Catalytic Proton Donor Research Articles

A Novel Subfamily GH13_46 of the α-Amylase Family GH13 Represented by the Cyclomaltodextrinase from Flavobacterium sp. No. 92.

In the CAZy database, the α-amylase family GH13 has already been divided into 45 subfamilies, with additional subfamilies still emerging. The presented in silico study was undertaken in an effort to propose a novel GH13 subfamily represented by the experimentally characterized cyclomaltodxtrinase from Flavobacterium sp. No. 92. Although most cyclomaltodextrinases have been classified in the subfamily GH13_20. This one has not been assigned any GH13 subfamily as yet. It possesses a non-specified immunoglobulin-like domain at its N-terminus mimicking a starch-binding domain (SBD) and the segment MPDLN in its fifth conserved sequence region (CSR) typical, however, for the subfamily GH13_36. The searches through sequence databases resulted in collecting a group of 108 homologs forming a convincing cluster in the evolutionary tree, well separated from all remaining GH13 subfamilies. The members of the newly proposed subfamily share a few exclusive sequence features, such as the "aromatic" end of the CSR-II consisting of two well-conserved tyrosines with either glycine, serine, or proline in the middle or a glutamic acid succeeding the catalytic proton donor in the CSR-III. Concerning the domain N of the representative cyclomaltodextrinase, docking trials with α-, β- and γ-cyclodextrins have indicated it may represent a new type of SBD. This new GH13 subfamily has been assigned the number GH13_46.

Structures of lactaldehyde reductase, FucO, link enzyme activity to hydrogen bond networks and conformational dynamics.

A group-III iron containing 1,2-propanediol oxidoreductase, FucO, (also known as lactaldehyde reductase) from Escherichia coli was examined regarding its structure-dynamics-function relationships in the catalysis of the NADH-dependent reduction of (2S)-lactaldehyde. Crystal structures of FucO variants in the presence or absence of cofactors have been determined, illustrating large domain movements between the apo and holo enzyme structures. Different structures of FucO variants co-crystallized with NAD+ or NADH together with substrate further suggest dynamic properties of the nicotinamide moiety of the coenzyme that are important for the reaction mechanism. Modelling of the native substrate (2S)-lactaldehyde into the active site can explain the stereoselectivity exhibited by the enzyme, with a critical hydrogen bond interaction between the (2S)-hydroxyl and the side-chain of N151, as well as the previously experimentally demonstrated pro-(R) selectivity in hydride transfer from NADH to the aldehydic carbon. Furthermore, the deuterium kinetic isotope effect of hydride transfer suggests that reduction chemistry is the main rate-limiting step for turnover which is not the case in FucO catalysed alcohol oxidation. We further propose that a water molecule in the active site - hydrogen bonded to a conserved histidine (H267) and the 2'-hydroxyl of the coenzyme ribose - functions as a catalytic proton donor in the protonation of the product alcohol. A hydrogen bond network of water molecules and the side-chains of amino acid residues D360 and H267 links bulk solvent to this proposed catalytic water molecule.

Insights to the Structural Basis for the Stereospecificity of the Escherichia coli Phytase, AppA.

AppA, the Escherichia coli periplasmic phytase of clade 2 of the histidine phosphatase (HP2) family, has been well-characterized and successfully engineered for use as an animal feed supplement. AppA is a 1D-6-phytase and highly stereospecific but transiently accumulates 1D-myo-Ins(2,3,4,5)P4 and other lower phosphorylated intermediates. If this bottleneck in liberation of orthophosphate is to be obviated through protein engineering, an explanation of its rather rigid preference for the initial site and subsequent cleavage of phytic acid is required. To help explain this behaviour, the role of the catalytic proton donor residue in determining AppA stereospecificity was investigated. Four variants were generated by site-directed mutagenesis of the active site HDT amino acid sequence motif containing the catalytic proton donor, D304. The identity and position of the prospective proton donor residue was found to strongly influence stereospecificity. While the wild-type enzyme has a strong preference for 1D-6-phytase activity, a marked reduction in stereospecificity was observed for a D304E variant, while a proton donor-less mutant (D304A) displayed exclusive 1D-1/3-phytase activity. High-resolution X-ray crystal structures of complexes of the mutants with a non-hydrolysable substrate analogue inhibitor point to a crucial role played by D304 in stereospecificity by influencing the size and polarity of specificity pockets A and B. Taken together, these results provide the first evidence for the involvement of the proton donor residue in determining the stereospecificity of HP2 phytases and prepares the ground for structure-informed engineering studies targeting the production of animal feed enzymes capable of the efficient and complete dephosphorylation of dietary phytic acid.

