Crystallization and preliminary X-ray diffraction studies of two thermostable alpha-galactosidases from glycoside hydrolase family 36.

alpha-Galactosidases from thermophilic organisms have gained interest owing to their applications in the sugar industry. The alpha-galactosidases AgaA, AgaB and AgaA A355E mutant from Geobacillus stearothermophilus have been overexpressed in Escherichia coli. Crystals of AgaB and AgaA A355E have been obtained by the vapour-diffusion method and synchrotron data have been collected to 2.0 and 2.8 A resolution, respectively. Crystals of AgaB belong to space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 87.5, b = 113.3, c = 161.6 A. Crystals of AgaA A355E belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 150.1, c = 233.2 A.

Crystal structure of the cellulase Cel9M enlightens structure/function relationships of the variable catalytic modules in glycoside hydrolases.

Cellulases cleave the beta-1.4 glycosidic bond of cellulose. They have been characterized as endo or exo and processive or nonprocessive cellulases according to their action mode on the substrate. Different types of these cellulases may coexist in the same glycoside hydrolase family, which have been classified according to their sequence homology and catalytic mechanism. The bacterium C. celluloyticum produces a set of different cellulases who belong mostly to glycoside hydrolase families 5 and 9. As an adaptation of the organism to different macroscopic substrates organizations and to maximize its cooperative digestion, it is expected that cellulases of these families are active on the various macroscopic organizations of cellulose chains. The nonprocessive cellulase Cel9M is the shortest variant of family 9 cellulases (subgroup 9(C)) which contains only the catalytic module to interact with the substrate. The crystal structures of free native Cel9M and its complex with cellobiose have been solved to 1.8 and 2.0 A resolution, respectively. Other structurally known family 9 cellulases are the nonprocessive endo-cellulase Cel9D from C. thermocellum and the processive endo-cellulase Cel9A from T. fusca, from subgroups 9(B1) and 9(A), respectively, whose catalytic modules are fused to a second domain. These enzymes differ in their activity on substrates with specific macroscopic appearances. The comparison of the catalytic module of Cel9M with the two other known GH family 9 structures may give clues to explain its substrate profile and action mode.

Specificity modulation of barley alpha-amylase through biased random mutagenesis involving a conserved tripeptide in beta --> alpha loop 7 of the catalytic (beta/alpha)(8)-barrel domain.

The relative specificity and bond cleavage pattern of barley alpha-amylase 1 (AMY1) were dramatically changed by mutation in F(286)VD that connected beta-strand 7 of the catalytic (beta/alpha)(8)-barrel to a succeeding 3(10)-helix. This conserved tripeptide of the otherwise variable beta --> alpha segment 7 lacked direct ligand contact, but the nearby residues His290 and Asp291 participated in transition-state stabilization and catalysis. On the basis of sequences of glycoside hydrolase family 13, a biased random mutagenesis protocol was designed which encoded 174 putative F(286)VD variants of C95A-AMY1, chosen as the parent enzyme to avoid inactivating glutathionylation by the yeast host. The FVG, FGG, YVD, LLD, and FLE mutants showed 12-380 and 1.8-33% catalytic efficiency (k(cat)/K(m)) toward 2-chloro-4-nitrophenyl beta-D-maltoheptaoside and amylose DP17, respectively, and 0.5-50% activity for insoluble starch compared to that of C95A-AMY1. K(m) and k(cat) were decreased 2-9- and 1.3-83-fold, respectively, for the soluble substrates. The starch:oligosaccharide and amylose:oligosaccharide specificity ratios were 13-172 and 2.4-14 for mutants and 520 and 27 for C95A-AMY1, respectively. The FVG mutant released 4-nitrophenyl alpha-D-maltotrioside (PNPG(3)) from PNPG(5), whereas C95A-AMY1 produced PNPG and PNPG(2). The mutation thus favored interaction with the substrate aglycon part, while products from PNPG(6) reflected the fact that the mutation restored binding at subsite -6 which was lost in C95A-AMY1. The outcome of this combined irrational and rational protein engineering approach was evaluated considering structural accommodation of mutant side chains. FVG and FGG, present in the most active variants, represented novel sequences. This emphasized the worth of random mutagenesis and launched flexibility as a goal for beta --> alpha loop 7 engineering in family 13.

