Sphingadienine-1-phosphate levels are regulated by a novel glycoside hydrolase family 1 glucocerebrosidase widely distributed in seed plants

Long-chain base phosphates (LCBPs) such as sphingosine-1-phosphate and phytosphingosine-1-phosphate function as abscisic acid (ABA)-mediated signaling molecules that regulate stomatal closure in plants. Recently, a glycoside hydrolase family 1 (GH1) β-glucosidase, Os3BGlu6, was found to improve drought tolerance by stomatal closure in rice, but the biochemical functions of Os3BGlu6 have remained unclear. Here we identified Os3BGlu6 as a novel GH1 glucocerebrosidase (GCase) that catalyzes the hydrolysis of glucosylceramide to ceramide. Phylogenetic and enzymatic analyses showed that GH1 GCases are widely distributed in seed plants and that pollen or anthers of all seed plants tested had high GCase activity, but activity was very low in ferns and mosses. Os3BGlu6 had high activity for glucosylceramides containing (4E,8Z)-sphingadienine, and GCase activity in leaves, stems, roots, pistils, and anthers of Os3BGlu6-deficient rice mutants was completely absent relative to that of wild-type rice. The levels of ceramides containing sphingadienine were correlated with GCase activity in each rice organ and were significantly lower in Os3BGlu6-deficient rice mutants than in the wild type. The levels of LCBPs synthesized from ceramides, especially the levels of sphingadienine-1-phosphate, were also correlated with GCase activity in each rice organ and were significantly lower in Os3BGlu6-deficient rice mutants than in the wild type. These results indicate that Os3BGlu6 regulates the level of ceramides containing sphingadienine, influencing the regulation of sphingadienine-1-phosphate levels and subsequent improvement of drought tolerance via stomatal closure in rice.     

The crystal structure of rice (Oryza sativa L.) Os4BGlu12, an oligosaccharide and tuberonic acid glucoside-hydrolyzing β-glucosidase with significant thioglucohydrolase activity

Rice Os4BGlu12, a glycoside hydrolase family 1 (GH1) β-glucosidase, hydrolyzes β-(1,4)-linked oligosaccharides of 3–6 glucosyl residues and the β-(1,3)-linked disaccharide laminaribiose, as well as certain glycosides. The crystal structures of apo Os4BGlu12, and its complexes with 2,4-dinitrophenyl-2-deoxyl-2-fluoroglucoside (DNP2FG) and 2-deoxy-2-fluoroglucose (G2F) were solved at 2.50, 2.45 and 2.40 Å resolution, respectively. The overall structure of rice Os4BGlu12 is typical of GH1 enzymes, but it contains an extra disulfide bridge in the loop B region. The glucose ring of the G2F in the covalent intermediate was found in a ⁴C₁ chair conformation, while that of the noncovalently bound DNP2FG had a ¹S₃ skew boat, consistent with hydrolysis via a ⁴H₃ half-chair transition state. The position of the catalytic nucleophile (Glu393) in the G2F structure was more similar to that of the Sinapsis alba myrosinase G2F complex than to that in covalent intermediates of other O-glucosidases, such as rice Os3BGlu6 and Os3BGlu7 β-glucosidases. This correlated with a significant thioglucosidase activity for Os4BGlu12, although with 200- to 1200-fold lower k_cat/K_m values for S-glucosides than the comparable O-glucosides, while hydrolysis of S-glucosides was undetectable for Os3BGlu6 and Os3BGlu7.     

Recombinant Expression and Characterization of the Cytoplasmic Rice β-Glucosidase Os1BGlu4

The Os1BGlu4 β-glucosidase is the only glycoside hydrolase family 1 member in rice that is predicted to be localized in the cytoplasm. To characterize the biochemical function of rice Os1BGlu4, the Os1bglu4 cDNA was cloned and used to express a thioredoxin fusion protein in Escherichia coli. After removal of the tag, the purified recombinant Os1BGlu4 (rOs1BGlu4) exhibited an optimum pH of 6.5, which is consistent with Os1BGlu4''s cytoplasmic localization. Fluorescence microscopy of maize protoplasts and tobacco leaf cells expressing green fluorescent protein-tagged Os1BGlu4 confirmed the cytoplasmic localization. Purified rOs1BGlu4 can hydrolyze p-nitrophenyl (pNP)-β-d-glucoside (pNPGlc) efficiently (k _cat/K _m  =  17.9 mM⁻¹·s⁻¹), and hydrolyzes pNP-β-d-fucopyranoside with about 50% the efficiency of the pNPGlc. Among natural substrates tested, rOs1BGlu4 efficiently hydrolyzed β-(1,3)-linked oligosaccharides of degree of polymerization (DP) 2–3, and β-(1,4)-linked oligosaccharide of DP 3–4, and hydrolysis of salicin, esculin and p-coumaryl alcohol was also detected. Analysis of the hydrolysis of pNP-β-cellobioside showed that the initial hydrolysis was between the two glucose molecules, and suggested rOs1BGlu4 transglucosylates this substrate. At 10 mM pNPGlc concentration, rOs1BGlu4 can transfer the glucosyl group of pNPGlc to ethanol and pNPGlc. This transglycosylation activity suggests the potential use of Os1BGlu4 for pNP-oligosaccharide and alkyl glycosides synthesis.     

