyperthermophilic maltogenic amylase from Staphylothermus marinus

Staphylothermus marinus maltogenic amylase (SMMA) is a novel extreme thermophile maltogenic amylase with an optimal temperature of 100 °C, which hydrolyzes α-(1-4)-glycosyl linkages in cyclodextrins and in linear malto-oligosaccharides. This enzyme has a long N-terminal extension that is conserved among archaic hyperthermophilic amylases but is not found in other hydrolyzing enzymes from the glycoside hydrolase 13 family. The SMMA crystal structure revealed that the N-terminal extension forms an N' domain that is similar to carbohydrate-binding module 48, with the strand-loop-strand region forming a part of the substrate binding pocket with several aromatic residues, including Phe-95, Phe-96, and Tyr-99. A structural comparison with conventional cyclodextrin-hydrolyzing enzymes revealed a striking resemblance between the SMMA N' domain position and the dimeric N domain position in bacterial enzymes. This result suggests that extremophilic archaea that live at high temperatures may have adopted a novel domain arrangement that combines all of the substrate binding components within a monomeric subunit. The SMMA structure provides a molecular basis for the functional properties that are unique to hyperthermophile maltogenic amylases from archaea and that distinguish SMMA from moderate thermophilic or mesophilic bacterial enzymes.     

Structural elucidation of dextran degradation mechanism by streptococcus mutans dextranase belonging to glycoside hydrolase family 66

Dextranase is an enzyme that hydrolyzes dextran α-1,6 linkages. Streptococcus mutans dextranase belongs to glycoside hydrolase family 66, producing isomaltooligosaccharides of various sizes and consisting of at least five amino acid sequence regions. The crystal structure of the conserved fragment from Gln¹⁰⁰ to Ile⁷³² of S. mutans dextranase, devoid of its N- and C-terminal variable regions, was determined at 1.6 Å resolution and found to contain three structural domains. Domain N possessed an immunoglobulin-like β-sandwich fold; domain A contained the enzyme''s catalytic module, comprising a (β/α)₈-barrel; and domain C formed a β-sandwich structure containing two Greek key motifs. Two ligand complex structures were also determined, and, in the enzyme-isomaltotriose complex structure, the bound isomaltooligosaccharide with four glucose moieties was observed in the catalytic glycone cleft and considered to be the transglycosylation product of the enzyme, indicating the presence of four subsites, −4 to −1, in the catalytic cleft. The complexed structure with 4′,5′-epoxypentyl-α-d-glucopyranoside, a suicide substrate of the enzyme, revealed that the epoxide ring reacted to form a covalent bond with the Asp³⁸⁵ side chain. These structures collectively indicated that Asp³⁸⁵ was the catalytic nucleophile and that Glu⁴⁵³ was the acid/base of the double displacement mechanism, in which the enzyme showed a retaining catalytic character. This is the first structural report for the enzyme belonging to glycoside hydrolase family 66, elucidating the enzyme''s catalytic machinery.     

Structural and mechanistic insights into the interaction of cytochrome P4503A4 with bromoergocryptine, a type I ligand

Cytochrome P4503A4 (CYP3A4), a major human drug-metabolizing enzyme, is responsible for the oxidation and clearance of the majority of administered drugs. One of the CYP3A4 substrates is bromoergocryptine (BEC), a dopamine receptor agonist prescribed for the inhibition of prolactin secretion and treatment of Parkinson disease, type 2 diabetes, and several other pathological conditions. Here we present a 2.15 Å crystal structure of the CYP3A4-BEC complex in which the drug, a type I heme ligand, is bound in a productive mode. The manner of BEC binding is consistent with the in vivo metabolite analysis and identifies the 8′ and 9′ carbons of the proline ring as the primary sites of oxidation. The crystal structure predicts the importance of Arg²¹² and Thr²²⁴ for binding of the tripeptide and lysergic moieties of BEC, respectively, which we confirmed experimentally. Our data support a three-step BEC binding model according to which the drug binds first at a peripheral site without perturbing the heme spectrum and then translocates into the active site cavity, where formation of a hydrogen bond between Thr²²⁴ and the N1 atom of the lysergic moiety is followed by a slower conformational readjustment of the tripeptide group modulated by Arg²¹².     