Dissecting the low catalytic capability of flavin-dependent halogenases

Although flavin-dependent halogenases (FDHs) are attractive biocatalysts, their practical applications are limited because of their low catalytic efficiency. Here, we investigated the reaction mechanisms and structures of tryptophan 6-halogenase (Thal) from Streptomyces albogriseolus using stopped-flow, rapid-quench flow, quantum/mechanics molecular mechanics calculations, crystallography, and detection of intermediate (hypohalous acid [HOX]) liberation. We found that the key flavin intermediate, C4a-hydroperoxyflavin (C4aOOH-FAD), formed by Thal and other FDHs (tryptophan 7-halogenase [PrnA] and tryptophan 5-halogenase [PyrH]), can react with I−, Br−, and Cl− but not F− to form C4a-hydroxyflavin and HOX. Our experiments revealed that I− reacts with C4aOOH-FAD the fastest with the lowest energy barrier and have shown for the first time that a significant amount of the HOX formed leaks out as free HOX. This leakage is probably a major cause of low product coupling ratios in all FDHs. Site-saturation mutagenesis of Lys79 showed that changing Lys79 to any other amino acid resulted in an inactive enzyme. However, the levels of liberated HOX of these variants are all similar, implying that Lys79 probably does not form a chloramine or bromamine intermediate as previously proposed. Computational calculations revealed that Lys79 has an abnormally lower pKa compared with other Lys residues, implying that the catalytic Lys may act as a proton donor in catalysis. Analysis of new X-ray structures of Thal also explains why premixing of FDHs with reduced flavin adenine dinucleotide generally results in abolishment of C4aOOH-FAD formation. These findings reveal the hidden factors restricting FDHs capability which should be useful for future development of FDHs applications.

Identification and characterization of a novel bifunctional cellulase/hemicellulase from a soil metagenomic library.

Microbes, especially the uncultured microbes, have been considered as an important resource for discovery of novel cellulases. In this study, a novel bifunctional cellulase/hemicellulase (ZFYN184) was identified by functional screening of a soil metagenomic library. Sequence analysis indicated that ZFYN184 shared at best 39% identity with glycoside hydrolase family 44 (GH44) proteins and contained a glutamic acid residue at 235 acting as the catalytic proton donor in hydrolysis of polysaccharides. The recombinant ZFYN184 was expressed in Escherichia coli BL21 (DE3), and the biochemical profiles of the enzyme, including optimum pH and temperature, pH and thermal stabilities, tolerance to various additives, and substrate specificity, were determined. ZFYN184 possessed strong endo-β-1,4-glucanase and endo-1,4-β-mannanase activities, as well as weak xylanase activity, while all these hydrolytic activities were derived from a single catalytic domain in this GH44 enzyme. KEY POINTS: • Discovery a novel bifunctional glycosyl hydrolase from uncultured microorganism. • ZFYN184 contains a single catalytic domain belonged to GH44.

In silico analysis of the \u03b1-amylase family GH57: eventual subfamilies reflecting enzyme specificities