Structural Insights into Cellulolytic and Chitinolytic Enzymes Revealing Crucial Residues of Insect β-N-acetyl-D-hexosaminidase

The chemical similarity of cellulose and chitin supports the idea that their corresponding hydrolytic enzymes would bind β-1,4-linked glucose residues in a similar manner. A structural and mutational analysis was performed for the plant cellulolytic enzyme BGlu1 from Oryza sativa and the insect chitinolytic enzyme OfHex1 from Ostrinia furnacalis. Although BGlu1 shows little amino-acid sequence or topological similarity with OfHex1, three residues (Trp⁴⁹⁰, Glu³²⁸, Val³²⁷ in OfHex1, and Trp³⁵⁸, Tyr¹³¹ and Ile¹⁷⁹ in BGlu1) were identified as being conserved in the +1 sugar binding site. OfHex1 Glu³²⁸ together with Trp⁴⁹⁰ was confirmed to be necessary for substrate binding. The mutant E328A exhibited a 8-fold increment in K _m for (GlcNAc)₂ and a 42-fold increment in K _i for TMG-chitotriomycin. A crystal structure of E328A in complex with TMG-chitotriomycin was resolved at 2.5 Å, revealing the obvious conformational changes of the catalytic residues (Glu³⁶⁸ and Asp³⁶⁷) and the absence of the hydrogen bond between E328A and the C3-OH of the +1 sugar. V327G exhibited the same activity as the wild-type, but acquired the ability to efficiently hydrolyse β-1,2-linked GlcNAc in contrast to the wild-type. Thus, Glu³²⁸ and Val³²⁷ were identified as important for substrate-binding and as glycosidic-bond determinants. A structure-based sequence alignment confirmed the spatial conservation of these three residues in most plant cellulolytic, insect and bacterial chitinolytic enzymes.     

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.     

Crystal Structure and Characterization of the Glycoside Hydrolase Family 62 α-l-Arabinofuranosidase from Streptomyces coelicolor

α-l-Arabinofuranosidase, which belongs to the glycoside hydrolase family 62 (GH62), hydrolyzes arabinoxylan but not arabinan or arabinogalactan. The crystal structures of several α-l-arabinofuranosidases have been determined, although the structures, catalytic mechanisms, and substrate specificities of GH62 enzymes remain unclear. To evaluate the substrate specificity of a GH62 enzyme, we determined the crystal structure of α-l-arabinofuranosidase, which comprises a carbohydrate-binding module family 13 domain at its N terminus and a catalytic domain at its C terminus, from Streptomyces coelicolor. The catalytic domain was a five-bladed β-propeller consisting of five radially oriented anti-parallel β-sheets. Sugar complex structures with l-arabinose, xylotriose, and xylohexaose revealed five subsites in the catalytic cleft and an l-arabinose-binding pocket at the bottom of the cleft. The entire structure of this GH62 family enzyme was very similar to that of glycoside hydrolase 43 family enzymes, and the catalytically important acidic residues found in family 43 enzymes were conserved in GH62. Mutagenesis studies revealed that Asp²⁰² and Glu³⁶¹ were catalytic residues, and Trp²⁷⁰, Tyr⁴⁶¹, and Asn⁴⁶² were involved in the substrate-binding site for discriminating the substrate structures. In particular, hydrogen bonding between Asn⁴⁶² and xylose at the nonreducing end subsite +2 was important for the higher activity of substituted arabinofuranosyl residues than that for terminal arabinofuranoses.     

