The first crystal structure of L-threonine dehydrogenase

L-threonine dehydrogenase (TDH) is an enzyme that catalyzes the oxidation of L-threonine to 2-amino-3-ketobutyrate. We solved the first crystal structure of a medium chain L-threonine dehydrogenase from a hyperthermophilic archaeon, Pyrococcus horikoshii (PhTDH), by the single wavelength anomalous diffraction method using a selenomethionine-substituted enzyme. This recombinant PhTDH is a homo-tetramer in solution. Three monomers of PhTDHs were located in the crystallographic asymmetric unit, however, the crystal structure exhibits a homo-tetramer structure with crystallographic and non-crystallographic 222 symmetry in the cell. Despite the low level of sequence identity to a medium-chain NAD(H)-dependent alcohol dehydrogenase (ADH) and the different substrate specificity, the overall folds of the PhTDH monomer and tetramer are similar to those of the other ADH. Each subunit is composed of two domains: a nicotinamide cofactor (NAD(H))-binding domain and a catalytic domain. The NAD(H)-binding domain contains the alpha/beta Rossmann fold motif, characteristic of the NAD(H)-binding protein. One molecule of PhTDH contains one zinc ion playing a structural role. This metal ion exhibits coordination with four cysteine ligands and some of the ligands are conserved throughout the structural zinc-containing ADHs and TDHs. However, the catalytic zinc ion that is coordinated at the bottom of the cleft in the case of ADH was not observed in the crystal of PhTDH. There is a significant difference in the orientation of the catalytic domain relative to the coenzyme-binding domain that results in a larger interdomain cleft.     

Three-dimensional structures of apo- and holo-L-alanine dehydrogenase from Mycobacterium tuberculosis reveal conformational changes upon coenzyme binding

l-Alanine dehydrogenase from Mycobacterium tuberculosis catalyzes the NADH-dependent reversible conversion of pyruvate and ammonia to l-alanine. Expression of the gene coding for this enzyme is up-regulated in the persistent phase of the organism, and alanine dehydrogenase is therefore a potential target for pathogen control by antibacterial compounds. We have determined the crystal structures of the apo- and holo-forms of the enzyme to 2.3 and 2.0 Å resolution, respectively. The enzyme forms a hexamer of identical subunits, with the NAD-binding domains building up the core of the molecule and the substrate-binding domains located at the apical positions of the hexamer. Coenzyme binding stabilizes a closed conformation where the substrate-binding domains are rotated by about 16° toward the dinucleotide-binding domains, compared to the open structure of the apo-enzyme. In the structure of the abortive ternary complex with NAD+ and pyruvate, the substrates are suitably positioned for hydride transfer between the nicotinamide ring and the C2 carbon atom of the substrate. The approach of the nucleophiles water and ammonia to pyruvate or the reaction intermediate iminopyruvate, respectively, is, however, only possible through conformational changes that make the substrate binding site more accessible. The crystal structures identified the conserved active-site residues His96 and Asp270 as potential acid/base catalysts in the reaction. Amino acid replacements of these residues by site-directed mutagenesis led to inactive mutants, further emphasizing their essential roles in the enzymatic reaction mechanism.     

The 3.0 A resolution crystal structure of glycosomal pyruvate phosphate dikinase from Trypanosoma brucei