Glycoside hydrolases (GHs) have been classified in the CAZy database into 153 GH families. Currently, there might be four α-amylase families: the main family GH13, the family GH57 with related GH119 and, eventually, also GH126. The family GH57 was established in 1996 as the second and smaller α-amylase family. In addition to α-amylase, it contains 4-α-glucanotransferase, α-glucan branching enzyme, amylopullulanase, dual-specificity amylopullulanase–cyclomaltodextrinase, non-specified amylase, maltogenic amylase and α-galactosidase. The family GH57 enzymes employ the retaining reaction mechanism, share five typical conserved sequence regions and possess catalytic (β/α)7-barrel succeeded by a four-helix bundle with the catalytic machinery consisting of catalytic nucleophile and proton donor (glutamic acid and aspartic acid at strands β4 and β7, respectively). The present bioinformatics study delivers a detailed sequence comparison of 1602 family GH57 sequences with the aim to highlight the uniqueness of each enzyme’s specificity and all eventual protein groups. This was achieved by creating the evolutionary tree focused on both the enzyme specificities and taxonomical origin. The substantial increase of numbers of sequences from recent comparisons done more than 5 years ago has allowed to refine the details of the sequence logos for the individual enzyme specificities. The study identifies a new evolutionary distinct group of α-galactosidase-related enzymes with until-now-undefined enzyme specificity but positioned on the evolutionary tree on a branch adjacent to α-galactosidases. The specificity of α-galactosidase is, moreover, the only one of the entire family GH57 for which there is no structural support for the proposal of the proton donor based on sequence analysis. The analysis also suggests a few so-called “like” protein groups related to some family GH57 enzyme specificities but lacking one or both catalytic residues.

Characterization of Thermotoga thermarum DSM 5069 α-Glucuronidase and Synergistic Degradation of Xylan

α-Glucuronidases are capable of breaking down the α-1,2-glycosidic bonds of 4-O-methyl-D-glucuronic acid residues. As an accessory enzyme, α-glucuronidase plays a vital role in xylan degradation. The recombinant α-glucuronidase from Thermotoga thermarum DSM 5069 was heterologously expressed in the Escherichia coli system, purified, and characterized. The purified enzyme exhibited optimal activity toward aldouronic acids at pH 6.5 and 80 °C. It was fairly thermostable and maintained 98% residual activity after incubation at 65 °C for 2.0 h. The kinetic parameters Km, Vmax, and kcat were 3.02 ± 0.16 mM, 88 ± 2 µmol min-1 mg-1, and 117 s-1, respectively. TtAguA had an apparent activation energy of 59.0 kJ/mol. By structure simulation and mutation analyses, Glu288 was identified as the catalytic proton donor, and Asp367 and Glu395 were likely nucleophile bases. The xylan degradation by endoxylanase Xyn10A was enhanced by approximately 10% in the presence of TtAguA.

A New Approach in the Active Site Investigation of an Inverting β-d-Xylosidase from Thermobifida fusca TM51

The catalytic amino acid residues of the β-D-xylosidase (EC 3.2.1.37; GH43), from Thermobifida fusca TM51 (TfBXyl43), were investigated by direct chemical modifications. The pH dependence curves of the kinetic parameters (k(cat) and k(cat)/K(M)) gave pK values for the free enzyme (5.55 ± 0.19; 6.44 ± 0.19), and pK values of for the enzyme-substrate complex (4.85 ± 0.23; 7.60 ± 0.28) respectively, by using an artificial substrate p-nitrophenyl-β-D-xylopyranoside (pNP-Xyl). The detailed inhibition studies demonstrated well the hydrophobic character of the glycon binding site. Carbodiimide-mediated chemical modifications of the enzyme with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) in the presence of glycine methyl ester supports the conclusion that a carboxylate residue can be fundamental in the catalytic process. We have also synthesized and tested N-bromoacetyl-β-D-xylopyranosylamine (NBAXA) for TfBXyl43 as an affinity label, which also inactivated the enzyme irreversible. The pH dependence studies in both cases of inactivation revealed that the modified group is the catalytic proton donor (NBAXA pK(A) = 6.68 ± 0,1; EDAC pK(A) = 7.42 ± 0.22) which displays its essential role in the hydrolytic process. The β-D-xylopyranosylazide as competitive inhibitor protected the enzyme in all cases against the inactivation, suggesting that the chemical modification which has an impact on the activity took place in the active center. Changing of the enzyme conformation was followed by CD spectroscopy, as a result of the NBAXA inactivation. Our study is important because to our knowledge no similar investigations were made in the case of an inverting β-D-xylosidase.

