Structure and mechanism of a bacterial haloalcohol dehalogenase: a new variation of the short-chain dehydrogenase/reductase fold without an NAD(P)H binding site.

Haloalcohol dehalogenases are bacterial enzymes that catalyze the cofactor-independent dehalogenation of vicinal haloalcohols such as the genotoxic environmental pollutant 1,3-dichloro-2-propanol, thereby producing an epoxide, a chloride ion and a proton. Here we present X-ray structures of the haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1, and complexes of the enzyme with an epoxide product and chloride ion, and with a bound haloalcohol substrate mimic. These structures support a catalytic mechanism in which Tyr145 of a Ser-Tyr-Arg catalytic triad deprotonates the haloalcohol hydroxyl function to generate an intramolecular nucleophile that substitutes the vicinal halogen. Haloalcohol dehalogenases are related to the widespread family of NAD(P)H-dependent short-chain dehydrogenases/reductases (SDR family), which use a similar Ser-Tyr-Lys/Arg catalytic triad to catalyze reductive or oxidative conversions of various secondary alcohols and ketones. Our results reveal the first structural details of an SDR-related enzyme that catalyzes a substitutive dehalogenation reaction rather than a redox reaction, in which a halide-binding site is found at the location of the NAD(P)H binding site. Structure-based sequence analysis reveals that the various haloalcohol dehalogenases have likely originated from at least two different NAD-binding SDR precursors.

Crystallization of quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni: crystals with unique optical properties.

Quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni is a functional electron-transfer protein containing both a haem c and a pyrroloquinoline quinone cofactor. The enzyme has been crystallized at 277 K using polyethylene glycol 6000 as precipitant. The crystals belong to space group C2, with unit-cell parameters a = 98.1, b = 74.3, c = 92.2 A, beta = 105.9 degrees. A native data set with a resolution of 2.44 A resolution has been collected. The approximate orientation of the haem group with respect to the unit-cell axes has been determined from the optical properties of the crystals.

X-ray structure of bovine pancreatic phospholipase A2 at atomic resolution.

Using synchrotron radiation and a CCD camera, X-ray data have been collected from wild-type bovine pancreatic phospholipase A(2) at 100 K to 0.97 A resolution allowing full anisotropic refinement. The final model has a conventional R factor of 9.44% for all reflections, with a mean standard uncertainty for the positional parameters of 0.031 A as calculated from inversion of the full positional least-squares matrix. At 0.97 A resolution, bovine pancreatic phospholipase A(2) reveals for the first time that its rigid scaffolding does not preclude flexibility, which probably plays an important role in the catalytic process. Functionally important regions (the interfacial binding site and calcium-binding loop) are located at the molecular surface, where conformational variability is more pronounced. A cluster of 2-methyl-2,4-pentanediol molecules is present at the entrance of the hydrophobic channel that leads to the catalytic site and mimics the fatty-acid chains of a substrate analogue. Bovine pancreatic phospholipase A(2) at atomic resolution is compared with previous crystallographic structures and with models derived from nuclear magnetic resonance studies. Given the high structural similarity among extracellular phospholipases A(2) observed so far at lower resolution, the results arising from this structural analysis are expected to be of general validity for this class of enzymes.

Crystal structures of intermediates in the dehalogenation of haloalkanoates by L-2-haloacid dehalogenase.

The L-2-haloacid dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic dehalogenation of small L-2-haloalkanoates to their corresponding D-2-hydroxyalkanoates, with inversion of the configuration at the C(2) atom. The structure of the apoenzyme at pH 8 was refined at 1.5-A resolution. By lowering the pH, the catalytic activity of the enzyme was considerably reduced, allowing the crystal structure determination of the complexes with L-2-monochloropropionate and monochloroacetate at 1.7 and 2.1 A resolution, respectively. Both complexes showed unambiguous electron density extending from the nucleophile Asp(8) to the C(2) atom of the dechlorinated substrates corresponding to a covalent enzyme-ester reaction intermediate. The halide ion that is cleaved off is found in line with the Asp(8) Odelta1-C(2) bond in a halide-stabilizing cradle made up of Arg(39), Asn(115), and Phe(175). In both complexes, the Asp(8) Odelta2 carbonyl oxygen atom interacts with Thr(12), Ser(171), and Asn(173), which possibly constitute the oxyanion hole in the hydrolysis of the ester bond. The carboxyl moiety of the substrate is held in position by interactions with Ser(114), Lys(147), and main chain NH groups. The L-2-monochloropropionate CH(3) group is located in a small pocket formed by side chain atoms of Lys(147), Asn(173), Phe(175), and Asp(176). The size and position of the pocket explain the stereospecificity and the limited substrate specificity of the enzyme. These crystallographic results demonstrate that the reaction of the enzyme proceeds via the formation of a covalent enzyme-ester intermediate at the nucleophile Asp(8).

