Báo cáo khoa học: Catalytic reaction mechanism of Pseudomonas stutzeri L-rhamnose isomerase deduced from X-ray structures doc

Catalytic reaction mechanism of Pseudomonas stutzeri L-rhamnose isomerase deduced from X-ray structures Hiromi Yoshida1, Masatsugu Yamaji1,2, Tomohiko Ishii2, Ken Izumori3 and Shigehiro Kamitori1 Life Science Research Center and Faculty of Medicine, Kagawa University, Japan Faculty of Technology, Kagawa University, Japan Rare Sugar Research Center and Faculty of Agriculture, Kagawa University, Japan Keywords catalytic mechanism; hydride-shift; L-rhamnose isomerase; rare sugar; X-ray structure Correspondence S Kamitori, Life Science Research Center and Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan Fax: +81 87 891 2421 Tel: +81 87 891 2421 E-mail: kamitori@med.kagawa-u.ac.jp (Received 27 October 2009, revised December 2009, accepted 15 December 2009) doi:10.1111/j.1742-4658.2009.07548.x l-Rhamnose isomerase (l-RhI) catalyzes the reversible isomerization of l-rhamnose to l-rhamnulose Pseudomonas stutzeri l-RhI, with a broad substrate specificity, can catalyze not only the isomerization of l-rhamnose, but also that between d-allose and d-psicose For the aldose–ketose isomerization by l-RhI, a metal-mediated hydride-shift mechanism has been proposed, but the catalytic mechanism is still not entirely understood To elucidate the entire reaction mechanism, the X-ray structures of P stutzeri l-RhI in an Mn2+-bound form, and of two inactive mutant forms of P stutzeri l-RhI (S329K and D327N) in a complex with substrate ⁄ product, were determined The structure of the Mn2+-bound enzyme indicated that the catalytic site interconverts between two forms with the displacement of the metal ion to recognize both pyranose and furanose ring substrates Solving the structures of S329K–substrates allowed us to examine the metal-mediated hydride-shift mechanism of l-RhI in detail The structural analysis of D327N–substrates and additional modeling revealed Asp327 to be responsible for the ring opening of furanose, and a water molecule coordinating with the metal ion to be involved in the ring opening of pyranose Structured digital abstract l MINT-7384817: L-RhI (uniprotkb:Q75WH8) and L-RhI (uniprotkb:Q75WH8) bind (MI:0407) by X-ray crystallography (MI:0114) Introduction l-Rhamnose isomerase (l-RhI), which catalyzes the reversible isomerization of l-rhamnose to l-rhamnulose, has been found to be involved in the metabolism of l-rhamnose in Escherichia coli (Fig 1A) [1,2], and the X-ray structure of E coli l-RhI was determined [3] Pseudomonas stutzeri l-RhI, with a broad substrate specificity compared with E coli l-RhI, can catalyze not only the isomerization of l-rhamnose, but also that between d-allose and d-psicose (Fig 1A) [4–6] As d-allose and d-psicose are ‘rare sugars’, existing in small amounts in nature, P stutzeri l-RhI is exploited for industrial applications in rare sugar production We have reported the structures of P stutzeri l-RhI in complexes with substrates (l-rhamnose and d-allose), revealing a unique catalytic site recognizing both l-rhamnose and d-allose [7] l-RhI has structural homology with d-xylose isomerase (d-XI), in spite of the low sequence identity (13– 17%) between them Both have a large domain with a (b ⁄ a)8 barrel and an additional small domain Abbreviations CSD, Cambridge Structure Database; D-XI, D-xylose isomerase; L-RhI, L-rhamnose isomerase; PDB, Protein Data Bank FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS 1045 Catalytic mechanism of L-rhamnose isomerase H O OH H OH H OH H Yoshida et al A CH2OH H3C HO HO O O H OH HO H HO H HO OH H HO H CH3 (α-L-rhamnopyranose) L-rhamnose H H H OH H H CH3 OH OH (β-L-rhamnulofuranose) L-rhamnulose OH 6 CH O OH CH2OH O OH CH2OH HO O OH OH OH OH H OH OH H CH OH O OH OH CH2OH (β-D-allopyranose) O H D-allose OH OH CH2OH CH2OH OH (α-D-psicofuranose) D-psicose “Catalytic” Mn2+ B H H O O H O Base Base H O H H O H “Structural” Mn2+ ene-diol mechanism Hydride-shift mechanism composed of a-helices, which form a homotetramer, and each subunit has two adjacent metal ions at the catalytic site: one a ‘structural metal ion’ to aid substrate binding, and the other a ‘catalytic metal ion’ to help with the catalytic reaction [8] Many structural studies of d-XI have been performed to understand its catalytic reaction mechanism [8–19] Two types of mechanism, the ene-diol mechanism [9] and the hydride-shift mechanism [8,11–13,16], have been proposed for the aldose–ketose isomerization of d-XI based on the X-ray structures According to the enediol mechanism, two bases transfer a proton from O2 to O1, and a proton from C1 to C2, respectively, producing ketose from aldose, as shown in Fig 1B During the reaction, the ene-diol intermediate is stabilized by the metal ion In the structures of d-XIs, a water molecule coordinating to the metal ion was thought to act as a base to transfer a proton from O2 to O1, but a suitable base to transfer a proton from C1 to C2 has been never found It was also reported that protons of C1 and ⁄ or C2 not exchange with solvent [20] Thus, the hydride-shift mechanism was proposed and generally