Crystal Structure of α-1,4-Glucan Lyase,a Unique Glycoside Hydrolase Family Member with a Novel Catalytic Mechanism

α-1,4-Glucan lyase (EC 4.2.2.13) from the red seaweed Gracilariopsis lemaneiformis cleaves α-1,4-glucosidic linkages in glycogen, starch, and malto-oligosaccharides, yielding the keto-monosaccharide 1,5-anhydro-d-fructose. The enzyme belongs to glycoside hydrolase family 31 (GH31) but degrades starch via an elimination reaction instead of hydrolysis. The crystal structure shows that the enzyme, like GH31 hydrolases, contains a (β/α)₈-barrel catalytic domain with B and B′ subdomains, an N-terminal domain N, and the C-terminal domains C and D. The N-terminal domain N of the lyase was found to bind a trisaccharide. Complexes of the enzyme with acarbose and 1-dexoynojirimycin and two different covalent glycosyl-enzyme intermediates obtained with fluorinated sugar analogues show that, like GH31 hydrolases, the aspartic acid residues Asp⁵⁵³ and Asp⁶⁶⁵ are the catalytic nucleophile and acid, respectively. However, as a unique feature, the catalytic nucleophile is in a position to act also as a base that abstracts a proton from the C2 carbon atom of the covalently bound subsite −1 glucosyl residue, thus explaining the unique lyase activity of the enzyme. One Glu to Val mutation in the active site of the homologous α-glucosidase from Sulfolobus solfataricus resulted in a shift from hydrolytic to lyase activity, demonstrating that a subtle amino acid difference can promote lyase activity in a GH31 hydrolase.     

Kinetic Basis for the Conjugation of Auxin by a GH3 Family Indole-acetic Acid-Amido Synthetase

The GH3 family of acyl-acid-amido synthetases catalyze the ATP-dependent formation of amino acid conjugates to modulate levels of active plant hormones, including auxins and jasmonates. Initial biochemical studies of various GH3s show that these enzymes group into three families based on sequence relationships and acyl-acid substrate preference (I, jasmonate-conjugating; II, auxin- and salicylic acid-conjugating; III, benzoate-conjugating); however, little is known about the kinetic and chemical mechanisms of these enzymes. Here we use GH3-8 from Oryza sativa (rice; OsGH3-8), which functions as an indole-acetic acid (IAA)-amido synthetase, for detailed mechanistic studies. Steady-state kinetic analysis shows that the OsGH3-8 requires either Mg²⁺ or Mn²⁺ for maximal activity and is specific for aspartate but accepts asparagine as a substrate with a 45-fold decrease in catalytic efficiency and accepts other auxin analogs, including phenyl-acetic acid, indole butyric acid, and naphthalene-acetic acid, as acyl-acid substrates with 1.4–9-fold reductions in k_cat/K_m relative to IAA. Initial velocity and product inhibition studies indicate that the enzyme uses a Bi Uni Uni Bi Ping Pong reaction sequence. In the first half-reaction, ATP binds first followed by IAA. Next, formation of an adenylated IAA intermediate results in release of pyrophosphate. The second half-reaction begins with binding of aspartate, which reacts with the adenylated intermediate to release IAA-Asp and AMP. Formation of a catalytically competent adenylated-IAA reaction intermediate was confirmed by mass spectrometry. These mechanistic studies provide insight on the reaction catalyzed by the GH3 family of enzymes to modulate plant hormone action.     

The GH130 Family of Mannoside Phosphorylases Contains Glycoside Hydrolases That Target β-1,2-Mannosidic Linkages in Candida Mannan

The depolymerization of complex glycans is an important biological process that is of considerable interest to environmentally relevant industries. β-Mannose is a major component of plant structural polysaccharides and eukaryotic N-glycans. These linkages are primarily cleaved by glycoside hydrolases, although recently, a family of glycoside phosphorylases, GH130, have also been shown to target β-1,2- and β-1,4-mannosidic linkages. In these phosphorylases, bond cleavage was mediated by a single displacement reaction in which phosphate functions as the catalytic nucleophile. A cohort of GH130 enzymes, however, lack the conserved basic residues that bind the phosphate nucleophile, and it was proposed that these enzymes function as glycoside hydrolases. Here we show that two Bacteroides enzymes, BT3780 and BACOVA_03624, which lack the phosphate binding residues, are indeed β-mannosidases that hydrolyze β-1,2-mannosidic linkages through an inverting mechanism. Because the genes encoding these enzymes are located in genetic loci that orchestrate the depolymerization of yeast α-mannans, it is likely that the two enzymes target the β-1,2-mannose residues that cap the glycan produced by Candida albicans. The crystal structure of BT3780 in complex with mannose bound in the −1 and +1 subsites showed that a pair of glutamates, Glu²²⁷ and Glu²⁶⁸, hydrogen bond to O₁ of α-mannose, and either of these residues may function as the catalytic base. The candidate catalytic acid and the other residues that interact with the active site mannose are conserved in both GH130 mannoside phosphorylases and β-1,2-mannosidases. Functional phylogeny identified a conserved lysine, Lys¹⁹⁹ in BT3780, as a key specificity determinant for β-1,2-mannosidic linkages.     

