Crystal structure of UDP-galactose 4-epimerase from the hyperthermophilic archaeon Pyrobaculum calidifontis

The crystal structure of a highly thermostable UDP-galactose 4-epimerase (GalE) from the hyperthermophilic archaeon Pyrobaculum calidifontis was determined at a resolution of 1.8 Å. The asymmetric unit contained one subunit, and the functional dimer was generated by a crystallographic two-fold axis. Each monomer consisted of a Rossmann-fold domain with NAD bound and a carboxyl terminal domain. The overall structure of P. calidifontis GalE showed significant similarity to the structures of the GalEs from Escherichia coli, human and Trypanosoma brucei. However, the sizes of several surface loops were markedly smaller in P. calidifontis GalE than the corresponding loops in the other enzymes. Structural comparison revealed that the presence of an extensive hydrophobic interaction at the subunit interface is likely the main factor contributing to the hyperthermostability of the P. calidifontis enzyme. Within the NAD-binding site of P. calidifontis GalE, a loop (NAD-binding loop) tightly holds the adenine ribose moiety of NAD. Moreover, a deletion mutant lacking this loop bound NAD in a loose, reversible manner. Thus the presence of the NAD-binding loop in GalE is largely responsible for preventing the release of the cofactor from the holoenzyme.     

Molecular evolution and functional divergence of UDP-hexose 4-epimerases

UDP-glucose 4-epimerase (GalE) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) and/or the interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc) in sugar metabolism. GalEs belong to the short-chain dehydrogenase/reductase superfamily, use a conserved ‘transient keto intermediate’ mechanism and have variable substrate specificity. GalEs have been classified into three groups based on substrate specificity: group 1 prefers UDP-Glc/Gal, group 3 prefers UDP-GlcNAc/GalNAc, and group 2 has comparable activities for both types of the substrates. The phylogenetic relationship and structural basis for the specificities of GalEs revealed possible molecular evolution of UDP-hexose 4-epimerases in various organisms. Based on the recent advances in studies on GalEs and related enzymes, an updated view of their evolutional diversification is presented.     

Novel psychrophilic and thermolabile L-threonine dehydrogenase from psychrophilic Cytophaga sp. strain KUC-1

A psychrophilic bacterium, Cytophaga sp. strain KUC-1, that abundantly produces a NAD(+)-dependent L-threonine dehydrogenase was isolated from Antarctic seawater, and the enzyme was purified. The molecular weight of the enzyme was estimated to be 139,000, and that of the subunit was determined to be 35,000. The enzyme is a homotetramer. Atomic absorption analysis showed that the enzyme contains no metals. In these respects, the Cytophaga enzyme is distinct from other L-threonine dehydrogenases that have thus far been studied. L-Threonine and DL-threo-3-hydroxynorvaline were the substrates, and NAD(+) and some of its analogs served as coenzymes. The enzyme showed maximum activity at pH 9.5 and at 45 degrees C. The kinetic parameters of the enzyme are highly influenced by temperatures. The K(m) for L-threonine was lowest at 20 degrees C. Dead-end inhibition studies with pyruvate and adenosine-5'-diphosphoribose showed that the enzyme reaction proceeds via the ordered Bi Bi mechanism in which NAD(+) binds to an enzyme prior to L-threonine and 2-amino-3-oxobutyrate is released from the enzyme prior to NADH. The enzyme gene was cloned into Escherichia coli, and its nucleotides were sequenced. The enzyme gene contains an open reading frame of 939 bp encoding a protein of 312 amino acid residues. The amino acid sequence of the enzyme showed a significant similarity to that of UDP-glucose 4-epimerase from Staphylococcus aureus and belongs to the short-chain dehydrogenase-reductase superfamily. In contrast, L-threonine dehydrogenase from E. coli belongs to the medium-chain alcohol dehydrogenase family, and its amino acid sequence is not at all similar to that of the Cytophaga enzyme. L-Threonine dehydrogenase is significantly similar to an epimerase, which was shown for the first time. The amino acid residues playing an important role in the catalysis of the E. coli and human UDP-glucose 4-epimerases are highly conserved in the Cytophaga enzyme, except for the residues participating in the substrate binding.     