accepted, in which a hydride ion moves from C2 to C1, as shown in Fig 1B However, neutron-based studies of d-XIs suggest that the possibility of the enediol mechanism still remains [17,18] The structure of P stutzeri l-RhI without a suitable base to transfer a proton between C2 and C1 seems to support the metal-mediated hydride-shift mechanism 1046 Fig (A) Chemical reactions catalyzed by P stutzeri L-RhI with potential substrates (B) Two types of proposed catalytic mechanism for aldose–ketose isomerization, the ene-diol mechanism (left) and the hydrideshift mechanism (right) for aldose–ketose isomerization, but the catalytic reaction mechanism is still not entirely understood First, the mechanism for the ring opening of a substrate is unknown In d-XI, two adjacent residues (His53 and Asp56) are proposed to be responsible for the opening, but the pair is not conserved in l-RhI This suggests that l-RhI has a different ring opening mechanism from d-XI Second, as the enzymatic activity of P stutzeri l-RhI is strongly dependent on the metal ion species, the activity ratio being 100 : 35 : 19 : 10 for Mn2+, Cu2+, Co2+ and Zn2+ [6], the relationship between the species of metal ion and enzymatic activity needs to be elucidated In a previously reported structure of P stutzeri l-RhI, the bound metal ions were refined as Zn2+, as the atomic absorption spectrum of the purified enzyme showed the presence of Zn2+, Mn2+ and Ni2+ in a ratio of : : [7] The structure with a specific metal ion, Mn2+, would be required To obtain new insights into the overall catalytic reaction mechanism of P stutzeri l-RhI, we report here the X-ray structures of an unliganded P stutzeri l-RhI in the Mn2+-bound form, and two inactive mutant forms of P stutzeri l-RhI, with substitutions of Ser329 with Lys (S329K) and Asp327 with Asn (D327N), in a complex with a substrate ⁄ product The complex structures of D327N with d-psicose and l-rhamnulose are the first in which the bound substrate ⁄ product has a furanose ring conformation in the sugar isomerase FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS Catalytic mechanism of L-rhamnose isomerase H Yoshida et al Mol-C and Mol-D, with a noncrystallographic 222 symmetry, having four catalytic sites The pair Mol-A and Mol-B and ⁄ or Mol-C and Mol-D with a two-fold symmetry forms the accessible surface for substrate binding, as shown in Fig Phe66 in the loop region between the first b-strand and a-helix of Mol-A approaches the catalytic site of Mol-B to interact with a substrate, whereas no amino acid residue of Mol-A approaches the catalytic sites of Mol-C and Mol-D Phe66 (Mol-A) Mol-D Mol-B Structure of the enzyme in the Mn2+-bound form Mol-A As Zn2+, Mn2+ and Ni2+ were found to bind to the purified enzyme from the atomic absorption spectrum in a previously reported study [7], the entire removal of metal ions from the purified enzyme should be required to obtain the enzyme with a specific metal ion-bound form We successfully prepared the enzyme in a metal-free form by mm EDTA treatment, and the removal of metal ions was confirmed by X-ray analysis (Table S1, see Supporting information) By incubating this metal-free form with each specific metal ion, the Mn2+-, Cu2+-, Co2+- and Zn2+-bound forms could be obtained In all the enzymes, two metal ions bind to each component of the tetramer The Cu2+-, Co2+- and Zn2+bound forms have the same metal-coordinated structure in all four molecules (Mol-A, Mol-B, Mol-C and Mol-D); however, the Mn2+-bound form has two metal-coordinated structures, as shown in Fig The final electron density maps for Mn2+ ions in the two metal-coordinated structures are given in Fig S1 (see Supporting information) In Mol-A and ⁄ or Mol-D, the structural Mn2+ (Mn1) is coordinated by six coordination bonds from Glu219(OE), Asp254(OD), His281(ND), Asp327(OD) and two water molecules (W1 and W2), and the catalytic Mn2+ (Mn2) is Mol-C Phe66 (Mol-B) Fig Overall tetrameric structure of P stutzeri L-RhI The four molecules are colored in yellow (Mol-A), green (Mol-B), magenta (Mol-C) and light blue (Mol-D) The dark-colored part of each molecule represents the additional small domain The small spheres indicate metal ions Phe66 and the loop regions between the first b-strand and a-helix of Mol-A and Mol-B are indicated by a stick model, and black, respectively Results and Discussion Overall structure of P stutzeri l-RhI The overall structure of P stutzeri l-RhI has been reported previously [7] Briefly, the monomeric structure of P stutzeri l-RhI comprises a large domain (Phe50–Val356) with a (b ⁄ a)8 barrel fold, and an additional small domain (N-terminus–Lys49 and Asp357– C-terminus), and two metal ions (Mn2+) bind to the centre of the barrel to form the catalytic site The enzyme forms a tetramer comprising Mol-A, Mol-B, Asp289 Asp289 His257 Fig Stereoview of the two forms of metal-bound structure of P stutzeri L-RhI in the Mn2+-bound form The AD-form (Mol-A) is indicated by yellow carbon amino acid residues, black Mn2+ ions and red water molecules The BC-form (Mol-B) is indicated by green carbon amino acid residues, gray Mn2+ ions and pink water molecules Selected