Crystallization and preliminary X-ray analysis of a bacterial psychrophilic enzyme, phosphoglycerate kinase.

The glycolytic enzyme phosphoglycerate kinase (PGK) from the Antarctic microorganism Pseudomonas sp. TACII18 is a cold-adapted enzyme that displays a high specific activity at low temperatures and decreased thermostability relative to its mesophilic counterpart. Herein, the preliminary crystallization and structure solution of psychrophilic PGK in its native form and cocrystallized with 3-phosphoglyceric acid (3-PGA) and the ATP analogue adenylyl imidophosphate (AMP-PNP) is reported. The complexed form of PGK crystallized in 2-3 d at 290 K, whereas the native form of the enzyme required 8-12 months. Morphologically, both crystal forms are similar and X-ray diffraction experiments indicate that the crystals are isomorphous. The crystals diffracted to a resolution of 2.0 A and belong to the space group P3(2). with unit-cell parameters a = b = 58.5, c = 85.4 A.

Structure of cytochrome c7 from Desulfuromonas acetoxidans at 1.9 A resolution.

Multihaem cytochromes play a key role in electron-transport reactions in the periplasm of sulfate- and sulfur-reducing bacteria. The redox proteins grouped in the c3 superfamily also display metal-reducing activities, which make them interesting biotechnological tools. The crystal structure of the fully oxidized cytochrome c7 from Desulfuromonas acetoxidans has been solved by combined molecular-replacement and MAD methods. The structure has been refined at 1.9 A resolution to an R value of 19.1% (R(free) = 24.3%) and includes three haems and 116 water molecules. The protein displays the cytochrome c3 fold in a highly minimized form, while haem 2 and the surrounding protein environment are missing. The geometry of haem packing and of the haem axial ligands and propionates are described and compared with that of c3 cytochromes. The crystal structure is compared with the solution structure recently obtained by NMR methods and with its homologue cytochromes of the c3 superfamily. Comparison of the high number of available structures makes it possible to analyze the structural role of the few highly conserved residues, in addition to the cysteines and histidines that link the porphyrin rings and the Fe atoms to the protein chain.

Crystal structures of the cellulase Cel48F in complex with inhibitors and substrates give insights into its processive action.

Cellulase Cel48F from Clostridium cellulolyticum was described as a processive endo-cellulase. The active site is composed of a 25 A long tunnel which is followed by an open cleft. During the processive action, the cellulose substrate has to slide through the tunnel to continuously supply the leaving group site with sugar residues after the catalytic cleavage. To study this processive action in the tunnel, the native catalytic module of Cel48F and the inactive mutant E55Q, have been cocrystallized with cellobiitol, two thio-oligosaccharide inhibitors (PIPS-IG3 and IG4) and the cello-oligosaccharides cellobiose, -tetraose and -hexaose. Seven sub-sites in the tunnel section of the active center could be identified and three of the four previously reported sub-sites in the open cleft section were reconfirmed. The sub-sites observed for the thio-oligosaccharide inhibitors and oligosaccharides, respectively, were located at two different positions in the tunnel corresponding to a shift in the chain direction of about a half sugar subunit. These two positions have different patterns of stacking interactions with aromatic residues present in the tunnel. Multiple patterns are not observed in nonprocessive endo-cellulases, where only one sugar position is favored by aromatic stacking. It is therefore proposed that the aromatic residues serve as lubricating agents to reduce the sliding barrier in the processive action.

Functional aspects of the heme bound hemophore HasA by structural analysis of various crystal forms.

The protein HasA from the Gram negative bacteria Serratia marcescens is the first hemophore to be described at the molecular level. It participates to the shuttling of heme from hemoglobin to the outer membrane receptor HasR, which in turn releases it into the bacterium. HasR alone is also able to take up heme from hemoglobin but synergy with HasA increases the efficiency of the system by a factor of about 100. This iron acquisition system allows the bacteria to survive with hemoglobin as the sole iron source. Here we report the structures of a new crystal form of HasA diffracting up to 1.77A resolution as well as the refined structure of the trigonal crystal form diffracting to 3.2A resolution. The crystal structure of HasA at high resolution shows two possible orientations of the heme within the heme-binding pocket, which probably are functionally involved in the heme-iron acquisition process. The detailed analysis of the three known structures reveals the molecular basis regulating the relative affinity of the heme/hemophore complex.