Crystal structures of apo wild-type M. jannaschii tyrosyl-tRNA synthetase (TyrRS) and an engineered TyrRS specific for O-methyl-L-tyrosine

The Methanococcus jannaschii tRNA(Tyr)/TyrRS pair has been engineered to incorporate unnatural amino acids into proteins in E. coli. To reveal the structural basis for the altered specificity of mutant TyrRS for O-methyl-L-tyrosine (OMeTyr), the crystal structures for the apo wild-type and mutant M. jannaschii TyrRS were determined at 2.66 and 3.0 A, respectively, for comparison with the published structure of TyrRS complexed with tRNA(Tyr) and substrate tyrosine. A large conformational change was found for the anticodon recognition loop 257-263 of wild-type TyrRS upon tRNA binding in order to facilitate recognition of G34 of the anticodon loop through pi-stacking and hydrogen bonding interactions. Loop 133-143, which is close to the tRNA acceptor stem-binding site, also appears to be stabilized by interaction with the tRNA(Tyr). Binding of the substrate tyrosine results in subtle and cooperative movements of the side chains within the tyrosine-binding pocket. In the OMeTyr-specific mutant synthetase structure, the signature motif KMSKS loop and acceptor stem-binding loop 133-143 were surprisingly ordered in the absence of bound ATP and tRNA. The active-site mutations result in altered hydrogen bonding and steric interactions which favor binding of OMeTyr over L-tyrosine. The structure of the mutant and wild-type TyrRS now provide a basis for generating new active-site libraries to evolve synthetases specific for other unnatural amino acids.     

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'.     

Conformational diversity in NAD(H) and interacting transhydrogenase nicotinamide nucleotide binding domains

Transhydrogenase (TH) couples direct and stereospecific hydride transfer between NAD(H) and NADP(H), bound within soluble domains I and III, respectively, to proton translocation across membrane bound domain II. The cocrystal structure of Rhodospirillum rubrum TH domains I and III has been determined in the presence of limiting NADH, under conditions in which the subunits reach equilibrium during crystallization. The crystals contain three heterotrimeric complexes, dI(2)dIII, in the asymmetric unit. Multiple conformations of loops and side-chains, and NAD(H) cofactors, are observed in domain I pertaining to substrate/product exchange, and highlighting electrostatic interactions during the hydride transfer. Two interacting NAD(H)-NADPH pairs are observed where alternate conformations of the NAD(H) phosphodiester and conserved arginine side-chains are correlated. In addition, the stereochemistry of one NAD(H)-NADPH pair approaches that expected for nicotinamide hydride transfer reactions. The cocrystal structure exhibits non-crystallographic symmetry that implies another orientation for domain III, which could occur in dimeric TH. Superposition of the "closed" form of domain III (PDB 1PNO, chain A) onto the dI(2)dIII complex reveals a severe steric conflict of highly conserved loops in domains I and III. This overlap, and the overlap with a 2-fold related domain III, suggests that motions of loop D within domain III and of the entire domain are correlated during turnover. The results support the concept that proton pumping in TH is driven by the difference in binding affinity for oxidized and reduced nicotinamide cofactors, and in the absence of a difference in redox potential, must occur through conformational effects.     

Redox-sensitive loops D and E regulate NADP(H) binding in domain III and domain I-domain III interactions in proton-translocating Escherichia coli transhydrogenase.

Membrane-bound transhydrogenases are conformationally driven proton-pumps which couple an inward proton translocation to the reversible reduction of NADP+ by NADH (forward reaction). This reaction is stimulated by an electrochemical proton gradient, Delta p, presumably through an increased release of NADPH. The enzymes have three domains: domain II spans the membrane, while domain I and III are hydrophilic and contain the binding sites for NAD(H) and NADP(H), respectively. Separately expressed domain I and III together catalyze a very slow forward reaction due to tightly bound NADP(H) in domain III. With the aim of examining the mechanistic role(s) of loop D and E in domain III and intact cysteine-free Escherichia coli transhydrogenase by cysteine mutagenesis, the conserved residues beta A398, beta S404, beta I406, beta G408, beta M409 and beta V411 in loop D, and residue beta Y431 in loop E were selected. In addition, the previously made mutants betaD392C and betaT393C in loop D, and beta G430C and beta A432C in loop E, were included. All loop D and E mutants, especially beta I406C and beta G430C, showed increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild-type enzyme. Determination of values indicated that the former increase was due to a strongly increased dissociation of NADPH caused by an altered conformation of loops D and E. In contrast, the cysteine-free G430C mutant of the intact enzyme showed the same inhibition of both forward and reverse rates. Most domain III mutants also showed a decreased affinity for domain I. The results support an important and regulatory role of loops D and E in the binding of NADP(H) as well as in the interaction between domain I and domain III.     