interactions among amino acid residues, metal ions and water molecules are indicated by black (Mol-A) and gray (Mol-B) dotted lines Asp291 Asp291 Mn2 W5 W4 His257 Lys221 W6 Mn2 W5 W3 W7 W4 W6 W3 W7 Asp254 W2 Asp254 Asp327 W2 Asp327 Mn1 His281 Lys221 Mn1 W1 FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS Glu219 His281 W1 Glu219 1047 Catalytic mechanism of L-rhamnose isomerase H Yoshida et al coordinated by His257(NE), Asp289(OD1) and four water molecules (W2, W3, W4 and W5) The distance ˚ between Mn1 and Mn2 is 4.2 A, and a water molecule of W2 bridges the metal ions This metal-coordinated structure is equivalent to those found in the Cu2+-, Co2+- and Zn2+-bound forms (Fig S2, see Supporting information) This is denoted as the ‘AD-form’ A substrate binds to the catalytic site in the AD-form, as described later In Mol-B and ⁄ or Mol-C, Mn1 is coordinated in the same way as in Mol-A and Mol-D, but Mn2 is coordinated by His257(NE), Asp289(OD1), Asp289(OD2), Asp291(OD) and two water molecules (W6, W7) The distance between Mn1 and Mn2 is ˚ 5.2 A, and the water molecules W2 and W6 bridge the metal ions This metal-coordinated structure is denoted as the ‘BC-form’ A disordered catalytic metal ion was identified by the high-resolution X-ray structure of Streptomyces olivochromogenes d-XI, showing that the displacement of metal ions was involved in the catalytic reaction [16] Thus, it is likely that the positions of the catalytic metal ions of P stutzeri l-RhI also vary between the AD- and BC-forms Through metal-coordinated structural change from the BC- to AD-form, ˚ Mn2 moves by 1.90 A towards the substrate-accessible surface, accompanied by the movement of W7 to the position of W5, W6 to W3 and W3 to the solvent channel Mn2 in the AD-form attracts W2, leading to ˚ the movement of Mn1, His281 and W1 by 0.65 A to Mn2 The distance between Mn1 and Mn2 changes ˚ ˚ from 5.2 A (BC-form) to 4.2 A (AD-form) Tempera˚ ture factors of Mn2 (30.3, 26.9, 30.0 and 30.1 A2 for Mol-A, Mol-B, Mol-C and Mol-D, respectively) are significantly higher than those of Mn1 (14.6, 17.7, 22.2 ˚ and 15.5 A2), supporting the high mobility of Mn2 in the enzyme The displacement of Mn2 does not affect greatly the overall structure of the subunit The small movement of His281 causes side-chain conformational changes of neighboring Phe280 and Leu255, but no other significant structural differences between subunits of the AD- and BC-forms were found It is unclear why the AD-form is found in Mol-A ⁄ Mo-D and the BC-form in Mol-B ⁄ Mol-C Complex structure of S329K with the linear conformation substrate In previously reported structures of P stutzeri l-RhI in complexes with l-rhamnose and d-allose, there was some ambiguity in the electron density of the bound substrate, and it was difficult to discuss the precise conformation of the substrate [7] These X-ray structures and the structural comparison with Actinopla1048 nes missouriensis d-XI complexed with d-sorbose [19] showed that the substitution of Ser329 with Lys is effective in increasing the attractive interactions between a substrate and the enzyme without any spatial change of the other amino acid residues at the catalytic site, because A missouriensis d-XI has inherently Lys as a corresponding residue to Ser329, directing its side-chain group to the substrate We prepared a mutant form through the substitution of Ser329 with Lys (S329K), and successfully determined the structure of its complexes, S329K–d-psicose (ketose) and S329K–l-rhamnose (aldose) As expected, the substituted Lys forms a hydrogen bond with the substrate, stabilizing the complex The enzymatic activity of S329K is 2% of that of the wild-type enzyme The catalytic site structure of S329K–d-psicose is shown in Fig 4A, with the electron density of the bound d-psicose Clear electron density gave the precise conformation of d-psicose, as indicated in Table O1, O2 and O3 of d-psicose strongly coordinate with ˚ Mn1 and Mn2 with distances of 2.0–2.3 A, instead of W3, W2 and W1 in the AD-form (Figs 3, 4A) As a result of the strong metal coordination, two virtual five-membered rings of O1, C1, C2, O2 and Mn2, and of O2, C2, C3, O3 and Mn1, adopt an almost planar ˚ ˚ structure within 0.03 A and 0.1 A, respectively Lys221, Asp327 and Glu219 form a hydrogen bond with O1, O2 and O3, respectively, helping to fix the substrate in the appropriate conformation for the catalytic reaction There are still two water molecules (W4 and W5) coordinating with Mn2, and they too form hydrogen bonds with Asp291 W4 is thought to be a catalytic water molecule responsible for the proton transfer between O1 and O2, because it possibly forms hydrogen bonds with O1 and O2 of the substrate On the opposite side to W4 [re-face side of the carbonyl carbon (C2)], there is no base to transfer a proton between C1 and C2, but a space along the C1–C2 bond surrounded by Trp179, Lys221 and His257 This space is favorable for the hydride-shift between C1 and C2, because C1 and C2 are shielded from solvent access, to prevent a water molecule as a nucleophile attacking the carbonyl carbon The indole ring of Trp179 makes CH–p interaction with H1A