Structure and mechanism of soluble quinoprotein glucose dehydrogenase.

Soluble glucose dehydrogenase (s-GDH; EC 1.1.99.17) is a classical quinoprotein which requires the cofactor pyrroloquinoline quinone (PQQ) to oxidize glucose to gluconolactone. The reaction mechanism of PQQ-dependent enzymes has remained controversial due to the absence of comprehensive structural data. We have determined the X-ray structure of s-GDH with the cofactor at 2.2 A resolution, and of a complex with reduced PQQ and glucose at 1.9 A resolution. These structures reveal the active site of s-GDH, and show for the first time how a functionally bound substrate interacts with the cofactor in a PQQ-dependent enzyme. Twenty years after the discovery of PQQ, our results finally provide conclusive evidence for a reaction mechanism comprising general base-catalyzed hydride transfer, rather than the generally accepted covalent addition-elimination mechanism. Thus, PQQ-dependent enzymes use a mechanism similar to that of nicotinamide- and flavin-dependent oxidoreductases.

Crystallographic and kinetic evidence of a collision complex formed during halide import in haloalkane dehalogenase.

Haloalkane dehalogenase (DhlA) converts haloalkanes to their corresponding alcohols and halide ions. The rate-limiting step in the reaction of DhlA is the release of the halide ion. The kinetics of halide release have been analyzed by measuring halide binding with stopped-flow fluorescence experiments. At high halide concentrations, halide import occurs predominantly via the rapid formation of a weak initial collision complex, followed by transport of the ion to the active site. To obtain more insight in this collision complex, we determined the X-ray structure of DhlA in the presence of bromide and investigated the kinetics of mutants that were constructed on the basis of this structure. The X-ray structure revealed one bromide ion firmly bound in the active site and two bromide ions weakly bound on the surface of the enzyme. One of the weakly bound ions is close to Thr197 and Phe294, near the entrance of the earlier proposed tunnel for substrate import. Kinetic analysis of bromide import by the Thr197Ala and Phe294Ala mutants of DhlA at high halide concentration showed that the rate constants for halide binding no longer displayed a wild-type-like parabolic increase with increasing bromide concentrations. This is in agreement with an elimination or a decrease in affinity of the surface-located halide-binding site. Likewise, chloride binding kinetics of the mutants indicated significant differences with wild-type enzyme. The results indicate that Thr197 and Phe294 are involved in the formation of an initial collision complex for halide import in DhlA and provide experimental evidence for the role of the tunnel in substrate and product transport.

Active-site structure of the soluble quinoprotein glucose dehydrogenase complexed with methylhydrazine: a covalent cofactor-inhibitor complex.

Soluble glucose dehydrogenase (s-GDH) from the bacterium Acinetobacter calcoaceticus is a classical quinoprotein. It requires the cofactor pyrroloquinoline quinone (PQQ) to catalyze the oxidation of glucose to gluconolactone. The precise catalytic role of PQQ in s-GDH and several other PQQ-dependent enzymes has remained controversial because of the absence of comprehensive structural data. We have determined the crystal structure of a ternary complex of s-GDH with PQQ and methylhydrazine, a competitive inhibitor of the enzyme. This complex, refined at 1.5-A resolution to an R factor of 16.7%, affords a detailed view of a cofactor-binding site of s-GDH. Moreover, it presents the first direct observation of covalent PQQ adduct in the active-site of a PQQ-dependent enzyme, thereby confirming previous evidence that the C5 carbonyl group of the cofactor is the most reactive moiety of PQQ.

Haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 refined at 1.15 A resolution.