Specific inhibition of barley alpha-amylase 2 by barley alpha-amylase/subtilisin inhibitor depends on charge interactions and can be conferred to isozyme 1 by mutation.

alpha-Amylase 2 (AMY2) and alpha-amylase/subtilisin inhibitor (BASI) from barley bind with Ki = 0.22 nM. AMY2 is a (beta/alpha)8-barrel enzyme and the segment Leu116-Phe143 in domain B (Val89-Ile152), protruding at beta-strand 3 of the (beta/alpha)8-barrel, was shown using isozyme hybrids to be crucial for the specificity of the inhibitor for AMY2. In the AMY2-BASI crystal structure [F. Vallée, A. Kadziola, Y. Bourne, M. Juy, K. W. Rodenburg, B. Svensson & R. Haser (1998) Structure 6, 649-659] Arg128AMY2 forms a hydrogen bond with Ser77BASI, while Asp142AMY2 makes a salt-bridge with Lys140BASI. These two enzyme residues are substituted by glutamine and asparagine, respectively, to assess their contribution in binding of the inhibitor. These mutations were performed in the well-expressed, inhibitor-sensitive hybrid barley alpha-amylase 1 (AMY1)-(1-90)/AMY2-(90-403) with Ki = 0.33 nM, because of poor production of AMY2 in yeast. In addition Arg128, only found in AMY2, was introduced into an AMY1 context by the mutation T129R/K130P in the inhibitor-insensitive hybrid AMY1-(1-161)/AMY2-(161-403). The binding energy was reduced by 2.7-3.0 kcal.mol-1 as determined from Ki after the mutations R128Q and D142N. This corresponds to loss of a charged interaction between the protein molecules. In contrast, sensitivity to the inhibitor was gained (Ki = 7 microM) by the mutation T129R/K130P in the insensitive isozyme hybrid. Charge screening raised Ki 14-20-fold for this latter mutant, AMY2, and the sensitive isozyme hybrid, but only twofold for the R128Q and D142N mutants. Thus electrostatic stabilization was effectively introduced and lost in the different mutant enzyme-inhibitor complexes and rational engineering using an inhibitor recognition motif to confer binding to the inhibitor mimicking the natural AMY2-BASI complex.

Amylose chain behavior in an interacting context. III. Complete occupancy of the AMY2 barley alpha-amylase cleft and comparison with biochemical data.

In the first two papers of this series, the tools necessary to evaluate substrate ring deformations were developed, and then the modeling of short amylose fragments (maltotriose and maltopentaose) inside the catalytic site of barley alpha-amylase was performed. In this third paper, this docking has been extended to the whole catalytic cleft. A systematic approach to extend the substrate was used on the reducing side from the previous enzyme/pentasaccharide complex. However, due to the lack of an obvious subsite at the nonreducing side, an alternate protocol has been chosen that incorporates biochemical information on the enzyme and features on the substrate shape as well. As a net result, ten subsites have been located consistent with the distribution of Ajandouz et al. (E. H. Ajandouz, J. Abe, B. Svensson, and G. Marchis-Mouren, Biochimica Biophysica Acta, 1992, Vol. 1159, pp. 193-202) and corresponding binding energies were estimated. Among them, two extreme subsites (-6) and (+4), with stacking residues Y104 and Y211, respectively, have strong affinities with glucose rings added to the substrate. No other deformation has been found for the new glucose rings added to the substrate; therefore, only ring A of the DP 10 fragment has a flexible form when interacting with the inner stacking residues Y51. Global conservation of the helical shape of the substrate can be postulated in spite of its significant distortion at subsite (-1).

The crystal structure of HasA, a hemophore secreted by Serratia marcescens.