on the re-face side, and this may help the formation of a stable hydride ion (H)) Therefore, the presented X-ray structure most probably supports the hydride-shift mechanism for the isomerization reaction of P stutzeri l-RhI As O5 forms a van der Waals’ contact with C2 on the si-face side of C2, H1B cannot shift to C2, showing that l-RhI can strictly produce an aldose with a right-hand configuration at the 2-position in FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS Catalytic mechanism of L-rhamnose isomerase H Yoshida et al A His257 Glu219 Mn2 Asp291 His257 Lys221 Mn1 O3 Ser→Lys329 Mn1 Trp179 H1B O3 Ser→Lys329 O5 H1A O2 W4 Trp179 H1B O1 W5 Asp291 O2 W4 Glu219 Mn2 O1 H1A W5 Lys221 O5 O4 O4 O6 Asp327 Trp57 Trp57 His101 B His101 Lys221 Lys221 His257 His257 Glu219 Mn2 Fig Stereoview of the linear conformation substrate-binding structure of S329K: (A) D-psicose (orange carbon) and (B) L-rhamnose (blue carbon) with a simulated annealing omit map at the 4.0r contour level Selected interactions among amino acid residues, substrates, metal ions and water molecules are indicated by dotted lines Hydrogen atoms involved in a hydrideshift ride on C1 of D-psicose and C2 of L-rhamnose were identified by geometrical calculations O6 Asp327 Glu219 Mn2 O1 O1 H2 H2 W5 Asp291 Asp291 O2 W4 W5 Mn1 O2 W4 Trp179 Ser→Lys329 O4 Trp179 Mn1 O3 O3 Ser→Lys329 O5 O4 O5 Asp327 Asp327 His101 Trp57 His101 Trp57 Table Torsion angles (deg) of the bound substrate ⁄ product O1–C1–C2–C3 D-Psicose L-Rhamnose D-Psicofuranose L-Rhamnulofuranose C1–C2–C3–C4 C2–C3–C4–C5 C3–C4–C5–C6 C4–C5–C6–O6 )173 )159 )173 )178 55 43 96 122 70 168 29 )18 )173 )177 )144 146 64 ) )154 – Fischer’s projection through isomerization from the ketose to aldose His101 forms a hydrogen bond with O4, and Asp327 with O5, to recognize the hydroxyl groups at the 4- and 5-positions of d-psicose Trp57 exhibits hydrophobic interaction with C6 of the substrate, but O6 does not form a hydrogen bond with any amino acid residue This is because the inherent substrate of l-rhamnose is a deoxy-sugar without a hydroxyl group at the 6-position The substituted Lys329 forms hydrogen bonds with O5, Asp327 and W4 The hydrogen bond between Lys329 and the substrate may freeze the conformation of a substrate to stabilize the enzyme– substrate complex The hydrogen bond between the amino group of Lys329 and W4 possibly compensates for the negative charge of W4 as a hydroxyl ion, leading to the inactivation of W4 as a catalytic water molecule This may be why the enzymatic activity of S329K is 2% that of the wild-type enzyme The catalytic site structure of S329K–l-rhamnose, with the electron density of the bound l-rhamnose, is shown in Fig 4B Owing to H2, the virtual five-membered ring of O1, C1, C2, O2 and Mn2, and ⁄ or of O2, C2, C3, O3 and Mn1, does not form a planar FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS 1049 Catalytic mechanism of L-rhamnose isomerase H Yoshida et al structure, and the distances from O1, O2 and O3 to ˚ Mn1 and Mn2 are relatively long (2.3–2.6 A) compared with those found in S329K–d-psicose However, the interactions between O1, O2 and O3 of l-rhamnose and the enzyme, including metal ions, are almost identical to those found in the bound d-psicose H2 is located between Trp179 and His257 As there is a space on the re-face side of the carbonyl carbon (C1) surrounded by Trp179, Lys221 and His257, H2 can easily attack C1 from the re-face side on a hydride-shift The torsion angle around the C3–C4 bond of the bound substrate differs between l-rhamnose and d-psicose (Table 1), because the 4- and 5-positions in l-rhamnose have the opposite configuration to those in d-psicose The bound l-rhamnose forms hydrogen bonds between O4 and Asp327, and O5 and His101, whereas the bound d-psicose does so between O4 and His101, and O5 and Asp327 This means that P stutzeri l-RhI can recognize substrates with different configurations of C4 and C5 by using His101 and Asp327, and vice versa The substituted Lys329 forms hydrogen bonds with O4, Asp327 and W4, and Trp57 shows hydrophobic interaction with the substrate, as found in the complex with the bound d-psicose Trp57 more effectively recognizes the hydrophobic methyl group (C6) of l-rhamnose Complex structure of D327 with the ring conformation substrate In the structure of S329K–d-psicose, O5 and C2 of d-psicose form a van der Waals’ contact, and Asp327 is located within hydrogen bond-forming distance of both O2 and O5, suggesting that Asp327 acts as an acid–base catalyst in the ring opening of d-psicose We prepared a mutant form with the substitution of Asp327 with Asn (D327N), and tried to solve the X-ray structure of the complex in which a substrate with a ring conformation binds to the enzyme As expected, no enzymatic activity of D327N could be detected As shown in Fig 5A, d-psicose with a ring conformation was successfully found at the catalytic site of A Lys221 Lys221 Mn2 Mn2 W5 W5 O1 Glu219 Asp291 W4 Mn1 O5 O4 O6 Trp57 O1 Glu219 O2 W4 Mn1 O5 Trp179 O3 Asp→Asn327 Asp291 O2 Trp179 O3 O4 O6 Asp→Asn327 Trp57 His101 His101 B Lys221 Mn2 Mn2 O1 Glu219 W5 Asp291 Lys221 W4Mn1 O2 O5 O1 Glu219 W5 Asp291 W4 