Structure of a bacterial α-1,2-glucosidase defines mechanisms of hydrolysis and substrate specificity in GH65 family hydrolases

Glycoside hydrolase family 65 (GH65) comprises glycoside hydrolases (GHs) and glycoside phosphorylases (GPs) that act on α-glucosidic linkages in oligosaccharides. All previously reported bacterial GH65 enzymes are GPs, whereas all eukaryotic GH65 enzymes known are GHs. In addition, to date, no crystal structure of a GH65 GH has yet been reported. In this study, we use biochemical experiments and X-ray crystallography to examine the function and structure of a GH65 enzyme from Flavobacterium johnsoniae (FjGH65A) that shows low amino acid sequence homology to reported GH65 enzymes. We found that FjGH65A does not exhibit phosphorolytic activity, but it does hydrolyze kojibiose (α-1,2-glucobiose) and oligosaccharides containing a kojibiosyl moiety without requiring inorganic phosphate. In addition, stereochemical analysis demonstrated that FjGH65A catalyzes this hydrolytic reaction via an anomer-inverting mechanism. The three-dimensional structures of FjGH65A in native form and in complex with glucose were determined at resolutions of 1.54 and 1.40 Å resolutions, respectively. The overall structure of FjGH65A resembled those of other GH65 GPs, and the general acid catalyst Glu⁴⁷² was conserved. However, the amino acid sequence forming the phosphate-binding site typical of GH65 GPs was not conserved in FjGH65A. Moreover, FjGH65A had the general base catalyst Glu⁶¹⁶ instead, which is required to activate a nucleophilic water molecule. These results indicate that FjGH65A is an α-1,2-glucosidase and is the first bacterial GH found in the GH65 family.     

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).     

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.     

Identification and Characterization of γ-Glutamylamine Cyclotransferase,an Enzyme Responsible for γ-Glutamyl-?-lysine Catabolism

γ-Glutamylamine cyclotransferase (GGACT) is an enzyme that converts γ-glutamylamines to free amines and 5-oxoproline. GGACT shows high activity toward γ-glutamyl-ϵ-lysine, derived from the breakdown of fibrin and other proteins cross-linked by transglutaminases. The enzyme adopts the newly identified cyclotransferase fold, observed in γ-glutamylcyclotransferase (GGCT), an enzyme with activity toward γ-glutamyl-α-amino acids (Oakley, A. J., Yamada, T., Liu, D., Coggan, M., Clark, A. G., and Board, P. G. (2008) J. Biol. Chem. 283, 22031–22042). Despite the absence of significant sequence identity, several residues are conserved in the active sites of GGCT and GGACT, including a putative catalytic acid/base residue (GGACT Glu⁸²). The structure of GGACT in complex with the reaction product 5-oxoproline provides evidence for a common catalytic mechanism in both enzymes. The proposed mechanism, combined with the three-dimensional structures, also explains the different substrate specificities of these enzymes. Despite significant sequence divergence, there are at least three subfamilies in prokaryotes and eukaryotes that have conserved the GGCT fold and GGCT enzymatic activity.     

Glutamate 90 at the Luminal Ion Gate of Sarcoplasmic Reticulum Ca2+-ATPase Is Critical for Ca2+ Binding on Both Sides of the Membrane

The roles of Ser⁷², Glu⁹⁰, and Lys²⁹⁷ at the luminal ends of transmembrane helices M1, M2, and M4 of sarcoplasmic reticulum Ca²⁺-ATPase were examined by transient and steady-state kinetic analysis of mutants. The dependence on the luminal Ca²⁺ concentration of phosphorylation by P_i (“Ca²⁺ gradient-dependent E2P formation”) showed a reduction of the apparent affinity for luminal Ca²⁺ in mutants with alanine or leucine replacement of Glu⁹⁰, whereas arginine replacement of Glu⁹⁰ or Ser⁷² allowed E2P formation from P_i even at luminal Ca²⁺ concentrations much too small to support phosphorylation in wild type. The latter mutants further displayed a blocked dephosphorylation of E2P and an increased rate of conversion of the ADP-sensitive E1P phosphoenzyme intermediate to ADP-insensitive E2P as well as insensitivity of the E2·BeF₃⁻ complex to luminal Ca²⁺. Altogether, these findings, supported by structural modeling, indicate that the E2P intermediate is stabilized in the mutants with arginine replacement of Glu⁹⁰ or Ser⁷², because the positive charge of the arginine side chain mimics Ca²⁺ occupying a luminally exposed low affinity Ca²⁺ site of E2P, thus identifying an essential locus (a “leaving site”) on the luminal Ca²⁺ exit pathway. Mutants with alanine or leucine replacement of Glu⁹⁰ further displayed a marked slowing of the Ca²⁺ binding transition as well as slowing of the dissociation of Ca²⁺ from Ca₂E1 back toward the cytoplasm, thus demonstrating that Glu⁹⁰ is also critical for the function of the cytoplasmically exposed Ca²⁺ sites on the opposite side of the membrane relative to where Glu⁹⁰ is located.     