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 (4)C(1) chair to B(O,3) boat distortion that facilitates catalysis in two different ways. It promotes oxidation at C(4) (step 1) by aligning the enzymatic base Tyr(147) with the reactive substrate hydroxyl and it brings the carboxylate group at C(5) 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 Tyr(147) stabilizes the enolate of decarboxylated C(4) keto species ((2)H(1) half-chair) that is then protonated from the Si face at C(5), involving water coordinated by Glu(120). Arg(277), which is positioned by a salt-link interaction with Glu(120), closes up the catalytic site and prevents release of the UDP-4-keto-pentose and NADH intermediates. Hydrogenation of the C(4) keto group by NADH, assisted by Tyr(147) as catalytic proton donor, yields UDP-xylose adopting the relaxed (4)C(1) chair conformation (step 3).

Covalent modifications of the catalytic tyrosine in octahaem cytochromecnitrite reductase and their effect on the enzyme activity

Octahaem cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens (TvNiR), like the previously characterized pentahaem nitrite reductases (NrfAs), catalyzes the six-electron reductions of nitrite to ammonia and of sulfite to sulfide. The active site of both TvNiR and NrfAs is formed bythe lysine-coordinated haem and His, Tyr and Arg residues. The distinguishing structural feature of TvNiR is the presence of a covalent bond between the CE2 atom of the catalytic Tyr303 and the S atom of Cys305, which might be responsible for the higher nitrite reductase activity of TvNiR compared with NrfAs. In the present study, a new modified form of the enzyme (TvNiRb) that contains an additional covalent bond between Tyr303 CE1 and Gln360 CG is reported. Structures of TvNiRb in complexes with phosphate (1.45 Å resolution) and sulfite (1.8 Å resolution), the structure of TvNiR in a complex with nitrite (1.83 Å resolution) and several additional structures were determined. The formation of the second covalent bond by Tyr303 leads to a decrease in both the nitrite and sulfite reductase activities of the enzyme. Tyr303 is located at the exit from the putative proton-transport channel to the active site, which is absent in NrfAs. This is an additional argument in favour of the involvement of Tyr303 as a proton donor in catalysis. The changes in the activity of cytochrome c nitrite reductases owing to the formation of Tyr-Cys and Tyr-Gln bonds may beassociated with changes in the pK(a) value of the catalytic tyrosine.

Identification of Catalytic Residues and Mechanistic Analysis of Family GH82 ι-Carrageenases

Marine polysaccharide degrading enzymes, and iota-carrageenases in particular, have received little attention in the past, although their substrate specificity is of interest for biotechnological applications. This is mostly a consequence of the lack of data about their occurrence in the marine environment. Recent metagenomic data mining and the genome sequencing of a marine bacterium, Zobellia galactanivorans, led to the identification of three new iota-carrageenase genes belonging to the glycoside hydrolase family GH82. The additional sequences helped to identify potential candidate residues as catalytic proton donor and nucleophile. We have identified the catalytic key residues experimentally by site-directed mutagenesis and subsequent kinetic analysis for the iota-carrageenase from Alteromonas fortis CgiA1_Af. The kinetic analyses of the purified mutant enzymes confirm that E245 plays the role of the catalytic proton donor and D247 the general base that activates the catalytic water molecule. The point mutations of three other residues, namely, Q222, H281, and Q310 in A. fortis, located in proximity of the active site also affect the enzyme activity. Our results indicate that E310 plays a role in stabilizing the substrate intermediate conformation, while H281 is involved in substrate binding and appears to be crucial for maintaining the protonation state of the catalytic proton donor E245. The third residue, Q222, that bridges the catalytic water molecule and a chloride ion, plays a crucial role in structuring the water network in the active site of A. fortis iota-carrageenase.

Substitution of Glu122 by Glutamine Revealed the Function of the Second Water Molecule as a Proton Donor in the Binuclear Metal Enzyme Creatininase