Mn1 Trp179 O5 O3 O4 Trp179 O3 O4 Asp→Asn327 Asp→Asn327 His101 Trp57 1050 O2 His101 Trp57 Fig Stereoview of the ring conformation substrate-binding structure of D327N: (A) D-psicose (orange carbon) and (B) L-rhamnulose (blue carbon) with a simulated annealing omit map at the 4.0r contour level Selected interactions among amino acid residues, substrate, metal ions and water molecules are indicated by dotted lines FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS H Yoshida et al D327N, and its precise conformation is indicated in Table The bound d-psicose adopts a five-membered ring structure with a-anomer (a-d-psicofuranose), having a half-chair conformation; C2, C3, C5 and O5 ˚ ˚ form a plane within 0.08 A, and C4 deviates by 0.47 A from the plane O1, O2 and O3 coordinate with Mn1 ˚ and Mn2 at distances of 2.0–2.6 A Lys221, Glu219 and His101 form hydrogen bonds with O1, O3 and O4, respectively O6 does not form a hydrogen bond with an amino acid residue, as found in S329K–d-psicose a-d-Psicofuranose is sandwiched between Trp57 and Trp179, and the indole ring of Trp179 forms a nicely stacking interaction with a furanose ring It is difficult to identify the NE and OE atoms of the substituted Asn327 at the present resolution The torsion angle around the CB–CG bond of 43° in Asn327 is significantly different from that of 8° found in Asp327 of the wild-type enzyme, and the coordina˚ ˚ tion distance to Mn1 becomes 2.6 A from 2.2 A If OE of Asn327 coordinates with Mn1, the lone pair electrons of OE are not directed to Mn1, but, if NE does, it can direct its lone pair electrons to Mn1 In addition, the opposite atom to the metal coordination of Asn327 forms a hydrogen bond with a secondary amino group of Trp57 Thus, we determined the positions of NE and OE atoms, as shown in Fig 5A The amino group (NE) of Asn327 forms hydrogen bonds possibly with O2 and O5 of a substrate to prevent ring opening of the substrate and to help stabilize the enzyme–substrate complex Moreover, Asp327 at its original position is expected to be located within hydrogen bond-forming distance of O2 and O5, acting as an acid–base catalyst for ring opening of a substrate To elucidate the six-membered ring (pyranose ring) structure of l-rhamnose, we also carried out X-ray structure determination of D327N–l-rhamnose However, unexpectedly, a product, l-rhamnulose with a five-membered ring structure (b-l-rhamnulofuranose), was found at the catalytic site of D327N, as shown in Fig 5B This means that D327N can achieve the ring opening of l-rhamnose followed by the isomerization of aldose to ketose After the production of l-rhamnulose, the O5 nucleophile attacks C2 (carbonyl carbon) to form a hemiacetal, b-l-rhamnulofuranose As the enzymatic activity of D327N towards l-rhamnose could not be detected with a cystein–carbazole assay measuring the amount of ketose produced [5,21], hydrogen bonds formed by Asn327 could allow a product to anchor at the catalytic site The bound b-l-rhamnulofuranose adopts a halfchair conformation, but the ring conformation is dif- Catalytic mechanism of L-rhamnose isomerase ferent from that of the bound a-d-psicofuranose, as shown in Fig 5B and Table In b-l-rhamnulofura˚ nose, C2, C3, C4 and O5 form a plane within 0.11 A, ˚ from the plane Owing to and C5 deviates by 0.30 A this ring puckering, b-l-rhamnulofuranose shows almost identical interaction with the enzyme as the bound a-d-psicofuranose, in spite of the different configurations of C4 and C5 l-rhamnose and d-allose are expected to adopt the six-membered ring structures with 1C4 and 4C1 chair conformations, respectively, because the C6 group should be equatorial (Fig 1A) Indeed, their crystal structures showed a-l-rhamnopyranose with a 1C4 chair conformation [22] and b-d-allopyranose with a C1 chair conformation [23] To elucidate how the enzyme recognizes a variety of pyranose ring conformations, the modeling of plausible pyranose-bound structures was performed by the least-squares fitting of O1, O2, O3, O4 and O5 between the bound a-d-psicofuranose and b-d-allopyranose, and between the bound b-l-rhamnulofuranose and a-l-rhamnopyranose, as shown in Fig In both models, the bound substrate is located in the hydrophobic pocket formed by Trp57, Phe131, Trp179 and Phe66* from Mol-B without any steric hindrance from amino acid residues of the enzyme Trp179 nicely stacks with a pyranose ring O2 and O3 coordinate with Mn1, but O1 is unlikely to coordinate with Mn2 in the AD-form because O1 of b-d-allopyranose and ⁄ or C1 of a-l-rhamnopyranose is too close to Mn2 Thus, a substrate with a pyranose ring is thought to bind to the catalytic site in the BC-form, although O1 of the substrate is not within coordination distance of Mn2 in the BC-form Asp327 cannot form a hydrogen bond with O1 and O5 of the substrate, suggesting that it is not involved in the opening of a pyranose ring As W4 possibly forms hydrogen bonds with O1 and O5 of both substrates, it may act as an acid–base catalyst in ring opening This is supported by the finding that the complex structure of D327N–l-rhamnulose (furanose) was obtained by incubating D327N with l-rhamnose (pyranose) The