Structural basis for three-step sequential catalysis by the cholesterol side chain cleavage enzyme CYP11A1

Mitochondrial cytochrome P450 11A1 (CYP11A1 or P450 11A1) is the only known enzyme that cleaves the side chain of cholesterol, yielding pregnenolone, the precursor of all steroid hormones. Pregnenolone is formed via three sequential monooxygenation reactions that involve the progressive production of 22R-hydroxycholesterol (22HC) and 20α,22R-dihydroxycholesterol, followed by the cleavage of the C20-C22 bond. Herein, we present the 2.5-Å crystal structure of CYP11A1 in complex with the first reaction intermediate, 22HC. The active site cavity in CYP11A1 represents a long curved tube that extends from the protein surface to the heme group, the site of catalysis. 22HC occupies two-thirds of the cavity with the 22R-hydroxyl group nearest the heme, 2.56 Å from the iron. The space at the entrance to the active site is not taken up by 22HC but filled with ordered water molecules. The network formed by these water molecules allows the “soft” recognition of the 22HC 3β-hydroxyl. Such a mode of 22HC binding suggests shuttling of the sterol intermediates between the active site entrance and the heme group during the three-step reaction. Translational freedom of 22HC and torsional motion of its aliphatic tail are supported by solution studies. The CYP11A1–22HC co-complex also provides insight into the structural basis of the strict substrate specificity and high catalytic efficiency of the enzyme and highlights conserved structural motifs involved in redox partner interactions by mitochondrial P450s.     

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.     

Structures of the Bacillus subtilis Glutamine Synthetase Dodecamer Reveal Large Intersubunit Catalytic Conformational Changes Linked to a Unique Feedback Inhibition Mechanism

Glutamine synthetase (GS), which catalyzes the production of glutamine, plays essential roles in nitrogen metabolism. There are two main bacterial GS isoenzymes, GSI-α and GSI-β. GSI-α enzymes, which have not been structurally characterized, are uniquely feedback-inhibited by Gln. To gain insight into GSI-α function, we performed biochemical and cellular studies and obtained structures for all GSI-α catalytic and regulatory states. GSI-α forms a massive 600-kDa dodecameric machine. Unlike other characterized GS, the Bacillus subtilis enzyme undergoes dramatic intersubunit conformational alterations during formation of the transition state. Remarkably, these changes are required for active site construction. Feedback inhibition arises from a hydrogen bond network between Gln, the catalytic glutamate, and the GSI-α-specific residue, Arg⁶², from an adjacent subunit. Notably, Arg⁶² must be ejected for proper active site reorganization. Consistent with these findings, an R62A mutation abrogates Gln feedback inhibition but does not affect catalysis. Thus, these data reveal a heretofore unseen restructuring of an enzyme active site that is coupled with an isoenzyme-specific regulatory mechanism. This GSI-α-specific regulatory network could be exploited for inhibitor design against Gram-positive pathogens.     

The crystal complex of phosphofructokinase-2 of Escherichia coli with fructose-6-phosphate: kinetic and structural analysis of the allosteric ATP inhibition

Substrate inhibition by ATP is a regulatory feature of the phosphofructokinases isoenzymes from Escherichia coli (Pfk-1 and Pfk-2). Under gluconeogenic conditions, the loss of this regulation in Pfk-2 causes substrate cycling of fructose-6-phosphate (fructose-6-P) and futile consumption of ATP delaying growth. In the present work, we have broached the mechanism of ATP-induced inhibition of Pfk-2 from both structural and kinetic perspectives. The crystal structure of Pfk-2 in complex with fructose-6-P is reported to a resolution of 2 Å. The comparison of this structure with the previously reported inhibited form of the enzyme suggests a negative interplay between fructose-6-P binding and allosteric binding of MgATP. Initial velocity experiments show a linear increase of the apparent K_0.5 for fructose-6-P and a decrease in the apparent k_cat as a function of MgATP concentration. These effects occur simultaneously with the induction of a sigmoidal kinetic behavior (n_H of approximately 2). Differences and resemblances in the patterns of fructose-6-P binding and the mechanism of inhibition are discussed for Pfk-1 and Pfk-2, as an example of evolutionary convergence, because these enzymes do not share a common ancestor.