Creatininase is a binuclear zinc enzyme and catalyzes the reversible conversion of creatinine to creatine. It exhibits an open–closed conformational change upon substrate binding, and the differences in the conformations of Tyr121, Trp154, and the loop region containing Trp174 were evident in the enzyme–creatine complex when compared to those in the ligand-free enzyme. We have determined the crystal structure of the enzyme complexed with a 1-methylguanidine. All subunits in the complex existed as the closed form, and the binding mode of creatinine was estimated. Site-directed mutagenesis revealed that the hydrophobic residues that show conformational change upon substrate binding are important for the enzyme activity.We propose a catalytic mechanism of creatininase in which two water molecules have significant roles. The first molecule is a hydroxide ion (Wat1) that is bound as a bridge between the two metal ions and attacks the carbonyl carbon of the substrate. The second molecule is a water molecule (Wat2) that is bound to the carboxyl group of Glu122 and functions as a proton donor in catalysis. The activity of the E122Q mutant was very low and it was only partially restored by the addition of ZnCl2 or MnCl2. In the E122Q mutant, kcat is drastically decreased, indicating that Glu122 is important for catalysis. X-ray crystallographic study and the atomic absorption spectrometry analysis of the E122Q mutant–substrate complex revealed that the drastic decrease of the activity of the E122Q was caused by not only the loss of one Zn ion at the Metal1 site but also a critical function of Glu122, which most likely exists for a proton transfer step through Wat2.

Theory and computation show that Asp463 is the catalytic proton donor in human endoplasmic reticulum α-(1→2)-mannosidase I

It has been difficult to identify the proton donor and nucleophilic assistant/base of endoplasmic reticulum α-(1→2)-mannosidase I, a member of glycoside hydrolase Family 47, which cleaves the glycosidic bond between two α-(1→2)-linked mannosyl residues by the inverting mechanism, trimming Man 9GlcNAc 2 to Man 8GlcNAc 2 isomer B. Part of the difficulty is caused by the enzyme’s use of a water molecule to transmit the proton that attacks the glycosidic oxygen atom. We earlier used automated docking to conclusively determine that Glu435 in the yeast enzyme (Glu599 in the corresponding human enzyme) is the nucleophilic assistant. The commonly accepted proton donor has been Glu330 in the human enzyme (Glu132 in the yeast enzyme). However, for theoretical reasons this conclusion is untenable. Theory, automated docking of α- d- 3 S 1-mannopyranosyl-(1→2)-α- d- 4 C 1-mannopyranose and water molecules associated with candidate proton donors, and estimation of dissociation constants of the latter have shown that the true proton donor is Asp463 in the human enzyme (Asp275 in the yeast enzyme).

Pyridine Nucleotide Complexes with Bacillus anthracis Coenzyme A-Disulfide Reductase: A Structural Analysis of Dual NAD(P)H Specificity

We have recently reported that CoASH is the major low-molecular weight thiol in Bacillus anthracis [Nicely, N. I. , Parsonage, D., Paige, C., Newton, G. L., Fahey, R. C., Leonardi, R., Jackowski, S., Mallett, T. C., and Claiborne, A. (2007) Biochemistry 46, 3234-3245], and we have now characterized the kinetic and redox properties of the B. anthracis coenzyme A-disulfide reductase (CoADR, BACoADR) and determined the crystal structure at 2.30 A resolution. While the Staphylococcus aureus and Borrelia burgdorferi CoADRs exhibit strong preferences for NADPH and NADH, respectively, B. anthracis CoADR can use either pyridine nucleotide equally well. Sequence elements within the respective NAD(P)H-binding motifs correctly reflect the preferences for S. aureus and Bo. burgdorferi CoADRs, but leave questions as to how BACoADR can interact with both pyridine nucleotides. The structures of the NADH and NADPH complexes at ca. 2.3 A resolution reveal that a loop consisting of residues Glu180-Thr187 becomes ordered and changes conformation on NAD(P)H binding. NADH and NADPH interact with nearly identical conformations of this loop; the latter interaction, however, involves a novel binding mode in which the 2'-phosphate of NADPH points out toward solvent. In addition, the NAD(P)H-reduced BACoADR structures provide the first view of the reduced form (Cys42-SH/CoASH) of the Cys42-SSCoA redox center. The Cys42-SH side chain adopts a new conformation in which the conserved Tyr367'-OH and Tyr425'-OH interact with the nascent thiol(ate) on the flavin si-face. Kinetic data with Y367F, Y425F, and Y367,425F BACoADR mutants indicate that Tyr425' is the primary proton donor in catalysis, with Tyr367' functioning as a cryptic alternate donor in the absence of Tyr425'.