hydrophobic pocket recognizing the sugar seems to be able to accept various sugar ring conformations, for example pyranose and ⁄ or furanose ring, the 1C4 and ⁄ or 4C1 chair conformation, and a- and ⁄ or b-anomer For isomerization to occur, a substrate first needs to coordinate Mn1 with O2 and O3 on the same side of the sugar ring, and the enzyme may strictly recognize the configuration of the 2- and 3-positions at this stage However, other anomers of furanoses, b-d-psicofuranose and a-l-rhamnulofuranose, in which O2 and O3 are on the opposite side of the sugar ring to FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS 1051 Catalytic mechanism of L-rhamnose isomerase H Yoshida et al Lys221 Lys221 Asp289 His257 Asp289 Phe66* His257 Phe66* Trp179 Trp179 Mn2 Mn2 O1 O2 Glu219 W4 Asp254 O2 O5 Mn1 O3 Asp291 Asp291 Phe131 O4 O6 O1 O2 W4 Asp254O2 O5 Mn1 O3 Glu219 O4 O6 Phe131 His281 His281 Asp327 Asp327 His101 His101 Trp57 Trp57 Fig Stereoview of the modeling structure of pyranose ring conformation substrates, D-allose (orange carbon) and L-rhamnose (blue carbon), binding to the catalytic site of P stutzeri L-RhI (Mol-A), with Mn2+ (black) Phe66* shown by green carbons belongs to Mol-B Two Mn2+ ions in the BC-form are also superimposed by gray spheres A B H O H O Mn2 Mn O OH H H C H O H HO– O H H Mn HO– O Mn1 Mn O AD-form – O D OH Mn H O O H – O H H O H H O – O – BC-form O H H O Mn1 Mn O O O Asp327 BC-form H O H OH O OH – O Asp291 Asp291 Trp179 O OH OH – AD-form H O O H H Mn Mn1 O O OH Asp327 F Mn2 Mn O H H HN OH Asp291 Asp327 E H OH Mn2 H – OH Mn1 Mn O AD-form Asp327 OH O O – Asp291 H O O Mn2 Mn O OH Mn1 Mn Asp291 H O H OH Mn2 O Mn2 Mn –O HN H O H OH Mn Mn1 OH H O OH – O Asp291 AD-form H Trp179 OH OH Asp327 Asp327 Fig The proposed catalytic reaction mechanism of P stutzeri L-RhI The catalytic water molecule (W4) is highlighted by a red ellipsoid Yellow and green circles represent metal ions in two metal coordination forms, AD- and BC-forms, respectively each other, seem to be poorly recognized as a substrate by the enzyme The catalytic reaction mechanism From the results of the X-ray structural study, we deduced the catalytic reaction mechanism of P stutzeri l-RhI, as shown in Fig 1052 Before the binding of a substrate, the catalytic site is expected to interconvert between two forms (AD- and BC-forms) with the displacement of Mn2 (Fig 7A, D) In the BC-form, a catalytic water molecule (W4) is activated to become a hydroxyl ion (OH)) by coordinating with Mn2 Asp291 helps the activation of W4 by removing a proton, because the pKa value of Asp291 is expected to be increased in the AD-form FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS Catalytic mechanism of L-rhamnose isomerase H Yoshida et al Table X-ray data collection and refinement statistics Rmerge = RR|Ii ) | ⁄ R Values in parentheses are of the high-resolution bin ˚ ˚ ˚ ˚ (1.86–1.80 A for wild-type–Mn, 1.66–1.60 A for S329K–D-psicose, 2.02–1.95 A for S329K–L-rhamnose, 1.97–1.90 A for D327N–D-psicose and ˚ 1.76–1.70 A for D327N–L-rhamnulose) Data set Beam line ˚ Wavelength (A) Temperature (K) ˚ Resolution (A) No of measured refs No of unique refs Completeness (%) Rmerge Io ⁄ r(Io) Space group Cell dimensions ˚ A (A) ˚ B (A) ˚ C (A) b (deg) ˚ Resolution (A) Completeness (%) Rfactor Rfree ˚ Rmsd bond lengths (A) Rmsd bond angles (deg) No of amino acids No of solvent molecules No of ligand Mn2+ Sugar Ramachandran plot (%)favored ⁄ additional ˚ B-factor (A2) Protein Sugar Mn1 (Structural metal ion) Mn2 (Catalytic metal ion) Solvent PDB code Mn2+-bound form S329K–D-psicose S329K–L-rhamnose D327N–D-psicose D327N–L-rhamnulose KEK PF BL-6A 0.978 100 50–1.80 560 757 150 765 99.9 (99.9) 0.065 (0.284) 11.0 (5.3) P21 KEK PF BL-17A 1.0000 100 50–1.60 766 234 208 180 99.7 (99.2) 0.045 (0.124) 19.9 (10.7) P21 SPring-8 BL-38B1 1.0000 100 50–1.95 370 304 112 393 97.2 (96.5) 0.061 (0.246) 11.0 (3.1) P21 KEK PF BL-6A 0.978 100 50–1.90 441 658 128 987 96.7 (96.8) 0.083 (0.347) 8.8 (3.4) P21 KEK PF BL-6A 0.978 100 50–1.70 698 734 185 271 100.0 (99.9) 0.064 (0.335) 11.5 (4.1) P21 78.647 105.115 102.526 102.799 43.36–1.80 96.6 0.165 0.197 0.005 1.2 Mol-A 420 Mol-B 418 Mol-C 418 Mol-D 418 1770 90.9 ⁄ 8.5 74.626 104.643 108.424 107.349 46.39–1.60 98.8 0.163 0.184 0.004 1.2 Mol-A 421 Mol-B 420 Mol-C 426 Mol-D 418 2101 90.9 ⁄ 8.5 75.018 104.842 109.936 106.917 47.01–1.96 94.0 0.183 0.225 0.005 1.1 Mol-A 421 Mol-B 420 Mol-C 426 Mol-D 426 1259 90.9 ⁄ 8.4 74.762 104.732 115.035 108.242 42.15–1.90 92.3 0.176 0.211 0.005 1.1 Mol-A 421 Mol-B 421 Mol-C 428 Mol-D 419 1436 90.5 ⁄ 8.9 74.700 104.634 115.096 108.142 42.12–1.70 96.2 0.165 0.188 0.005 1.2 Mol-A 421 Mol-B 421 Mol-C 428 Mol-D 419 1899 91.1 ⁄ 8.4 13.1 10.8 16.1 13.6 12.3 21.8 3ITV 20.7 37.5 25.3 32.0 24.8 3ITT 14.5 26.2 18.4 17.3 20.8 3ITO 14.7 13.5 11.4 10.0 24.8 3ITL 17.5 29.3 22.3 3ITX compared with the BC-form, where Asp291 coordinates directly with Mn2 to stabilize its ionization state From the presented X-ray structures, it is difficult to determine whether or not W4 is a hydroxyl ion (OH)) However, high-resolution X-ray crystallography and neutron diffraction studies of d-XI clearly show