Crystal structure and enzyme kinetics of the (S)-specific 1-phenylethanol dehydrogenase of the denitrifying bacterium strain EbN1

(S)-1-Phenylethanol dehydrogenase (PED) from the denitrifying bacterium strain EbN1 catalyzes the NAD+-dependent, stereospecific oxidation of (S)-1-phenylethanol to acetophenone and the biotechnologically interesting reverse reaction. This novel enzyme belongs to the short-chain alcohol dehydrogenase/aldehyde reductase family. The coding gene (ped) was heterologously expressed in Escherichia coli and the purified protein was crystallized. The X-ray structures of the apo-form and the NAD+-bound form were solved at a resolution of 2.1 and 2.4 A, respectively, revealing that the enzyme is a tetramer with two types of hydrophobic dimerization interfaces, similar to beta-oxoacyl-[acyl carrier protein] reductase (FabG) from E. coli. NAD+-binding is associated with a conformational shift of the substrate binding loop of PED from a crystallographically unordered "open" to a more ordered "closed" form. Modeling the substrate acetophenone into the active site revealed the structural prerequisites for the strong enantioselectivity of the enzyme and for the catalytic mechanism. Studies on the steady-state kinetics of PED indicated a highly positive cooperativity of both catalytic directions with respect to the substrates. This is contrasted by the behavior of FabG. Moreover, PED exhibits extensive regulation on the enzyme level, being inhibited by elevated concentrations of substrates and products, as well as the wrong enantiomer of 1-phenylethanol. These regulatory properties of PED are consistent with the presence of a putative "transmission module" between the subunits. This module consists of the C-terminal loops of all four subunits, which form a special interconnected structural domain and mediate close contact of the subunits, and of a phenylalanine residue in each subunit that reaches out between substrate-binding loop and C-terminal domain of an adjacent subunit. These elements may transmit the substrate-induced conformational change of the substrate binding loop from one subunit to the others in the tetrameric complex and thus mediate the cooperative behavior of PED.     

Cosubstrate-induced dynamics of D-3-hydroxybutyrate dehydrogenase from Pseudomonas putida

D-3-Hydroxybutyrate dehydrogenase from Pseudomonas putida belongs to the family of short-chain dehydrogenases/reductases. We have determined X-ray structures of the D-3-hydroxybutyrate dehydrogenase from Pseudomonas putida, which was recombinantly expressed in Escherichia coli, in three different crystal forms to resolutions between 1.9 and 2.1 A. The so-called substrate-binding loop (residues 187-210) was partially disordered in several subunits, in both the presence and absence of NAD(+). However, in two subunits, this loop was completely defined in an open conformation in the apoenzyme and in a closed conformation in the complex structure with NAD(+). Structural comparisons indicated that the loop moves as a rigid body by about 46 degrees . However, the two small alpha-helices (alphaFG1 and alphaFG2) of the loop also re-orientated slightly during the conformational change. Probably, the interactions of Val185, Thr187 and Leu189 with the cosubstrate induced the conformational change. A model of the binding mode of the substrate D-3-hydroxybutyrate indicated that the loop in the closed conformation, as a result of NAD(+) binding, is positioned competent for catalysis. Gln193 is the only residue of the substrate-binding loop that interacts directly with the substrate. A translation, libration and screw (TLS) analysis of the rigid body movement of the loop in the crystal showed significant librational displacements, describing the coordinated movement of the substrate-binding loop in the crystal. NAD(+) binding increased the flexibility of the substrate-binding loop and shifted the equilibrium between the open and closed forms towards the closed form. The finding that all NAD(+) -bound subunits are present in the closed form and all NAD(+) -free subunits in the open form indicates that the loop closure is induced by cosubstrate binding alone. This mechanism may contribute to the sequential binding of cosubstrate followed by substrate.     

L-threonine dehydrogenase from Escherichia coli K-12: thiol-dependent activation by Mn2+