The role of residues R97 and Y331 in modulating the pH optimum of an insect beta-glycosidase of family 1.

The activity of the digestive beta-glycosidase from Spodoptera frugiperda (Sfbetagly50, pH optimum 6.2) depends on E399 (pKa = 4.9; catalytic nucleophile) and E187 (pKa = 7.5; catalytic proton donor). Homology modelling of the Sfbetagly50 active site confirms that R97 and Y331 form hydrogen bonds with E399. Site-directed mutagenesis showed that the substitution of R97 by methionine or lysine increased the E399 pKa by 0.6 or 0.8 units, respectively, shifting the pH optima of these mutants to 6.5. The substitution of Y331 by phenylalanine increased the pKa of E399 and E187 by 0.7 and 1.6 units, respectively, and displaced the pH optimum to 7.0. From the observed deltapKa it was calculated that R97 and Y331 contribute 3.4 and 4.0 kJ.mol(-1), respectively, to stabilization of the charged E399, thus enabling it to be the catalytic nucleophile. The substitution of E187 by D decreased the pKa of residue 187 by 0.5 units and shifted the pH optimum to 5.8, suggesting that an electrostatic repulsion between the deprotonated E399 and E187 may increase the pKa of E187, which then becomes the catalytic proton donor. In short the data showed that a network of noncovalent interactions among R97, Y331, E399 and E187 controls the Sfbetagly50 pH optimum. As those residues are conserved among the family 1 beta-glycosidases, it is proposed here that similar interactions modulate the pH optimum of all family 1 beta-glycosidases.

Crystal structures of human prostatic acid phosphatase in complex with a phosphate ion and alpha-benzylaminobenzylphosphonic acid update the mechanistic picture and offer new insights into inhibitor design.

The X-ray crystal structure of human prostatic acid phosphatase (PAP) in complex with a phosphate ion has been determined at 2.4 A resolution. This structure offers a snapshot of the final intermediate in the catalytic mechanism and does not support the role of Asp 258 as a proton donor in catalysis. A total of eight hydrogen bonds serve to strongly bind the phosphate ion within the active site. Bound PEG molecules from the crystallization matrix have allowed the identification of a channel within the molecule that likely plays a role in molecular recognition and in macromolecular substrate selectivity. Additionally, the structure of PAP in complex with a phosphate derivative, alpha-benzylaminobenzylphosphonic acid, a potent inhibitor (IC(50) = 4 nM), has been determined to 2.9 A resolution. This structure gives new insight into the determinants of binding hydrophobic ligands within the active site and allows us to explain PAP's preference for aromatic substrates.

Structure of the Humicola insolens cellobiohydrolase Cel6A D416A mutant in complex with a non-hydrolysable substrate analogue, methyl cellobiosyl-4-thio-beta-cellobioside, at 1.9 A.

The enzymatic degradation of cellulose continues to be one of the most important enzyme-catalysed reactions. Glycoside hydrolases from family GH-6 hydrolyse cellulose with inversion of the configuration of the anomeric carbon. Whilst the catalytic proton donor has been clearly identified (Asp226 in Humicola insolens Cel6A), the identification and even the existence of a potential Brønsted base remains unclear. Equally controversial is the role of surface-loop flexibility. Here, the structure of the D416A mutant of the H. insolens cellobiohydrolase Cel6A in complex with a non-hydrolysable thiooligosaccharide methyl cellobiosyl-4-thio-beta-cellobioside at 1.9 A resolution is presented. Substrate distortion in the -1 subsite, to a (2)S(0) skew-boat conformation, is observed, similar to that seen in the analogous Trichoderma reesei Cel6A structure [Zou et al. (1999), Structure, 7, 1035-1045], but the active-centre N-terminal loop of the H. insolens enzyme is found in a more open conformation than described for previous structures.

Engineering water to act as an active site acid catalyst in a soluble fumarate reductase.