that the catalytic water molecule is activated as a hydroxyl ion (OH)) concurrently with the displacement of a catalytic metal ion [16] It is probable that W4 is activated through the metal-coordinated structural change from the BC- to the AD-form in l-RhI A ketose with a furanose ring binds to the AD-form (Fig 7B), and O1, O2 and O3 coordinate with Mn1 and Mn2 Asp327 acts as an acid–base catalyst in ring opening, helping to transfer a proton from O2 to O5 After the ring has been opened, the catalytic water molecule (W4) mediates the transfer of a proton from O1 to O2, and a hydride (H1A), shielded by Trp179 from solvent access, attacks C2, producing an aldose with a hydroxyl group (O2) having a right-handed configuration in Fischer’s projection (Fig 7C) An aldose with a pyranose ring probably binds to the BC-form (Fig 7E), because O1 seems to be too close to Mn2 in the ADform, supposing that O2 and O3 coordinate with Mn1, FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS 1053 Catalytic mechanism of L-rhamnose isomerase H Yoshida et al as shown in Fig The catalytic water molecule (W4) may act as an acid–base catalyst in ring opening, after which Mn2 moves to a position in the AD-form to activate W4, and mediates the transfer of a proton from O2 to O1, to permit a hydride (H2) to attack C1, giving a ketose (Fig 7F) Interconversion between the two forms (AD- and BC-forms) with the displacement of Mn2 is very important to the recognition of both pyranose and furanose ring substrates In the Mn2+-bound form, the structural energy of the two forms is thought to be almost equal, allowing easy interconversion between them, because the two conformers were found equally in the X-ray structure However, for Cu2+-, Co2+and Zn2+-bound enzymes, the AD-form may be more stable than the BC-form, and interconversion does not occur easily This may be why P stutzeri l-RhI has maximum enzymatic activity in the presence of Mn2+ Based on the structure of S olivochromogenes d-XI complexed with a-d-glucopyranose, which is a suitable substrate, as is d-xylose, a pair of adjacent residues (His53 and Asp56) was proposed to be involved in ring opening [16] However, there is no corresponding pair of His and Asp residues in P stutzeri l-RhI, only a His residue (His101) This may be a result of a difference in metal coordination with a ring conformation between d-XI and l-RhI The two hydroxyl groups coordinating with Mn1 must be in the right-handed configuration in Fischer’s projection, otherwise steric hindrance occurs between the bound substrate and Trp179 (Figs 4, 5) In d-XI, O2 and O4 in the righthanded configuration coordinate with Mn1, and O1 and O2 coordinate with Mn2 when the hydride-shift occurs However, in the binding of a ring conformation, O3 and O4 coordinate with Mn1, because O2 and O4 in a ring conformation are too far from each other to coordinate with Mn1 Consequently, O5 of a ring conformation substrate is close to His53 (His101 in l-RhI) His53 attaches a proton to O5 and the water molecule forming a hydrogen bond to Asp56 removes a proton from O1, which is followed by ring opening On changing to a linear conformation, the metal coordination structure changes drastically so that O2 (instead of O3) coordinates to Mn1, with O4 However, in l-RhI, the bound substrate with a ring conformation inherently coordinates to Mn1 with O2 and O3, and the metal coordination structure is not changed as much by ring opening O5 of the ring conformation is close to Asp327 (furanose) or W4 (pyranose) not His101 Therefore, it could be that Asp327 and the catalytic water molecule (W4) are responsible for the ring opening of furanose and pyranose, respectively, in l-RhI 1054 Materials and methods Site-directed mutagenesis and purification of the enzyme Mutant forms of P stutzeri l-RhI were prepared using recombinant E coli JM 109 cells Site-directed mutagenesis was carried out using a plasmid, pOI-02, encoding the l-RhI gene [6] and the Quick Change Kit (Stratagene, La Jolla, CA, USA) for the construction of S329K and D327N The oligonucleotides used were: for S329K: forward primer, 5¢-GATCGACCAGAAGCACAACGTC AC-3¢; reverse primer, 5¢-GTGACGTTGTGCTTCTGGTC GATC-3¢; for D327N: forward primer, 5¢-CCACATGAT CAACCAGTCGC-3¢; reverse primer, 5¢-GCGACTGGTT GATCATGTGG-3¢ The mutant forms were purified in the same way as wild-type P stutzeri l-RhI, as reported previously [7] The enzymatic activity (Vmax for l-rhamnose as a substrate) of the mutant enzymes was measured by a cystein–carbazole assay, detecting the amount of ketose (l-rhamnulose) produced, using the calorimetric method [5,21] Protein preparation for crystallization The purified enzyme was dialyzed against a buffer solution (5 mm Tris ⁄ HCl and mm EDTA, pH 8.0) to remove bound metal ions retained from the culture medium After the buffer had been replaced with mm Hepes, pH 8.0, by dialysis to remove EDTA, the enzyme solution was concentrated to 1–2 mgỈmL)1 to prepare P stutzeri l-RhI in metal-free form For the preparation of the enzyme in metal-bound form, the enzyme solution was incubated in the presence of mm MnCl2, CuSO4, CoCl2 or ZnSO4 for 20 at 20 °C Each sample