The crystal structure of the glycosomal enzyme pyruvate phosphate dikinase from the African protozoan parasite Trypanosoma brucei has been solved to 3.0 A resolution by molecular replacement. The search model was the 2.3 A resolution structure of the Clostridium symbiosum enzyme. Due to different relative orientations of the domains and sub-domains in the two structures, molecular replacement could be achieved only by positioning these elements (four bodies altogether) sequentially in the asymmetric unit of the P2(1)2(1)2 crystal, which contains one pyruvate phosphate dikinase (PPDK) subunit. The refined model, comprising 898 residues and 188 solvent molecules per subunit, has a crystallographic residual index Rf = 0.245 (cross-validation residual index Rfree = 0.291) and displays satisfactory stereochemistry. Eight regions, comprising a total of 69 amino acid residues at the surface of the molecule, are disordered in this crystal form. The PPDK subunits are arranged around the crystallographic 2-fold axis as a dimer, analogous to that observed in the C. symbiosum enzyme. Comparison of the two structures was carried out by superposition of the models. Although the fold of each domain or sub-domain is similar, the relative orientations of these constitutive elements are different in the two structures. The trypanosome enzyme is more "bent" than the bacterial enzyme, with bending increasing from the center of the molecule (close to the molecular 2-fold axis) towards the periphery where the N-terminal domain is located. As a consequence of this increased bending and of the differences in relative positions of subdomains, the nucleotide-binding cleft in the amino-terminal domain is wider in T. brucei PPDK: the N-terminal fragment of the amino-terminal domain is distant from the catalytic, phospho-transfer competent histidine 482 (ca 10 A away). Our observations suggest that the requirements of domain motion during enzyme catalysis might include widening of the nucleotide-binding cleft to allow access and departure of the AMP or ATP ligand.     

Structure and mechanism of the RuvB Holliday junction branch migration motor

The RuvB hexamer is the chemomechanical motor of the RuvAB complex that migrates Holliday junction branch-points in DNA recombination and the rescue of stalled DNA replication forks. The 1.6 A crystal structure of Thermotoga maritima RuvB together with five mutant structures reveal that RuvB is an ATPase-associated with diverse cellular activities (AAA+-class ATPase) with a winged-helix DNA-binding domain. The RuvB-ADP complex structure and mutagenesis suggest how AAA+-class ATPases couple nucleotide binding and hydrolysis to interdomain conformational changes and asymmetry within the RuvB hexamer implied by the crystallographic packing and small-angle X-ray scattering in solution. ATP-driven domain motion is positioned to move double-stranded DNA through the hexamer and drive conformational changes between subunits by altering the complementary hydrophilic protein- protein interfaces. Structural and biochemical analysis of five motifs in the protein suggest that ATP binding is a strained conformation recognized both by sensors and the Walker motifs and that intersubunit activation occurs by an arginine finger motif reminiscent of the GTPase-activating proteins. Taken together, these results provide insights into how RuvB functions as a motor for branch migration of Holliday junctions.     

Crystal structures of mouse class II alcohol dehydrogenase reveal determinants of substrate specificity and catalytic efficiency

The structure of mouse class II alcohol dehydrogenase (ADH2) has been determined in a binary complex with the coenzyme NADH and in a ternary complex with both NADH and the inhibitor N-cyclohexylformamide to 2.2 A and 2.1 A resolution, respectively. The ADH2 dimer is asymmetric in the crystal with different orientations of the catalytic domains relative to the coenzyme-binding domains in the two subunits, resulting in a slightly different closure of the active-site cleft. Both conformations are about half way between the open apo structure and the closed holo structure of horse ADH1, thus resembling that of ADH3. The semi-open conformation and structural differences around the active-site cleft contribute to a substantially different substrate-binding pocket architecture as compared to other classes of alcohol dehydrogenase, and provide the structural basis for recognition and selectivity of alcohols and quinones. The active-site cleft is more voluminous than that of ADH1 but not as open and funnel-shaped as that of ADH3. The loop with residues 296-301 from the coenzyme-binding domain is short, thus opening up the pocket towards the coenzyme. On the opposite side, the loop with residues 114-121 stretches out over the inter-domain cleft. A cavity is formed below this loop and adds an appendix to the substrate-binding pocket. Asp301 is positioned at the entrance of the pocket and may control the binding of omega-hydroxy fatty acids, which act as inhibitors rather than substrates. Mouse ADH2 is known as an inefficient ADH with a slow hydrogen-transfer step. By replacing Pro47 with His, the alcohol dehydrogenase activity is restored. Here, the structure of this P47H mutant was determined in complex with NADH to 2.5 A resolution. His47 is suitably positioned to act as a catalytic base in the deprotonation of the substrate. Moreover, in the more closed subunit, the coenzyme is allowed a position closer to the catalytic zinc. This is consistent with hydrogen transfer from an alcoholate intermediate where the Pro/His replacement focuses on the function of the enzyme.     

The crystal structure of the beta-lactamase of Streptomyces albus G at 0.3 nm resolution.