Crystal structure of glutamyl-queuosine tRNAAsp synthetase complexed with L-glutamate: structural elements mediating tRNA-independent activation of glutamate and glutamylation of tRNAAsp anticodon

Glutamyl-queuosine tRNA^Asp synthetase (Glu-Q-RS) from Escherichia coli is a paralog of the catalytic core of glutamyl-tRNA synthetase (GluRS) that catalyzes glutamylation of queuosine in the wobble position of tRNA^Asp. Despite important structural similarities, Glu-Q-RS and GluRS diverge strongly by their functional properties. The only feature common to both enzymes consists in the activation of Glu to form Glu-AMP, the intermediate of transfer RNA (tRNA) aminoacylation. However, both enzymes differ by the mechanism of selection of the cognate amino acid and by the mechanism of its activation. Whereas GluRS selects l-Glu and activates it only in the presence of the cognate tRNA^Glu, Glu-Q-RS forms Glu-AMP in the absence of tRNA. Moreover, while GluRS transfers the activated Glu to the 3′ accepting end of the cognate tRNA^Glu, Glu-Q-RS transfers the activated Glu to Q34 located in the anticodon loop of the noncognate tRNA^Asp. In order to gain insight into the structural elements leading to distinct mechanisms of amino acid activation, we solved the three-dimensional structure of Glu-Q-RS complexed to Glu and compared it to the structure of the GluRS·Glu complex. Comparison of the catalytic site of Glu-Q-RS with that of GluRS, combined with binding experiments of amino acids, shows that a restricted number of residues determine distinct catalytic properties of amino acid recognition and activation by the two enzymes. Furthermore, to explore the structural basis of the distinct aminoacylation properties of the two enzymes and to understand why Glu-Q-RS glutamylates only tRNA^Asp among the tRNAs possessing queuosine in position 34, we performed a tRNA mutational analysis to search for the elements of tRNA^Asp that determine recognition by Glu-Q-RS. The analyses made on tRNA^Asp and tRNA^Asn show that the presence of a C in position 38 is crucial for glutamylation of Q34. The results are discussed in the context of the evolution and adaptation of the tRNA glutamylation system.     

Structural and Enzymatic Characterization of Os3BGlu6, a Rice β-Glucosidase Hydrolyzing Hydrophobic Glycosides and (1→3)- and (1→2)-Linked Disaccharides