Crystals of the 35 kDa protein haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 diffract to 1.15 A resolution at cryogenic temperature using synchrotron radiation. Blocked anisotropic least-squares refinement with SHELXL gave a final conventional R factor of 10.51% for all reflections in the 15-1.15 A resolution range. The estimated r.m.s. errors of the model are 0.026 and 0.038 A for protein atoms and all atoms, respectively. The structure comprises all 310 amino acids, with 28 side chains and two peptide bonds in multiple conformations, two covalently linked Pb atoms, 601 water molecules, seven glycerol molecules, one sulfate ion and two chloride ions. Water molecules accounting for alternative solvent structure are modelled with a fixed occupancy of 0.5. The structure is described in detail and compared with previously reported dehalogenase structures refined at 1.9-2.3 A resolution. An analysis of the protein's geometry and stereochemistry reveals eight mean values of bond lengths and angles which deviate significantly from the Engh & Huber parameters, a wide spread in the main-chain omega torsion angle around its ideal value of 180 (6) degrees and a role for C-HcO interactions in satisfying the hydrogen-bond acceptor capacity of main-chain carbonyl O atoms in the central beta-sheet.

The 1.7 A crystal structure of the apo form of the soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus reveals a novel internal conserved sequence repeat.

The crystal structure of a dimeric apo form of the soluble quinoprotein glucose dehydrogenase (s-GDH) from Acinetobacter calcoaceticus has been solved by multiple isomorphous replacement followed by density modification, and was subsequently refined at 1. 72 A resolution to a final crystallographic R-factor of 16.5% and free R-factor of 20.8% [corrected]. The s-GDH monomer has a beta-propeller fold consisting of six four-stranded anti-parallel beta-sheets aligned around a pseudo 6-fold symmetry axis. The enzyme binds three calcium ions per monomer, two of which are located in the dimer interface. The third is bound in the putative active site, where it may bind and functionalize the pyrroloquinoline quinone (PQQ) cofactor. A data base search unexpectedly showed that four uncharacterized protein sequences are homologous to s-GDH with many residues in the putative active site absolutely conserved. This indicates that these homologs may have a similar structure and that they may catalyze similar PQQ-dependent reactions.A structure-based sequence alignment of the six four-stranded beta-sheets in s-GDH's beta-propeller fold shows an internally conserved sequence repeat that gives rise to two distinct conserved structural motifs. The first structural motif is found at the corner of the short beta-turn between the inner two beta-strands of the beta-sheets, where an Asp side-chain points back into the beta-sheet to form a hydrogen-bond with the OH/NH of a Tyr/Trp side-chain in the same beta-sheet. The second motif involves an Arg/Lys side-chain in the C beta-strand of one beta-sheet, which forms a bidentate salt-bridge with an Asp/Glu in the CD loop of the next beta-sheet. These intra and inter-beta-sheet hydrogen-bonds are likely to contribute to the stability of the s-GDH beta-propeller fold.

The x-ray structure of epoxide hydrolase from Agrobacterium radiobacter AD1. An enzyme to detoxify harmful epoxides.

Epoxide hydrolases catalyze the cofactor-independent hydrolysis of reactive and toxic epoxides. They play an essential role in the detoxification of various xenobiotics in higher organisms and in the bacterial degradation of several environmental pollutants. The first x-ray structure of one of these, from Agrobacterium radiobacter AD1, has been determined by isomorphous replacement at 2.1-A resolution. The enzyme shows a two-domain structure with the core having the alpha/beta hydrolase-fold topology. The catalytic residues, Asp107 and His275, are located in a predominantly hydrophobic environment between the two domains. A tunnel connects the back of the active-site cavity with the surface of the enzyme and provides access to the active site for the catalytic water molecule, which in the crystal structure, has been found at hydrogen bond distance to His275. Because of a crystallographic contact, the active site has become accessible for the Gln134 side chain, which occupies a position mimicking a bound substrate. The structure suggests Tyr152/Tyr215 as the residues involved in substrate binding, stabilization of the transition state, and possibly protonation of the epoxide oxygen.

Kinetic analysis and X-ray structure of haloalkane dehalogenase with a modified halide-binding site.