The crystal structure of the beta-lactamase of Streptomyces albus G has been solved at 0.3 nm resolution by X-ray-diffraction methods. The enzyme is a typical two-domain protein. One domain consists of five alpha-helices, and the other is five-stranded beta-sheet with alpha-helices on both sides of the sheet. The active-site serine residue (Ser-48) is within a cleft located between the two domains.     

Phenylalanine dehydrogenase from Rhodococcus sp. M4: high-resolution X-ray analyses of inhibitory ternary complexes reveal key features in the oxidative deamination mechanism

The molecular structures of recombinant L-phenylalanine dehydrogenase from Rhodococcus sp. M4 in two different inhibitory ternary complexes have been determined by X-ray crystallographic analyses to high resolution. Both structures show that L-phenylalanine dehydrogenase is a homodimeric enzyme with each monomer composed of distinct globular N- and C-terminal domains separated by a deep cleft containing the active site. The N-terminal domain binds the amino acid substrate and contributes to the interactions at the subunit:subunit interface. The C-terminal domain contains a typical Rossmann fold and orients the dinucleotide. The dimer has overall dimensions of approximately 82 A x 75 A x 75 A, with roughly 50 A separating the two active sites. The structures described here, namely the enzyme.NAD+.phenylpyruvate, and enzyme. NAD+.beta-phenylpropionate species, represent the first models for any amino acid dehydrogenase in a ternary complex. By analysis of the active-site interactions in these models, along with the currently available kinetic data, a detailed chemical mechanism has been proposed. This mechanism differs from those proposed to date in that it accounts for the inability of the amino acid dehydrogenases, in general, to function as hydroxy acid dehydrogenases.     

X-ray structure of nucleoside diphosphate kinase.

The X-ray structure of a point mutant of nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum has been determined to 2.2 A resolution. The enzyme is a hexamer made of identical subunits with a novel mononucleotide binding fold. Each subunit contains an alpha/beta domain with a four stranded, antiparallel beta-sheet. The topology is different from adenylate kinase, but identical to the allosteric domain of Escherichia coli ATCase regulatory subunits, which bind mononucleotides at an equivalent position. Dimer contacts between NDP kinase subunits within the hexamer are similar to those in ATCase. Trimer contacts involve a large loop of polypeptide chain that bears the site of the Pro----Ser substitution in Killer of prune (K-pn) mutants of the highly homologous Drosophila enzyme. Properties of Drosophila NDP kinase, the product of the awd developmental gene, and of the human enzyme, the product of the nm23 genes in tumorigenesis, are discussed in view of the three-dimensional structure and of possible interactions of NDP kinase with other nucleotide binding proteins.     

Crystal structure of D-erythronate-4-phosphate dehydrogenase complexed with NAD

Pyridoxal-5′-phosphate (the active form of vitamin B6) is an essential cofactor in many enzymatic reactions. While animals lack any of the pathways for de novo synthesis and salvage of vitamin B6, it is synthesized by two distinct biosynthetic routes in bacteria, fungi, parasites, and plants. One of them is the PdxA/PdxJ pathway found in the γ subdivision of proteobacteria. It depends on the pdxB gene, which encodes erythronate-4-phosphate dehydrogenase (PdxB), a member of the d-isomer specific 2-hydroxyacid dehydrogenase superfamily. Although three-dimensional structures of other functionally related dehydrogenases are available, no structure of PdxB has been reported. To provide the missing structural information and to gain insights into the catalytic mechanism, we have determined the first crystal structure of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa in the ligand-bound state. It is a homodimeric enzyme consisting of 380-residue subunits. Each subunit consists of three structural domains: the lid domain, the nucleotide-binding domain, and the C-terminal dimerization domain. The latter domain has a unique fold and is largely responsible for dimerization. Interestingly, two subunits of the dimeric enzyme are bound with different combinations of ligands in the crystal and they display significantly different conformations. Subunit A is bound with NAD and a phosphate ion, while subunit B, with a more open active site cleft, is bound with NAD and l(+)-tartrate. Our structural data allow a detailed understanding of cofactor and substrate recognition, thus providing substantial insights into PdxB catalysis.     