Glycoside hydrolase family 1 (GH1) β-glucosidases play roles in many processes in plants, such as chemical defense, alkaloid metabolism, hydrolysis of cell wall-derived oligosaccharides, phytohormone regulation, and lignification. However, the functions of most of the 34 GH1 gene products in rice (Oryza sativa) are unknown. Os3BGlu6, a rice β-glucosidase representing a previously uncharacterized phylogenetic cluster of GH1, was produced in recombinant Escherichia coli. Os3BGlu6 hydrolyzed p-nitrophenyl (pNP)-β-d-fucoside (k_cat/K_m = 67 mm⁻¹ s⁻¹), pNP-β-d-glucoside (k_cat/K_m = 6.2 mm⁻¹ s⁻¹), and pNP-β-d-galactoside (k_cat/K_m = 1.6 mm⁻¹s⁻¹) efficiently but had little activity toward other pNP glycosides. It also had high activity toward n-octyl-β-d-glucoside and β-(1→3)- and β-(1→2)-linked disaccharides and was able to hydrolyze apigenin β-glucoside and several other natural glycosides. Crystal structures of Os3BGlu6 and its complexes with a covalent intermediate, 2-deoxy-2-fluoroglucoside, and a nonhydrolyzable substrate analog, n-octyl-β-d-thioglucopyranoside, were solved at 1.83, 1.81, and 1.80 Å resolution, respectively. The position of the covalently trapped 2-F-glucosyl residue in the enzyme was similar to that in a 2-F-glucosyl intermediate complex of Os3BGlu7 (rice BGlu1). The side chain of methionine-251 in the mouth of the active site appeared to block the binding of extended β-(1→4)-linked oligosaccharides and interact with the hydrophobic aglycone of n-octyl-β-d-thioglucopyranoside. This correlates with the preference of Os3BGlu6 for short oligosaccharides and hydrophobic glycosides.β-Glucosidases (EC 3.2.1.21) have a wide range of functions in plants, including acting in cell wall remodeling, lignification, chemical defense, plant-microbe interactions, phytohormone activation, activation of metabolic intermediates, and release of volatiles from their glycosides (Esen, 1993). They fulfill these roles by hydrolyzing the glycosidic bond at the nonreducing terminal glucosyl residue of a glycoside or an oligosaccharide, thereby releasing Glc and an aglycone or a shortened carbohydrate. The aglycone released from the glycoside may be a monolignol, a toxic compound, or a compound that further reacts to release a toxic component, an active phytohormone, a reactive metabolic intermediate, or a volatile scent compound (Brzobohatý et al., 1993; Dharmawardhama et al., 1995; Reuveni et al., 1999; Lee et al., 2006; Barleben et al., 2007; Morant et al., 2008). Indeed, the wide range of glucosides of undocumented functions found in plants suggests that many β-glucosidase functions may remain to be discovered.Plant β-glucosidases fall into related families that have been classified as glycosyl hydrolase (GH) families GH1, GH3, and GH5 (Henrissat, 1991; Coutinho and Henrissat, 1998, 1999). Of these, GH1 has been most thoroughly documented and shown to comprise a gene family encoding 40 putative functional GHs in Arabidopsis (Arabidopsis thaliana) and 34 in rice (Oryza sativa) in addition to a few pseudogenes (Xu et al., 2004; Opassiri et al., 2006). In addition to β-glucosidases, plant GH1 members include β-mannosidases (Mo and Bewley, 2002), β-thioglucosidases (Burmeister et al., 1997), and disaccharidases such as primeverosidase (Mizutani et al., 2002) as well as hydroxyisourate hydrolase, which hydrolyzes the internal bond in a purine ring rather than a glycosidic bond (Raychaudhuri and Tipton, 2002). The specificity for the glycone in GH1 enzymes varies. Some enzymes are quite specific for β-d-glucosides or β-d-mannosides, while many accept either β-d-glucosides or β-d-fucosides, and some also hydrolyze β-d-galactosides, β-d-xylosides, and α-l-arabinoside (Esen, 1993). However, most GH1 enzymes are thought to hydrolyze glucosides in the plant, and it is the aglycone specificity that determines the functions of most GH1 enzymes.Aglycone specificity of GH1 β-glucosidases ranges from rather broad to absolutely specific for one substrate and is not obvious from sequence similarity. For instance, maize (Zea mays) ZmGlu1 β-glucosidase hydrolyzes a range of glycosides, including its natural substrate, 2-O-β-d-glucopyranosyl-4-dihydroxy-1,4-benzoxazin-3-one (DIMBOAGlc), but not dhurrin, whereas sorghum (Sorghum bicolor) Dhr1, which is 72% identical to ZmGlu1, only hydrolyzes its natural cyanogenic substrate dhurrin (Verdoucq et al., 2003). Similarly, despite sharing over 80% amino acid sequence identity, the legume isoflavonoid β-glucosidases dalcochinase from Dalbergia cochinchinensis and Dnbglu2 from Dalbergia nigrescens hydrolyze each other''s natural substrate very poorly (Chuankhayan et al., 2007). Thus, small differences in the amino acid sequence surrounding the active site may be expected to account for significant differences in substrate specificity.GH1 is classified in GH clan A, which consists of GH families whose members have a (β/α)₈-barrel structure with the catalytic acid/base on strand 4 of the β-barrel and the catalytic nucleophile on strand 7 (Henrissat et al., 1995; Jenkins et al., 1995). As such, all GH1 enzymes have similar overall structures, but it has been noted that four variable loops at the C-terminal end of the β-barrel strands, designated