Free iron availability is strongly limited in vertebrate hosts, making the iron acquisition by siderophores inappropriate. Pathogenic bacteria have developed various ways to use the host's iron from iron-containing proteins. Serratia marcescens can use the iron from hemoglobin through the secretion of a hemophore called HasA, which takes up the heme from hemoglobin and shuttles it to the receptor HasR, which in turn, releases heme into the bacterium. We report here the first crystal structure of such a hemophore, bound to a heme group at two different pH values and at a resolution of 1.9 A. The structure reveals a new original fold and suggests a hypothetical mechanism for both heme uptake and release.

Key role of phenylalanine 20 in cytochrome c3: structure, stability, and function studies.

Aromatic residues in c-type cytochromes might have an important function in the folding and/or electron transferring properties of the molecule. In the tetraheme cytochrome c3 (Mr 13 000) from Desulfovibrio vulgaris Hildenborough, Phe20, is located between heme 1 and heme 3 with its aromatic ring close and almost parallel to the ring plane of heme 1. We replaced this residue by a nonaromatic hydrophobe residue, leucine, and analyzed the effects in terms of functional, structural, and physicochemical properties. While the F20L replacement did not have any strong effects on the heme region stability, a decrease of the thermostability of the whole molecule was observed. In the same way, the four macroscopic redox potentials were affected by the mutation as well as the flexibility of the surface loop around heme 4. The F20L replacement itself and/or this structural modification might be responsible for the loss of the intermolecular cooperativity between F20L cytochrome c3 molecules.

Structures of the psychrophilic Alteromonas haloplanctis alpha-amylase give insights into cold adaptation at a molecular level.

BACKGROUND: . Enzymes from psychrophilic (cold-adapted) microorganisms operate at temperatures close to 0 degreesC, where the activity of their mesophilic and thermophilic counterparts is drastically reduced. It has generally been assumed that thermophily is associated with rigid proteins, whereas psychrophilic enzymes have a tendency to be more flexible. RESULTS: . Insights into the cold adaptation of proteins are gained on the basis of a psychrophilic protein's molecular structure. To this end, we have determined the structure of the recombinant form of a psychrophilic alpha-amylase from Alteromonas haloplanctis at 2.4 A resolution. We have compared this with the structure of the wild-type enzyme, recently solved at 2.0 A resolution, and with available structures of their mesophilic counterparts. These comparative studies have enabled us to identify possible determinants of cold adaptation. CONCLUSIONS: . We propose that an increased resilience of the molecular surface and a less rigid protein core, with less interdomain interactions, are determining factors of the conformational flexibility that allows efficient enzyme catalysis in cold environments.

Crystal structure of oxidized trimethylamine N-oxide reductase from Shewanella massilia at 2.5 A resolution.

The periplasmic trimethylamine N-oxide (TMAO) reductase from the marine bacteria Shewanella massilia is involved in a respiratory chain, having trimethylamine N-oxide as terminal electron acceptor. This molybdoenzyme belongs to the dimethyl sulfoxide (DMSO) reductase family, but has a different substrate specificity than its homologous enzyme. While the DMSO reductases reduce a broad spectra of organic S-oxide and N-oxide compounds, TMAO reductase from Shewanella massilia reduces only TMAO as the natural compound. The crystal structure was solved by molecular replacement with the coordinates of the DMSO reductase from Rhodobacter sphaeroides. The overall fold of the protein structure is essentially the same as the DMSO reductase structures, organized into four domains. The molybdenum coordination sphere is closest to that described in the DMSO reductase of Rhodobacter capsulatus. The structural differences found in the protein environment of the active site could be related to the differences in substrate specificity of these enzymes. In close vicinity of the molybdenum ion a tyrosine residue is missing in the TMAO reductase, leaving a greater space accessible to the solvent. This tyrosine residue has contacts to the oxo groups in the DMSO reductase structures. The arrangement and number of charged residues lining the inner surface of the funnel-like entrance to the active site, is different in the TMAO reductase than in the DMSO reductases from Rhodobacter species. Furthermore a surface loop at the top of the active-site funnel, for which no density was present in the DMSO reductase structures, is well defined in the oxidized form of the TMAO reductase structure, and is located on the border of the funnel-like entrance of the active center.

Crystallization of the catalytic domain of Clostridium cellulolyticum CeLF cellulase in the presence of a newly synthesized cellulase inhibitor.