Haloalkane dehalogenase (DhlA) catalyzes the hydrolysis of haloalkanes via an alkyl-enzyme intermediate. Trp175 forms a halogen/halide-binding site in the active-site cavity together with Trp125. To get more insight in the role of Trp175 in DhlA, we mutated residue 175 and explored the kinetics and X-ray structure of the Trp175Tyr enzyme. The mutagenesis study indicated that an aromatic residue at position 175 is important for the catalytic performance of DhlA. Pre-steady-state kinetic analysis of Trp175Tyr-DhlA showed that the observed 6-fold increase of the Km for 1,2-dibromoethane (DBE) results from reduced rates of both DBE binding and cleavage of the carbon-bromine bond. Furthermore, the enzyme isomerization preceding bromide release became 4-fold faster in the mutant enzyme. As a result, the rate of hydrolysis of the alkyl-enzyme intermediate became the main determinant of the kcat for DBE, which was 2-fold higher than the wild-type kcat. The X-ray structure of the mutant enzyme at pH 6 showed that the backbone structure of the enzyme remains intact and that the tyrosine side chain lies in the same plane as Trp175 in the wild-type enzyme. The Clalpha-stabilizing aromatic rings of Tyr175 and Trp125 are 0.7 A further apart and due to the smaller size of the mutated residue, the volume of the cavity has increased by one-fifth. X-ray structures of mutant and wild-type enzyme at pH 5 demonstrated that the Tyr175 side chain rotated away upon binding of an acetic acid molecule, leaving one of its oxygen atoms hydrogen bonded to the indole nitrogen of Trp125 only. These structural changes indicate a weakened interaction between residue 175 and the halogen atom or halide ion in the active site and help to explain the kinetic changes induced by the Trp175Tyr mutation.

Three-dimensional structure of L-2-haloacid dehalogenase from Xanthobacter autotrophicus GJ10 complexed with the substrate-analogue formate.

The L-2-haloacid dehalogenase from the 1,2-dichloroethane degrading bacterium Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic dehalogenation of small L-2-haloalkanoic acids to yield the corresponding D-2-hydroxyalkanoic acids. Its crystal structure was solved by the method of multiple isomorphous replacement with incorporation of anomalous scattering information and solvent flattening, and was refined at 1.95-A resolution to an R factor of 21.3%. The three-dimensional structure is similar to that of the homologous L-2-haloacid dehalogenase from Pseudomonas sp. YL (1), but the X. autotrophicus enzyme has an extra dimerization domain, an active site cavity that is completely shielded from the solvent, and a different orientation of several catalytically important amino acid residues. Moreover, under the conditions used, a formate ion is bound in the active site. The position of this substrate-analogue provides valuable information on the reaction mechanism and explains the limited substrate specificity of the Xanthobacter L-2-haloacid dehalogenase.

The raw starch binding domain of cyclodextrin glycosyltransferase from Bacillus circulans strain 251.

The E-domain of cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19) from Bacillus circulans strain 251 is a putative raw starch binding domain. Analysis of the maltose-dependent CGTase crystal structure revealed that each enzyme molecule contained three maltose molecules, situated at contact points between protein molecules. Two of these maltoses were bound to specific sites in the E-domain, the third maltose was bound at the C-domain. To delineate the roles in raw starch binding and cyclization reaction kinetics of the two maltose binding sites in the E-domain, we replaced Trp-616 and Trp-662 of maltose binding site 1 and Tyr-633 of maltose binding site 2 by alanines using site-directed mutagenesis. Purified mutant CGTases were characterized with respect to raw starch binding and cyclization reaction kinetics on both soluble and raw starch. The results show that maltose binding site 1 is most important for raw starch binding, whereas maltose binding site 2 is involved in guiding linear starch chains into the active site. beta-Cyclodextrin causes product inhibition by interfering with catalysis in the active site and the function of maltose binding site 2 in the E-domain. CGTase mutants in the E-domain maltose binding site 1 could no longer be crystallized as maltose-dependent monomers. Instead, the W616A mutant CGTase protein was successfully crystallized as a carbohydrate-independent dimer; its structure has been refined to 2.2 A resolution. The three-dimensional structure shows that, within the error limits, neither the absence of carbohydrates nor the W616A mutation caused significant further conformational changes. The modified starch binding and cyclization kinetic properties observed with the mutant CGTase proteins thus can be directly related to the amino acid replacements.

Crystallization and preliminary crystallographic analysis of endo-1,4-beta-xyalanase I from Aspergillus niger.

A family G xylanase from Aspergillus niger has been crystallized using the vapor-diffusion method. Several crystal forms could be obtained using various sodium salts as precipitants. Three of the crystal forms belong to space groups P21, P2(1)2(1)2(1) and P4(3) and have cell parameters of approximately a = b = 85.1, c = 113.6 A and alpha = beta = gamma = 90 degrees. These crystal forms can be converted into one another by flash freezing or macroseeding. A fourth crystal form is cubic (space group P2(1)3) with unit-cell axes of a = b = c = 112.3 A. Data sets for three of the four crystal forms have been collected, extending to a maximum resolution of 2.4 A. The structures of the monoclinic and orthorhombic crystals have been solved by molecular replacement by combining the crystallographic information of the different crystal forms. Refinement of the orthorhombic crystal form is now in progress.