A, B, C, and D, account for much of the differences in the active site architecture (Sanz-Aparicio et al., 1998). The similar structures with great diversity in substrate specificity make plant GH1 enzymes an ideal model system to investigate the structural basis of substrate specificity. To date, seven plant β-glucosidase structures have been reported, including three closely related chloroplastic enzymes from maize (Czjzek et al., 2000, 2001), sorghum (Verdoucq et al., 2004), and wheat (Triticum aestivum; Sue et al., 2006), the cytoplasmic strictosidine β-glucosidase from Rauvolfia serpentine (Barleben et al., 2007), and the secreted enzymes white clover (Trifolium repens) cyanogenic β-glucosidase (Barrett et al., 1995), white mustard (Sinapsis alba) myrosinase (thioglucosidase; Burmeister et al., 1997), and rice Os3BGlu7 (BGlu1; Chuenchor et al., 2008). These enzymes hydrolyze substrates with a range of structures, but they cannot account for the full range of β-glucosidase substrates available in plants, and determining the structural differences that bring about substrate specificity differences in even closely related GH1 enzymes has proven tricky (Verdoucq et al., 2003, 2004; Sue et al., 2006; Chuenchor et al., 2008).Amino acid sequence-based phylogenetic analysis of GH1 enzymes encoded by the rice genome showed that there are eight clusters containing both rice and Arabidopsis proteins that are more closely related to each other than they are to enzymes from the same plants outside the clusters (Fig. 1; Opassiri et al., 2006). In addition, there are a cluster of sixteen putative β-glucosidases and a cluster of myrosinases in Arabidopsis without any closely related rice counterparts. Comparison with characterized GH1 enzymes from other plants reveals other clusters of related enzymes not found in rice or Arabidopsis, including the chloroplastic enzymes, from which the maize, sorghum, and wheat structures are derived, and the cytoplasmic metabolic enzymes, from with the strictosidine hydrolase structure is derived (Fig. 1). Therefore, although the known structures provide good tools for molecular modeling of plant enzymes, most rice and Arabidopsis GH1 enzymes lack a close correspondence in sequence and functional evolution to these structures, suggesting that the variable loops that determine the active site may be different. It would be useful, therefore, to know the structures and substrate specificities of representative members of each of the eight clusters seen in rice and Arabidopsis. To begin to acquire this information, we have expressed Os3BGlu6, a member of cluster At/Os 1 in Figure 1, characterized its substrate specificity, and determined its structure alone and in complex with a glycosyl intermediate and a nonhydrolyzable substrate analog.Open in a separate windowFigure 1.Simplified phylogenetic tree of the amino acid sequences of eukaryotic GH1 proteins with known structures and those of rice and Arabidopsis GH1 gene products. The protein sequences of the eukaryotic proteins with known structures are marked with four-character PDB codes for one of their structures, including Trifolium repens cyanogenic β-glucosidase (1CBG; Barrett et al., 1995), Sinapsis alba myrosinase (1MYR; Burmeister et al., 1997), Zea mays ZmGlu1 β-glucosidase (1E1F; Czjzek et al., 2000), Sorghum bicolor Dhr1 dhurrinase (1V02; Verdoucq et al., 2004), Triticum aestivum β-glucosidase (2DGA; Sue et al., 2006), Rauvolfia serpentina strictosidine β-glucosidase (2JF6; Barleben et al., 2007), and Oryza sativa Os3BGlu7 (BGlu1) β-glucosidase (2RGL; Chuenchor et al., 2008) from plants, along with Brevicoryne brassicae myrosinase (1WCG; Husebye et al., 2005), Homo sapiens cytoplasmic (Klotho) β-glucosidase (2E9M; Hayashi et al., 2007), and Phanerochaete chrysosporium (2E3Z; Nijikken et al., 2007), while those encoded in the Arabidopsis and rice genomes are labeled with the systematic names given by Xu et al. (2004) and Opassiri et al. (2006), respectively. One or two example proteins from each plant are given for each of the eight clusters of genes shared by Arabidopsis (At) and rice (Os) and the Arabidopsis-specific clusters At I (β-glucosidases) and At II (myrosinases), with the number of Arabidopsis or rice enzymes in each cluster given in parentheses. These sequences were aligned with all of the Arabidopsis and rice sequences in ClustalX (Thompson et al., 1997), the alignment was manually edited, all but representative sequences were removed, and the tree was calculated by the neighbor-joining method, bootstrapped with 1,000 trials, and then drawn with TreeView (Page, 1996). The grass plastid β-glucosidases, which are not represented in Arabidopsis and rice, are shown in the group marked “Plastid.” Percentage bootstrap reproducibility values are shown on internal branches where they are greater than 60%. Except those marked by asterisks, all external branches represent groups with 100% bootstrap reproducibility. To avoid excess complexity, those groups of sequences marked with asterisks are not monophyletic and represent more branches within the designated cluster than are shown. For a complete phylogenetic analysis of Arabidopsis and rice GH1 proteins, see Opassiri et al. (2006).     