was concentrated to 20 mgỈmL)1 with a Microcon YM-10 filter (Millipore, Billerica, MA, USA) Crystals of P stutzeri l-RhI in metal-free and metal-bound forms, and mutant enzymes, were grown by the vapor diffusion method using a protein solution (20 mgỈmL)1) and a reservoir solution [7–8% (w ⁄ v) polyethylene glycol 20 000 and 50 mm Mes buffer (pH 6.3)] Crystals of complexes with d-psicose and ⁄ or l-rhamnose were obtained by a soaking method, and incubation for 24–31 h with an additional 0.5 lL of 100 mm substrate solution Data collection and structural determination Crystals were flash-cooled in liquid nitrogen at 100 K and X-ray diffraction data were collected on the BL-6A and BL-17A beam lines in the Photon Factory (Tsukuba, Japan), and the BL38B1 beam line in SPring-8 Diffraction data were processed using the programs hkl2000 [24] and the ccp4 program suite [25] Data collection statistics and scaling results are listed in Table The initial phases were FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS H Yoshida et al determined by a molecular replacement method with the program molrep [26] in the ccp4 program suite, using the structure of wild-type P stutzeri l-RhI [Protein Data Bank (PDB) code 2HCV] as a probe model [7] Further model building was performed with the programs coot [27] in the ccp4 program suite, and x-fit [28] in the xtalview program system [29], and the structure was refined using the programs refmac5 [30] and cns [31] Water molecules were gradually introduced if the peaks above 3.5r in the (Fo ) Fc) electron density map were in the range of a hydrogen bond In a Ramachandran plot [32], the number of residues in most favored regions was determined by the program procheck [33] Refinement statistics are listed in Table Data collection and refinement statistics of the enzymes in metal-free and Cu2+-, Co2+- and Zn2+-bound forms, and their metal-binding structures, are given in Table S1 with the PDB code Figures 2–4 were illustrated by the program pymol [34] Modeling of plausible pyranose-bound structures From the Cambridge Structure Database (CSD) [35], the atomic coordinates of b-d-allopyranose (CSD record COKBIN) and a-l-rhamonopyranose (CSD record RHAMAH12) were obtained In the structure of b-d-allopyranose, the torsion angle of C4–C5–C6–O6 was changed from 45° to 180°, because the bound a-d-psicofuranose has a torsion angle of 154° (trans conformation) to avoid unusual short contacts with amino acid residues The structure was further optimized by mopac2002 [36] in the Winmopac system (Fujitsu Ltd., Japan) [37] using the AM1 Hamiltonian The optimized structures were added to the catalytic site of the enzyme by the least-squares fitting of O1, O2, O3, O4 and O5 with the bound a-d-psicofuranose and ⁄ or b-lrhamnulofuranose in the enzyme using a locally developed program, because the hydroxyl groups of a substrate are expected to mainly interact with the enzyme Figure was illustrated by the program pymol [33] PDB accession numbers The atomic coordinates and structure factors of P stutzeri l-RhI in Mn2+-bound form (PDB code 3ITX), S329K– d-psicose (PDB code 3ITV), S329K–l-rhamnose (PDB code 3ITT), D327N–d-psicose (PDB code 3ITO) and D327N– l-rhamnulose (PDB code 3ITL) have been deposited in the Protein Data Bank, Research Collaboration for Structural Bioinformatics, Rutgers University, New Brunswick, NJ, USA Acknowledgements This study was supported in part by the National Project on Protein Structural and Functional Analyses, Catalytic mechanism of L-rhamnose isomerase the ‘intellectual-cluster’, and a Grant-in-Aid for Young Scientist (B) (19770085) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by the fund for Young Scientists 2007–8 and Characteristic Prior Research 2009 from Kagawa University This research was performed with the approval of the Photon Factory Advisory Committee and the National Laboratory for High Energy Physics, and SPring-8, Japan References Wilson DM & Ajl S (1957) Metabolism of l-rhamnose by Escherichia coli I l-Rhamnose isomerase J Bacteriol 73, 410–414 Moralejo P, Egan SM, Hidalgo E & Aguilar J (1993) Sequencing and characterization of a gene cluster encoding the enzymes for l-rhamnose metabolism in Escherichia coli J Bacteriol 175, 5585–5594 Korndorfer IP, Fessner WD & Matthews BW (2000) ă The structure of rhamnose isomerase from Escherichia coli and its relation with xylose 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version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 1045–1057 ª 2010 The Authors Journal compilation ª 2010 FEBS 1057 ... The catalytic reaction mechanism From the results of the X-ray structural study, we deduced the catalytic reaction mechanism of P stutzeri l-RhI, as shown in Fig 1052 Before the binding of a... overall catalytic reaction mechanism of P stutzeri l-RhI, we report here the X-ray structures of an unliganded P stutzeri l-RhI in the Mn2+-bound form, and two inactive mutant forms of P stutzeri. .. the ring conformation is dif- Catalytic mechanism of L-rhamnose isomerase ferent from that of the bound a-d-psicofuranose, as shown in Fig 5B and Table In b-l-rhamnulofura˚ nose, C2, C3, C4 and