The catalytic domain of the CeIF processive endocellulase, a family 48 glycosyl hydrolase from Clostridium cellulolyticum has been crystallized in the presence of a newly synthesized inhibitor (methyl 4-S-beta-cellobiosyl-4-thio-beta-cellobioside), by vapour diffusion, using PEG as a precipitant. The protein crystallizes in the orthorhombic P212121 space group and diffracts to a resolution of 2.0 A. The unit-cell parameters are a = 61.4, b = 84.5, c = 121.9 A.

The crystal structure of the processive endocellulase CelF of Clostridium cellulolyticum in complex with a thiooligosaccharide inhibitor at 2.0 A resolution.

The mesophilic bacterium Clostridium cellulolyticum exports multienzyme complexes called cellulosomes to digest cellulose. One of the three major components of the cellulosome is the processive endocellulase CelF. The crystal structure of the catalytic domain of CelF in complex with two molecules of a thiooligosaccharide inhibitor was determined at 2.0 A resolution. This is the first three-dimensional structure to be solved of a member of the family 48 glycosyl hydrolases. The structure consists of an (alpha alpha)6-helix barrel with long loops on the N-terminal side of the inner helices, which form a tunnel, and an open cleft region covering one side of the barrel. One inhibitor molecule is enclosed in the tunnel, the other exposed in the open cleft. The active centre is located in a depression at the junction of the cleft and tunnel regions. Glu55 is the proposed proton donor in the cleavage reaction, while the corresponding base is proposed to be either Glu44 or Asp230. The orientation of the reducing ends of the inhibitor molecules together with the chain translation through the tunnel in the direction of the active centre indicates that CelF cleaves processively cellobiose from the reducing to the non-reducing end of the cellulose chain.

Barley alpha-amylase bound to its endogenous protein inhibitor BASI: crystal structure of the complex at 1.9 A resolution.

BACKGROUND: Barley alpha-amylase is a 45 kDa enzyme which is involved in starch degradation during barley seed germination. The released sugars provide the plant embryo with energy for growth. The major barley alpha-amylase isozyme (AMY2) binds with high affinity to the endogenous inhibitor BASI (barley alpha-amylase/subtilisin inhibitor) whereas the minor isozyme (AMY1) is not inhibited. BASI is a 19.6 kDa bifunctional protein that can simultaneously inhibit AMY2 and serine proteases of the subtilisin family. This inhibitor may therefore prevent degradation of the endosperm starch during premature sprouting and protect the seed from attack by pathogens secreting proteases. RESULTS: The crystal structure of AMY2 in complex with BASI was determined and refined at 1.9 A resolution. BASI consists of a 12-stranded beta-barrel structure which belongs to the beta-trefoil fold family and inhibits AMY2 by sterically occluding access of the substrate to the active site of the enzyme. The AMY2-BASI complex is characterized by an unusual completely solvated calcium ion located at the protein-protein interface. CONCLUSIONS: The AMY2-BASI complex represents the first reported structure of an endogenous protein-protein complex from a higher plant. The structure of the complex throws light on the strict specificity of BASI for AMY2, and shows that domain B of AMY2 contributes greatly to the specificity of enzyme-inhibitor recognition. In contrast to the three-dimensional structures of porcine pancreatic alpha-amylase in complex with proteinaceous inhibitors, the AMY2-BASI structure reveals that the catalytically essential amino acid residues of the enzyme are not directly bound to the inhibitor. Binding of BASI to AMY2 creates a cavity, exposed to the external medium, that is ideally shaped to accommodate an extra calcium ion. This feature may contribute to the inhibitory effect, as the key amino acid sidechains of the active site are in direct contact with water molecules which are in turn ligated to the calcium ion.