Structural and Functional Characterization of the Clostridium perfringens N-Acetylmannosamine-6-phosphate 2-Epimerase Essential for the Sialic Acid Salvage Pathway

Pathogenic bacteria are endowed with an arsenal of specialized enzymes to convert nutrient compounds from their cell hosts. The essential N-acetylmannosamine-6-phosphate 2-epimerase (NanE) belongs to a convergent glycolytic pathway for utilization of the three amino sugars, GlcNAc, ManNAc, and sialic acid. The crystal structure of ligand-free NanE from Clostridium perfringens reveals a modified triose-phosphate isomerase (β/α)₈ barrel in which a stable dimer is formed by exchanging the C-terminal helix. By retaining catalytic activity in the crystalline state, the structure of the enzyme bound to the GlcNAc-6P product identifies the topology of the active site pocket and points to invariant residues Lys⁶⁶ as a putative single catalyst, supported by the structure of the catalytically inactive K66A mutant in complex with substrate ManNAc-6P. ¹H NMR-based time course assays of native NanE and mutated variants demonstrate the essential role of Lys⁶⁶ for the epimerization reaction with participation of neighboring Arg⁴³, Asp¹²⁶, and Glu¹⁸⁰ residues. These findings unveil a one-base catalytic mechanism of C2 deprotonation/reprotonation via an enolate intermediate and provide the structural basis for the development of new antimicrobial agents against this family of bacterial 2-epimerases.     

An Acyl-covalent Enzyme Intermediate of Lecithin:Retinol Acyltransferase

Synthesis of fatty acid retinyl esters determines systemic vitamin A levels and provides substrate for production of visual chromophore (11-cis-retinal) in vertebrates. Lecithin:retinol acyltransferase (LRAT), the main enzyme responsible for retinyl ester formation, catalyzes the transfer of an acyl group from the sn-1 position of phosphatidylcholine to retinol. To delineate the catalytic mechanism of this reaction, we expressed and purified a fully active, soluble form of this enzyme and used it to examine the possible formation of a transient acyl-enzyme intermediate. Detailed mass spectrometry analyses revealed that LRAT undergoes spontaneous, covalent modification upon incubation with a variety of phosphatidylcholine substrates. The addition of an acyl chain occurs at the Cys¹⁶¹ residue, indicating formation of a thioester intermediate. This observation provides the first direct experimental evidence of thioester intermediate formation that constitutes the initial step in the proposed LRAT catalytic reaction. Additionally, we examined the effect of increasing fatty acyl side chain length in phosphatidylcholine on substrate accessibility in this reaction, which provided insights into the function of the single membrane-spanning domain of LRAT. These observations are critical to understanding the catalytic mechanism of LRAT protein family members as well as other lecithin:acyltransferases wherein Cys residues are required for catalysis.     

Analysis of rice glycosyl hydrolase family 1 and expression of Os4bglu12 β-glucosidase

Background

Glycosyl hydrolase family 1 (GH1) β-glucosidases have been implicated in physiologically important processes in plants, such as response to biotic and abiotic stresses, defense against herbivores, activation of phytohormones, lignification, and cell wall remodeling. Plant GH1 β-glucosidases are encoded by a multigene family, so we predicted the structures of the genes and the properties of their protein products, and characterized their phylogenetic relationship to other plant GH1 members, their expression and the activity of one of them, to begin to decipher their roles in rice.

Results

Forty GH1 genes could be identified in rice databases, including 2 possible endophyte genes, 2 likely pseudogenes, 2 gene fragments, and 34 apparently competent rice glycosidase genes. Phylogenetic analysis revealed that GH1 members with closely related sequences have similar gene structures and are often clustered together on the same chromosome. Most of the genes appear to have been derived from duplications that occurred after the divergence of rice and Arabidopsis thaliana lineages from their common ancestor, and the two plants share only 8 common gene lineages. At least 31 GH1 genes are expressed in a range of organs and stages of rice, based on the cDNA and EST sequences in public databases. The cDNA of the Os4bglu12 gene, which encodes a protein identical at 40 of 44 amino acid residues with the N-terminal sequence of a cell wall-bound enzyme previously purified from germinating rice, was isolated by RT-PCR from rice seedlings. A thioredoxin-Os4bglu12 fusion protein expressed in Escherichia coli efficiently hydrolyzed β-(1,4)-linked oligosaccharides of 3–6 glucose residues and laminaribiose.

Conclusion

Careful analysis of the database sequences produced more reliable rice GH1 gene structure and protein product predictions. Since most of these genes diverged after the divergence of the ancestors of rice and Arabidopsis thaliana, only a few of their functions could be implied from those of GH1 enzymes from Arabidopsis and other dicots. This implies that analysis of GH1 enzymes in monocots is necessary to understand their function in the major grain crops. To begin this analysis, Os4bglu12 β-glucosidase was characterized and found to have high exoglucanase activity, consistent with a role in cell wall metabolism.     

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

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