Addition of 1 mM Mn2+ to all solutions in the final chromatographic step used to purify L-threonine dehydrogenase (L-threonine:NAD+ oxidoreductase, EC 1.1.1.103) from extracts of Escherichia coli K-12 routinely provides 30-40 mg of pure enzyme per 100 g wet weight of cells with specific activity = 20-30 units/mg. Enzyme dialyzed exhaustively against buffers containing Chelex-100 resin has a specific activity = 8 units/mg and contains 0.003 or 0.02 mol of Mn2+/mol of enzyme as determined by radiolabeling studies with 54Mn2+ or by atomic absorption spectroscopy, respectively. Dehydrogenase activity is completely abolished by low concentrations of either Hg2+ or Ag+; of a large spectrum of other metal ions tested, only Mn2+ and Cd2+ have an activating effect. Activation of threonine dehydrogenase by Mn2+ is thiol-dependent and is saturable with an activation Kd = 9.0 microM and a Vmax = 105 units/mg. Stoichiometry of Mn2+ binding was found to be 0.86 mol of Mn2+/mol of enzyme subunit with a dissociation constant (Kd) = 8.5 microM. Mn2+ appears to interact directly with threonine dehydrogenase; gel filtration studies with the dehydrogenase plus 54Mn2+ in the presence of either NAD+, NADH, L-threonine, or combinations thereof show that only Mn2+ coelutes with the enzyme whereas all other ligands elute in the salt front and the stoichiometry of the dehydrogenase-Mn2+ interaction is not affected in any instance. A theoretical curve fit to data for the pH-activity profile of Mn2+-saturated enzyme has a pKa = 7.95 for one proton ionization. The data establish L-threonine dehydrogenase of E. coli to be a metal ion activated enzyme.     

The role of tyrosine residues in the extracellular domain of the 5-hydroxytryptamine3 receptor

Aromatic residues play an important role in the ligand-binding domain of Cys loop receptors. Here we examine the role of the 11 tyrosines in this domain of the 5-HT3 receptor in ligand binding and receptor function by substituting them for alanine, for serine, and, for some residues, also for phenylalanine. The mutant receptors were expressed in HEK293 cells and Xenopus oocytes and examined using radioligand binding, Ca2+ imaging, electrophysiology, and immunochemistry. The data suggest that Tyr50 and Tyr91 are critical for receptor assembly and/or structure, Tyr141 is important for antagonist binding and/or the structure of the binding pocket, Tyr143 plays a critical role in receptor gating and/or agonist binding, and Tyr153 and Tyr234 are involved in ligand binding and/or receptor gating. Tyr73, Tyr88, Tyr94, Tyr167, and Tyr240 do not appear to play major roles either in the structure of the extracellular domain or in ligand binding. The data support the location of these residues on a model of 5-HT docked into the ligand-binding domain and also provide evidence for the structural similarity of the extracellular domain to AChBP and the homologous regions of other Cys loop ligand-gated ion channels.     

Cofactor-induced conformational rearrangements establish a catalytically competent active site and a proton relay conduit in FabG

beta-Ketoacyl-acyl carrier protein reductase (FabG) is a key component in the type II fatty acid synthase system. The structures of Escherichia coli FabG and the FabG[Y151F] mutant in binary complexes with NADP(H) reveal that mechanistically important conformational changes accompany cofactor binding. The active site Ser-Tyr-Lys triad is repositioned into a catalytically competent constellation, and a hydrogen bonded network consisting of ribose hydroxyls, the Ser-Tyr-Lys triad, and four water molecules creates a proton wire to replenish the tyrosine proton donated during catalysis. Also, a disordered loop in FabG forms a substructure in the complex that shapes the entrance to the active site. A key observation is that the nicotinamide portion of the cofactor is disordered in the FabG[Y151F].NADP(H) complex, and Tyr151 appears to be necessary for high-affinity cofactor binding. Biochemical data confirm that FabG[Y151F] is defective in NADPH binding. Finally, structural changes consistent with the observed negative cooperativity of FabG are described.     

Anion binding properties of reduced and oxidized iron-containing superoxide dismutase reveal no requirement for tyrosine 34

We report the first spectroscopic observation of substrate analogue binding to the reduced state of iron superoxide dismutase from Escherichia coli (Fe(2+)SOD) and demonstrate that the pH dependence reflects inhibition of anion binding by ionized Tyr34, not loss of a positive contribution on the part of Tyr34's labile proton. This can also explain the pH dependence of the K(M) of Fe(2+)SOD. Thus, it appears that substrate binding to Fe(2+)SOD occurs in the second sphere and is not strongly coupled to hydrogen bond donation. Parallel investigations of substrate analogue binding to the oxidized state (Fe(3+)SOD) confirm formation of a six-coordinate complex and resolve the apparent conflict with earlier nuclear magnetic relaxation dispersion (NMRD) results. Thus, we propose that two F(-) ions can bind to the oxidized Fe(3+)SOD active site, either displacing the coordinated solvent or lowering its exchange rate with bulk solvent. We show that neutral Tyr34's unfavorable effect on binding of the substrate analogue N(3)(-) can be ascribed to steric interference, as it does not apply to the smaller substrate analogues F(-) and OH(-). Finally, we report the first demonstration that HS(-) can act as a substrate analogue with regard both to redox reactivity with FeSOD and to ability to coordinate to the active site Fe(3+). Indeed, it forms a novel green complex. Thus, we have begun to evaluate the relative importance of different contributions that Tyr34 may make to substrate binding, and we have identified a novel, redox active substrate analogue that offers new possibilities for elucidating the mechanism of FeSOD.     