The ability of an arginine residue to function as the active site acid catalyst in the fumarate reductase family of enzymes is now well-established. Recently, a dual role for the arginine during fumarate reduction has been proposed [Mowat, C. G., Moysey, R., Miles, C. S., Leys, D., Doherty, M. K., Taylor, P., Walkinshaw, M. D., Reid, G. A., and Chapman, S. K. (2001) Biochemistry 40, 12292-12298] in which it acts both as a Lewis acid in transition-state stabilization and as a Brønsted acid in proton delivery. This proposal has led to the prediction that, if appropriately positioned, a water molecule would be capable of functioning as the active site Brønsted acid. In this paper, we describe the construction and kinetic and crystallographic analysis of the Q363F single mutant and Q363F/R402A double mutant forms of flavocytochrome c(3), the soluble fumarate reductase from Shewanella frigidimarina. Although replacement of the active site acid, Arg402, with alanine has been shown to eliminate fumarate reductase activity, this phenomenon is partially reversed by the additional substitution of Gln363 with phenylalanine. This Gln --> Phe substitution in the inactive R402A mutant enzyme was designed to "push" a water molecule close enough to the substrate C3 atom to allow it to act as a Brønsted acid. The 2.0 A resolution crystal structure of the Q363F/R402A mutant enzyme does indeed reveal the introduction of a water molecule at the correct position in the active site to allow it to act as the catalytic proton donor. The 1.8 A resolution crystal structure of the Q363F mutant enzyme shows a water molecule similarly positioned, which can account for its measured fumarate reductase activity. However, in this mutant enzyme Michaelis complex formation is impaired due to significant and unpredicted structural changes at the active site.

Understanding protein structure-function relationships in Family 47 alpha-1,2-mannosidases through computational docking of ligands.

Family 47 alpha-1,2-mannosidases are crucial enzymes involved in N-glycan maturation in the endoplasmic reticula and Golgi apparati of eukaryotic cells. High-resolution crystal structures of the human and yeast endoplasmic reticulum alpha-1,2-mannosidases have been recently determined, the former complexed with the inhibitors 1-deoxymannojirimycin and kifunensine, both of which bind in its active site in the unusual 1C4 conformation. However, unambiguous identification of the catalytic proton donor and nucleophile involved in glycoside bond hydrolysis was not possible from this structural information. In this work, alpha-D-galactose, alpha-D-glucose, and alpha-D-mannose were computationally docked in the active site in the energetically stable 4C1 conformation as well as in the 1C4 conformation to compare their interaction energetics. From these docked structures, a model for substrate and conformer selectivity based on the dimensions of the active site was proposed. Alpha-D-galactopyranosyl-(1-->2)-alpha-D-mannopyranose, alpha-D-glucopyranosyl-(1-->2)-alpha-D-mannopyranose, and alpha-D-mannopyranosyl-(1-->2)-alpha-D-mannopyranose were also docked into the active site with their nonreducing-end residues in the 1C4 and E4 (representing the transition state) conformations. Based on the docked structure of alpha-D-mannopyranosyl-E4-(1-->2)-alpha-D-mannopyranose, the catalytic acid and base are Glu132 and Glu435, respectively.

The concerted mechanism of photo-induced biprotonic transfer in 7-azaindole dimers: structure, quantum-theoretical analysis, and simultaneity principles.

Six stable dimer models for 7-azaindole (including the classic C(2h) doubly hydrogen-bonded, coplanar, centrosymmetric dimer) are considered to be observable in adiabatic nozzle jet molecular beams. They are analyzed by hybrid density functional theory (DFT), the MP2 ab initio method for the ground electronic state, and the single-excitation configuration interaction (CIS) (over frozen ground state optimized geometries obtained from DFT) excited state calculations, for global potential minima and proton-transfer potential energy curves. Three simultaneity principles are stated: (i) intermolecular coherent excitation molecular exciton simultaneity, (ii) intramolecular acid-base change simultaneity at the pyrrolo-N-H and aza-N proton-donor, proton-acceptor sites, and (iii) intermolecular simultaneity of catalytic proton-donor, proton-acceptor action. It is suggested that the formation of the classic C(2h) dimer of 7-azaindole, which is considered exclusively by previous researchers, can be formed from at least one of the several card-pack hydrogen-bonded dimers in a secondary slower step approaching a microsecond scale, instead of the picosecond events at the supersonic nozzle. It is proposed that the complexity of dimerization modes is the basis of the postexcitation, postionization diverse kinetic isotope results.