Ligand-induced conformational changes and a mechanism for domain closure in Aspergillus nidulans dehydroquinate synthase

In order to investigate systematically substrate and cofactor-induced conformational changes in the enzyme dehydroquinate synthase (DHQS), eight structures representing a series of differently liganded states have been determined in a total of six crystal forms. DHQS in the absence of the substrate analogue carbaphosphonate, either unliganded or in the presence of NAD or ADP, is in an open form where a relative rotation of 11-13 degrees between N and C-terminal domains occurs.Analysis of torsion angle difference plots between sets of structures reveals eight rearrangements that appear relevant to domain closure and a further six related to crystal packing. Overlapping 21 different copies of the individual N and C-terminal DHQS domains further reveals a series of pivot points about which these movements occur and illustrates the way in which widely separated secondary structure elements are mechanically inter-linked to form "composite elements", which propagate structural changes across large distances.This analysis has provided insight into the basis of DHQS ligand-initiated domain closure and gives rise to the proposal of an ordered sequence of events involving substrate binding, and local rearrangements within the active site that are propagated to the hinge regions, leading to closure of the active-site cleft.     

X-ray structure of RLI, an essential twin cassette ABC ATPase involved in ribosome biogenesis and HIV capsid assembly

The ABC ATPase RNase-L inhibitor (RLI) emerges as a key enzyme in ribosome biogenesis, formation of translation preinitiation complexes, and assembly of HIV capsids. To help reveal the structural mechanism of RLI, we determined the Mg2+-ADP bound crystal structure of the twin cassette ATPase of P. furiosus RLI at 1.9 A resolution and analyzed functional motifs in yeast in vivo. RLI shows similarities but also differences to known ABC enzyme structures. Twin nucleotide binding domains (NBD1 and NBD2) are arranged to form two composite active sites in their interface cleft, indicating they undergo the ATP-driven clamp-like motion of the NBDs of ABC transporters. An unusual "hinge" domain along the NBD1:NBD2 interface provides a frame for association and possibly ATP-driven conformational changes of the NBDs. Our results establish a first structural basis for ABC domain heterodimers and suggest that RLI may act as mechanochemical enzyme in ribosome and HIV capsid biogenesis.     

Crystal structure of prephenate dehydrogenase from Aquifex aeolicus. Insights into the catalytic mechanism

The enzyme prephenate dehydrogenase catalyzes the oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate for the biosynthesis of tyrosine. Prephenate dehydrogenases exist as either monofunctional or bifunctional enzymes. The bifunctional enzymes are diverse, since the prephenate dehydrogenase domain is associated with other enzymes, such as chorismate mutase and 3-phosphoskimate 1-carboxyvinyltransferase. We report the first crystal structure of a monofunctional prephenate dehydrogenase enzyme from the hyper-thermophile Aquifex aeolicus in complex with NAD+. This protein consists of two structural domains, a modified nucleotide-binding domain and a novel helical prephenate binding domain. The active site of prephenate dehydrogenase is formed at the domain interface and is shared between the subunits of the dimer. We infer from the structure that access to the active site is regulated via a gated mechanism, which is modulated by an ionic network involving a conserved arginine, Arg250. In addition, the crystal structure reveals for the first time the positions of a number of key catalytic residues and the identity of other active site residues that may participate in the reaction mechanism; these residues include Ser126 and Lys246 and the catalytic histidine, His147. Analysis of the structure further reveals that two secondary structure elements, beta3 and beta7, are missing in the prephenate dehydrogenase domain of the bifunctional chorismate mutase-prephenate dehydrogenase enzymes. This observation suggests that the two functional domains of chorismate mutase-prephenate dehydrogenase are interdependent and explains why these domains cannot be separated.     