Molecular structure of a barley alpha-amylase-inhibitor complex: implications for starch binding and catalysis.

alpha-Amylases are widely occurring, multidomain proteins with a catalytic (beta/alpha)8-barrel. In barley alpha-amylase, insight into the catalytic mechanism is gained from the X-ray crystal structure of its molecular complex with acarbose, a pseudotetrasaccharide that acts like a transition-state analogue and which is shown to bind at two specific regions of the enzyme. The structure of the complex has been refined to an R-factor of 15.1% for all observations with Fo>sigma(Fo) between 10 and 2.8 A resolution. A difference Fourier map produced after refinement of the native structure against the data of the acarbose complex clearly revealed density corresponding to two oligosaccharide-binding sites. One of these is defined as the surface-located starch granule-binding site characteristic of cereal alpha-amylases. It involves stacking of two acarbose rings on Trp276 and Trp277. The other binding region is the active site covering subsites -1, +1 and +2. Here, Glu204 is positioned to act in general acid/base catalysis protonating the glucosidic oxygen atom assisted by Asp289. A water molecule that bridges Glu204 and Asp289 is found at the entrance cavity containing a total of five water molecules. This water molecule is proposed to reprotonate Glu204 and supply the hydroxyl ion for nucleophilic attack on the glucosyl C1 atom. Asp 179 acts as the nucleophile that can bind covalently to the substrate intermediate after bond cleavage. The present complex structure together with the conservation of active-site residues among alpha-amylases and related enzymes, are consistent with a common catalytic mechanism for this class of retaining carbohydrases.

Crystal structures of the psychrophilic alpha-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor.

Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the alpha-amylase (AHA) secreted by this bacterium, in its native state to 2.0 angstroms resolution as well as in complex with Tris to 1.85 angstroms resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known alpha-amylases of various origins with a surprisingly greatest similarity to mammalian alpha-amylases. AHA contains a chloride ion which activates the hydrolytic cleavage of substrate alpha-1,4-glycosidic bonds. The chloride binding site is situated approximately 5 angstroms from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation. A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found approximately 22 angstroms from the active site. We found that this triad is equally present in other chloride dependent alpha-amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted alpha-amylase.

Structural and kinetic studies of the Y73E mutant of octaheme cytochrome c3 (Mr = 26 000) from Desulfovibrio desulfuricans Norway.

A combination of structural, kinetic, and interaction experiments has been used to study the role of a highly conserved aromatic residue, Tyr73, parallel to the sixth heme axial ligand of heme 4 in multiheme cytochrome c3 (Mr = 26 000), also called cytochrome cc3 or octaheme cytochrome, from Desulfovibrio desulfuricans Norway. This residue is expected to be involved in intermolecular electron transfer and protein-protein interaction, since heme 4 is described to be the interaction site between physiological partners. The kinetic experiments show that the Y73E replacement provokes no significant change in the electron-transfer reaction with the physiological partner, the [NiFeSe] hydrogenase, but that the protein-protein interaction between cytochrome c3 (Mr = 26 000) and hydrogenase is strongly affected by the mutation. The aromatic residue does not play a role in maintaining the axial heme ligand in a particular orientation, since the mutation did not affect the orientation of histidine 77, the sixth axial ligand of heme 4. The structural analysis by X-ray crystallography clearly shows that a rearrangement of the charged residues in the vicinity of the mutation site is responsible for the change in protein-protein interaction, which is of an electrostatic nature. Lys22 and Arg66, residues which are located at the interacting surface, are twisted toward the mutated position Glu73 in order to compensate for the negative charge and therefore are no longer accessible for the docking with a physiological partner. Tyr73 has instead a structural function and probably a role in maintaining the hydrophobic environment of the heme 4 cavity rather than a function in the intermolecular electron transfer with the physiological partners.

Crystallization and preliminary X-ray diffraction studies of alpha-amylase from the antarctic psychrophile Alteromonas haloplanctis A23.

A cold-active alpha-amylase was purified from culture supernatants of the antarctic psychrophile Alteromonas haloplanctis A23 grown at 4 degrees C. In order to contribute to the understanding of the molecular basis of cold adaptations, crystallographic studies of this cold-adapted enzyme have been initiated because a three-dimensional structure of a mesophilic counterpart, pig pancreatic alpha-amylase, already exists. alpha-Amylase from A. haloplanctis, which shares 53% sequence identity with pig pancreatic alpha-amylase, has been crystallized and data to 1.85 A have been collected. The space group is found to be C222(1) with a = 71.40 A, b = 138.88 A, and c = 115.66 A. Until now, a three-dimensional structure of a psychrophilic enzyme is lacking.