Structure of cyclodextrin glycosyltransferase complexed with a maltononaose inhibitor at 2.6 angstrom resolution. Implications for product specificity.

Crystals of the Y195F mutant of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 were subjected to a double soaking procedure, in which they were first soaked in a solution containing the inhibitor acarbose and subsequently in a solution containing maltohexaose. The refined structure of the resulting protein-carbohydrate complex has final crystallographic and free R-factors for data in the 8-2.6 angstrom resolution range of 15.0% and 21.5%, respectively, and reveals that a new inhibitor, composed of nine saccharide residues, is bound in the active site. The first four residues correspond to acarbose and occupy the same subsites near the catalytic residues as observed in the previously reported acarbose-enzyme complex [Strokopytov et al. (1995) Biochemistry 34, 2234-2240]. An oliogosaccharide consisting of five glucose residues has been coupled to the nonreducing end of acarbose. At the fifth residue the polysaccharide chain makes a sharp turn, allowing it to interact with residues Tyr89, Phe195, and Asn193 and a flexible loop formed by residues 145-148. On the basis of the refined model of the complex an explanation is given for the product specificity of CGTases.

Crystal structure at 2.3 A resolution and revised nucleotide sequence of the thermostable cyclodextrin glycosyltransferase from Thermonanaerobacterium thermosulfurigenes EM1.

The crystal structure of the cyclodextrin glycosyltransferase (CGTase) from the thermophilic microorganism Thermoanaerobacterium thermosulfurigenes EM1 has been elucidated at 2.3 A resolution. The final model consists of all 683 amino acid residues, two calcium ions and 343 water molecules, and has a crystallographic R-factor of 17.9% (Rfree 24.9%) with excellent stereochemistry. The overall fold of the enzyme is highly similar to that reported for mesophilic CGTases and differences are observed only at surface loop regions. Closer inspection of these loop regions and comparison with other CGTase structures reveals that especially loops 88-95, 335-339 and 534-539 possibly contribute with novel hydrogen bonds and apolar contacts to the stabilization of the enzyme. Other structural features that might confer thermostability to the T. thermosulfurigenes EM1 CGTase are the introduction of five new salt-bridges and three Gly to Ala/Pro substitutions. The abundance of Ser, Thr and Tyr residues near the active site and oligosaccharide binding sites might explain the increased thermostability of CGTase in the presence of starch, by allowing amylose chains to bind non-specifically to the protein. Additional stabilization of the A/E domain interface through apolar contacts involves residues Phe273 and Tyr187. No additional or improved calcium binding is observed in the structure, suggesting that the observed stabilization in the presence of calcium ions is caused by the reduced exchange of calcium from the protein to the solvent, rendering it less susceptible to unfolding. The 50% decrease in cyclization activity of the T. thermosulfurigenes EM1 CGTase compared with that of B. circulans strain 251 appears to be caused by the changes in the conformation and amino acid composition of the 88-95 loop. In the T. thermosulfurigenes EM1 CGTase there is no residue homologous to Tyr89, which was observed to take part in stacking interactions with bound substrate in the case of the B. circulans strain 251 CGTase. The lack of this interaction in the enzyme-substrate complex is expected to destabilize bound substrates prior to cyclization. Apparently, some catalytic functionality of CGTase has been sacrificed for the sake of structural stability by modifying loop regions near the active site.

Crystallization and preliminary X-ray analysis of L-2-haloacid dehalogenase from Xanthobacter autotrophicus GJ10.

Haloacid dehalogenases are enzymes that cleave carbon-chlorine or carbon-bromine bonds of 2-haloalkanoates. X-ray-quality crystals of L-2-haloacid dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 have been grown at room temperature from 20% PEG 8000, 200 mM sodium formate at pH 6.8-7.0, using macroseeding techniques. The crystals, which diffract in the X-ray beam up to 2.0 A resolution, belong to the spacegroup C2221. Cell parameters are a = 58.8 A, b = 93.1 A, c = 84.2 A. A native data set to 2.3 A has been collected, with a completeness of 97% and an Rsym of 6.0%.

Crystallographic studies of the interaction of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 with natural substrates and products.