Crystal structure of Bacillus sp. GL1 xanthan lyase,which acts on the side chains of xanthan

Xanthan lyase, a member of polysaccharide lyase family 8, is a key enzyme for complete depolymerization of a bacterial heteropolysaccharide, xanthan, in Bacillus sp. GL1. The enzyme acts exolytically on the side chains of the polysaccharide. The x-ray crystallographic structure of xanthan lyase was determined by the multiple isomorphous replacement method. The crystal structures of xanthan lyase and its complex with the product (pyruvylated mannose) were refined at 2.3 and 2.4 A resolution with final R-factors of 17.5 and 16.9%, respectively. The refined structure of the product-free enzyme comprises 752 amino acid residues, 248 water molecules, and one calcium ion. The enzyme consists of N-terminal alpha-helical and C-terminal beta-sheet domains, which constitute incomplete alpha(5)/alpha(5)-barrel and anti-parallel beta-sheet structures, respectively. A deep cleft is located in the N-terminal alpha-helical domain facing the interface between the two domains. Although the overall structure of the enzyme is basically the same as that of the family 8 lyases for hyaluronate and chondroitin AC, significant differences were observed in the loop structure over the cleft. The crystal structure of the xanthan lyase complexed with pyruvylated mannose indicates that the sugar-binding site is located in the deep cleft, where aromatic and positively charged amino acid residues are involved in the binding. The Arg(313) and Tyr(315) residues in the loop from the N-terminal domain and the Arg(612) residue in the loop from the C-terminal domain directly bind to the pyruvate moiety of the product through the formation of hydrogen bonds, thus determining the substrate specificity of the enzyme.     

Crystal structure of novel NADP-dependent 3-hydroxyisobutyrate dehydrogenase from Thermus thermophilus HB8

3-Hydroxyisobutyrate, a central metabolite in the valine catabolic pathway, is reversibly oxidized to methylmalonate semialdehyde by a specific dehydrogenase belonging to the 3-hydroxyacid dehydrogenase family. To gain insight into the function of this enzyme at the atomic level, we have determined the first crystal structures of the 3-hydroxyisobutyrate dehydrogenase from Thermus thermophilus HB8: holo enzyme and sulfate ion complex. The crystal structures reveal a unique tetrameric oligomerization and a bound cofactor NADP+. This bacterial enzyme may adopt a novel cofactor-dependence on NADP, whereas NAD is preferred in eukaryotic enzymes. The protomer folds into two distinct domains with open/closed interdomain conformations. The cofactor NADP+ with syn nicotinamide and the sulfate ion are bound to distinct sites located at the interdomain cleft of the protomer through an induced-fit domain closure upon cofactor binding. From the structural comparison with the crystal structure of 6-phosphogluconate dehydrogenase, another member of the 3-hydroxyacid dehydrogenase family, it is suggested that the observed sulfate ion and the substrate 3-hydroxyisobutyrate share the same binding pocket. The observed oligomeric state might be important for the catalytic function through forming the active site involving two adjacent subunits, which seems to be conserved in the 3-hydroxyacid dehydrogenases. A kinetic study confirms that this enzyme has strict substrate specificity for 3-hydroxyisobutyrate and serine, but it cannot distinguish the chirality of the substrates. Lys165 is likely the catalytic residue of the enzyme.     

Interdomain motion in liver alcohol dehydrogenase. Structural and energetic analysis of the hinge bending mode

A study of the hinge bending mode in the enzyme liver alcohol dehydrogenase is made by use of empirical energy functions. The enzyme is a dimer, with each monomer composed of a coenzyme binding domain and a catalytic domain with a large cleft between the two. Superposition of the apoenzyme and holoenzyme crystal structures is used to determine a rigid rotation axis for closing of the cleft. It is shown that a rigid body transformation of the apoenzyme to the holoenzyme structure corresponds to a 10 degrees rotation of the catalytic domain about this axis. The rotation is not along the least-motion path for closing of the cleft but instead corresponds to the catalytic domain coming closer to the coenzyme binding domain by a sliding motion. Estimation of the energy associated with the interdomain motion of the apoenzyme over a range of 90 degrees (-40 to 50 degrees, where 0 degrees corresponds to the minimized crystal structure) demonstrates that local structural relaxation makes possible large-scale rotations with relatively small energy increments. A variety of structural rearrangements associated with the domain motion are characterized. They involve the hinge region residues that provide the covalent connections between the two domains and certain loop regions that are brought into contact by the rotation. Differences between the energy minimized and the holoenzyme structures point to the existence of alternative conformations for loops and to the importance of the ligands in the structural rearrangements.     

The structure of bovine glutamate dehydrogenase provides insights into the mechanism of allostery.

BACKGROUND: Bovine glutamate dehydrogenase (boGDH) is a homohexameric, mitochondrial enzyme that reversibly catalyzes the oxidative deamination of L-glutamate to 2-oxoglutarate using either NADP(H) or NAD(H) with comparable efficacy. GDH represents a key enzymatic link between catabolic and biosynthetic pathways, and is therefore ubiquitous in both higher and lower organisms. Only mammalian GDH exhibits strong negative cooperativity with respect to the coenzyme, however, and is regulated by a large number of allosteric effectors. RESULTS: The atomic structure of boGDH in complex with NADH, glutamate, and the allosteric inhibitor GTP has been determined to 2.8 A resolution. The major difference between the bacterial and bovine GDH structures is the presence of an additional 'antenna' in boGDH that protrudes from each trimer, twisting counterclockwise along the threefold axis. NADH and glutamate are clearly observed in the active site, but the contacts differ slightly from those observed in Clostridium symbiosum GDH. A second, inhibitory NADH molecule lies buried in the core of the hexamer. Finally, two GTP molecules bind near the hinge region connecting the NAD(+)- and glutamate-binding domains. CONCLUSIONS: We propose that the antenna serves as an intersubunit communication conduit during negative cooperativity and allosteric regulation. GTP and NADH inhibit GDH by keeping the catalytic cleft in a closed conformation. In contrast, ADP probably binds to the back of the NAD(+)-binding domain and activates the enzyme by keeping the catalytic cleft open. Extensive contacts between antennae within the crystal lattice may represent hexamer interactions in solution and, perhaps, with other enzymes within the mitochondrial matrix.     

Subunit assembly and active site location in the structure of glutamate dehydrogenase.

The three-dimensional crystal structure of the NAD(+)-linked glutamate dehydrogenase from Clostridium symbiosum has been solved to 1.96 A resolution by a combination of isomorphous replacement and molecular averaging and refined to a conventional crystallographic R factor of 0.227. Each subunit in this multimeric enzyme is organised into two domains separated by a deep cleft. One domain directs the self-assembly of the molecule into a hexameric oligomer with 32 symmetry. The other domain is structurally similar to the classical dinucleotide binding fold but with the direction of one of the strands reversed. Difference Fourier analysis on the binary complex of the enzyme with NAD+ shows that the dinucleotide is bound in an extended conformation with the nicotinamide moiety deep in the cleft between the two domains. Hydrogen bonds between the carboxyamide group of the nicotinamide ring and the side chains of T209 and N240, residues conserved in all hexameric GDH sequences, provide a positive selection for the syn conformer of this ring. This results in a molecular arrangement in which the A face of the nicotinamide ring is buried against the enzyme surface and the B face is exposed, adjacent to a striking cluster of conserved residues including K89, K113, and K125. Modeling studies, correlated with chemical modification data, have implicated this region as the glutamate/2-oxoglutarate binding site and provide an explanation at the molecular level for the B type stereospecificity of the hydride transfer of GDH during the catalytic cycle.     

Large-scale networks of hydration water molecules around bovine beta-trypsin revealed by cryogenic X-ray crystal structure analysis.

The hydration structure of bovine beta-trypsin was investigated in cryogenic X-ray diffraction experiments. Three crystal forms of the enzyme inhibited by benzamidine with different molecular packing were selected to deduce the hydration structure for the entire surface of the enzyme. The crystal structures in all three of the crystal forms were refined at the resolution of 1.8 A at 100 K and 293 K. The number of hydration water molecules around the enzyme at 100 K was 1.5 to two times larger than that at 293 K, indicating that the motion of hydration water was quenched by cooling. In particular, the increase in the number of hydration water molecules was prominent on flat and electrostatically neutral surface areas. The water-to-protein mass ratio and the radius of gyration of a structural model of hydrated trypsin at 100 K was consistent with the results obtained by other experimental techniques for proteins in solution. Hydration water molecules formed aggregates of various shapes and dimensions, and some of the aggregates even covered hydrophobic residues by forming oligomeric arrangements. In addition, the aggregates brought about large-scale networks of hydrogen bonds. The networks covered a large proportion of the surface of trypsin like a patchwork, and mechanically linked several secondary structures of the enzyme. By merging the hydration structures of the three crystal forms at 100 K, a distribution function of hydration water molecules was introduced to approximate the static hydration structure of trypsin in solution. The function showed that the negatively charged active site of trypsin tended to be easily exposed to bulk solvent. This result is of interest with respect to the solvent shielding effect and the recognition of a positively charged substrate by trypsin.     