Asp-229, Glu-257, and Asp-328 constitute the catalytic residues in cyclodextrin glycosyl transferase from Bacillus circulans strain 251. Via site-directed mutagenesis constructed D229N, E257Q, and D328N mutant proteins showed a 4,000-60,000-fold reduction of cyclization activity. A D229N/E257Q double mutant showed a 700,000-fold reduction and was crystallized for use in soaking experiments with alpha-cyclodextrin. Crystal structures were determined of wild type CGTase soaked at elevated pH with alpha-cyclodextrin (resolution, 2.1 A) and maltoheptaose (2.4 A). In addition, structures at cryogenic temperature were solved of the unliganded enzyme (2.2 A) and of the D229N/E257Q mutant after soaking with alpha-cyclodextrin (2.6 A). In the crystals soaked in alpha-cyclodextrin and maltoheptaose, a maltotetraose molecule is observed to bind in the active site. Residue 229 is at hydrogen bonding distance from the C-6 hydroxyl group of the sugar, which after cleavage will contain the new reducing end. In the D229N/E257Q double mutant structure, two alpha-cyclodextrins are observed to replace two maltoses at the E-domain, thus providing structural information on product inhibition via binding to the enzyme's raw starch binding domain.

Structure of the 70-kDa soluble lytic transglycosylase complexed with bulgecin A. Implications for the enzymatic mechanism.

Bulgecins are O-sulfonated glycopeptides that are able to enhance the antibacterial activity of beta-lactam antibiotics. The 70-kDa soluble lytic transglycosylase (SLT70) from Escherichia coli forms a specific target of these compounds. Using X-ray crystallography, the three-dimensional structure of a complex of SLT70 with bulgecin A has been determined to 2.8-A resolution and refined to an R factor of 19.5%. The model contains all 618 amino acids of SLT70 and a single molecule of bound bulgecin, located in the active site of the enzyme. The glycopeptide inhibitor is bound in an extended conformation occupying sites analogous to the B, C, and D subsites of lysozyme. Upon binding of bulgecin, the three-stranded antiparallel beta-sheet in the C domain shows a pronounced shift toward the inhibitor. In subsite D, the proposed catalytic residue Glu478 forms a hydrogen bond to the hydroxymethyl oxygen of the proline part of bulgecin and interacts electrostatically with the proline NH2+ group. These interactions, in addition to the interactions observed for the 2-acetamido group of the N-acetylglucosamine residue bound in subsite C, may explain the strong inhibition of SLT70 activity by bulgecin, suggesting that bulgecin acts as an analogue of an oxocarbonium ion intermediate in the reaction catalyzed by SLT70. The structure of the SLT70--bulgecin A complex may be of assistance in the rational design of novel antibiotics.

Site-directed mutations in tyrosine 195 of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 affect activity and product specificity.

Tyrosine 195 is located in the center of the active site cleft of cyclodextrin glycosyltransferase (EC 2.4.1.19) from Bacillus circulans strain 251. Alignment of amino acid sequences of CGTases and alpha-amylases, and the analysis of the binding mode of the substrate analogue acarbose in the active site cleft [Strokopytov, B., et al. (1995) Biochemistry 34, (in press)], suggested that Tyr195 plays an important role in cyclization of oligosaccharides. Tyr195 therefore was replaced with Phe (Y195F), Trp (Y195W), Leu (Y195L), and Gly (Y195G). Mutant proteins were purified and crystallized, and their X-ray structures were determined at 2.5-2.6 angstrum resolution, allowing a detailed comparison of their biochemical properties and three-dimensional structures with those of the wild-type CGTase protein. The mutant proteins possessed significantly reduced cyclodextrin forming and coupling activities but were not negatively affected in the disproportionation and saccharifying reactions. Also under production process conditions, after a 45 h incubation with a 10% starch solution, the Y195W, Y195L, and Y195G mutants showed a lower overall conversion of starch into cyclodextrins. These mutants produced a considerable amount of linear maltooligosaccharides. The presence of aromatic amino acids (Tyr or Phe) at the Tyr195 position thus appears to be of crucial importance for an efficient cyclization reaction, virtually preventing the formation of linear products. Mass spectrometry of the Y195L reaction mixture, but not that of the other mutants and the wild type, revealed a shift toward the synthesis (in low yields) of larger products, especially of beta- and gamma- (but no alpha-) cyclodextrins and minor amounts of delta-, epsilon-, zeta- and eta-cyclodextrins.(ABSTRACT TRUNCATED AT 250 WORDS)