Structure of a stabilizing disulfide bridge mutant that closes the active-site cleft of T4 lysozyme.

The engineered disulfide bridge between residues 21 and 142 of phage T4 lysozyme spans the active-site cleft and can be used as a switch to control the activity of the enzyme (Matsumura, M. & Matthews, B.W., 1989, Science 243, 792-794). In the oxidized form the disulfide increases the melting temperature of the protein by 11 degrees C at pH 2. The crystal structure of this mutant lysozyme has been determined in both the reduced and oxidized forms. In the reduced form, the crystal structure of the mutant is shown to be extremely similar to that of wild type. In the oxidized form, however, the formation of the disulfide bridge causes the alpha-carbons of Cys 21 and Cys 142, on opposite sides of the active-site cleft, to move toward each other by 2.5 A. In association with this movement, the amino-terminal domain of the protein undergoes a rigid-body rotation of 5.1 degrees relative to the carboxy-terminal domain. This rotation occurs about an axis passing through the junction of the amino-terminal and carboxy-terminal domains and is also close to the axis that best fits the apparent thermal motion of the amino-terminal domain seen previously in crystals of wild-type lysozyme. Even though the engineered Cys 21-Cys 142 disulfide links together the amino-terminal and carboxy-terminal domains of T4 lysozyme, it does not reduce the apparent mobility of the one domain relative to the other. The pronounced "hinge-bending" mobility of the amino-terminal domain that is suggested by the crystallographic thermal parameters of wild-type lysozyme persists in the oxidized (and reduced) mutant structures. In the immediate vicinity of the introduced disulfide bridge the mutant structure is more mobile (or disordered) than wild type, so much so that the exact conformation of Cys 21 remains obscure. As with the previously described disulfide bridge between residues 9 and 164 of T4 lysozyme (Pjura, P.E., Matsumura, M., Wozniak, J.A., & Matthews, B.W., 1990, Biochemistry 29, 2592-2598), the engineered cross-link substantially enhances the stability of the protein without making the folded structure more rigid.     

The crystal structure of Thermotoga maritima maltosyltransferase and its implications for the molecular basis of the novel transfer specificity.

Maltosyltransferase (MTase) from the hyperthermophile Thermotoga maritima represents a novel maltodextrin glycosyltransferase acting on starch and malto-oligosaccharides. It catalyzes the transfer of maltosyl units from alpha-1,4-linked glucans or malto-oligosaccharides to other alpha-1,4-linked glucans, malto-oligosaccharides or glucose. It belongs to the glycoside hydrolase family 13, which represents a large group of (beta/alpha)(8) barrel proteins sharing a similar active site structure. The crystal structures of MTase and its complex with maltose have been determined at 2.4 A and 2.1 A resolution, respectively. MTase is a homodimer, each subunit of which consists of four domains, two of which are structurally homologous to those of other family 13 enzymes. The catalytic core domain has the (beta/alpha)(8) barrel fold with the active-site cleft formed at the C-terminal end of the barrel. Substrate binding experiments have led to the location of two distinct maltose-binding sites; one lies in the active-site cleft, covering subsites -2 and -1; the other is located in a pocket adjacent to the active-site cleft. The structure of MTase, together with the conservation of active-site residues among family 13 glycoside hydrolases, are consistent with a common double-displacement catalytic mechanism for this enzyme. Analysis of maltose binding in the active site reveals that the transfer of dextrinyl residues longer than a maltosyl unit is prevented by termination of the active-site cleft after the -2 subsite by the side-chain of Lys151 and the stretch of residues 314-317, providing an explanation for the strict transfer specificity of MTase.