Structure-based mutational analysis of the active site residues of -hydantoinase

We previously proposed the hydrophobic and bulky residues of the three loops, designated stereochemistry gate loops (SGLs), to constitute a hydrophobic substrate binding pocket of -hydantoinase from Bacillus stearothermophilus SD1. Simulation of substrate binding in the active site of -hydantoinase and sequence alignment of various -hydantoinases revealed the critical hydrophobic residues closely located around the exocyclic substituent of substrate. To evaluate the roles of these residues in substrate binding pocket, site-directed mutagenesis was performed specifically for Leu 65, Tyr 155, and Phe 159. When Tyr 155 was mutated to Phe and Glu, both mutants Y155F and Y155E were totally inactive for nonsubstituted hydantoin and -5-hydroxyphenyl hydantoin (HPH), which indicates that Tyr 155 is involved in substrate binding via a hydrogen bond with the hydantoinic ring. Furthermore, replacement of the hydrophobic residues Leu 65 and Phe 159 with Glu, a charged amino acid, resulted in a significant decrease in activity for nonsubstituted hydantoin, but not for HPH. The K_cat values of both mutants for nonsubstituted hydantoin also severely decreased, but a slight change in the K_cat values was observed towards HPH. These results suggest that the hydrophobic residues in SGLs play an essential role in substrate binding, and differentially interact according to the property of the exocyclic substituent.     

Specificity of the proton adenosinetriphosphatase of Escherichia coli for adenine, guanine, and inosine nucleotides in catalysis and binding

Specificity of the Escherichia coli proton ATPase for adenine, guanine, and inosine nucleotides in catalysis and binding was studied. MgADP, CaADP, MgGDP, and MgIDP were each good substrates for oxidative phosphorylation. The corresponding triphosphates were each substrates for hydrolysis and proton pumping. At 1 mM concentration, MgATP, MgGTP, and MgITP drove proton pumping with equal efficiency. At 0.1 mM concentration, MgATP was 4-fold more efficient than MgITP or MgGTP. Nucleotide-depleted soluble F1 could rebind to F1-depleted membranes and block proton conductivity through F0; rebound nucleotide-depleted F1 catalyzed pH gradient formation with MgATP, MgGTP, or MgITP. This showed that the nonexchangeable nucleotide sites on F1 need not be occupied by adenine nucleotide for proton pumping to occur. It was further shown that no nucleotide was tightly bound in the nonexchangeable sites of F1 during proton pumping driven by MgGTP in these reconstituted membranes, whereas adenine nucleotide was tightly bound when MgATP was the substrate. Nucleotide-depleted soluble F1 bound maximally 5.9 ATP, 3.2 GTP, and 3.6 ITP of which half the ATP and almost all of the GTP and ITP exchanged over a period of 30-240 min with medium ADP or ATP. Also, half of the bound ATP exchanged with medium GTP or ITP. These data showed that inosine and guanine nucleotides do not bind to soluble F1 in nonexchangeable fashion, in contrast to adenine nucleotides. Purified alpha-subunit from F1 bound ATP at a single site but showed no binding of GTP nor ITP, supporting previous suggestions that the non-exchangeable sites in intact F1 are on alpha-subunits.     

Structures of Escherichia coli branched-chain amino acid aminotransferase and its complexes with 4-methylvalerate and 2-methylleucine: induced fit and substrate recognition of the enzyme.

The following three-dimensional structures of three forms of Escherichia coli branched-chain amino acid aminotransferase (eBCAT) have been determined by the X-ray diffraction method: the unliganded pyridoxal 5'-phosphate (PLP) form at a 2.1 A resolution, and the two complexes with the substrate analogues, 4-methylvalerate (4-MeVA) as the Michaelis complex model and 2-methylleucine (2-MeLeu) as the external aldimine model at 2.4 A resolution. The enzyme is a trimer of dimers, and each subunit consists of small and large domains, and the interdomain loop. The active site is formed by the residues at the domain interface and those from two loops of the other subunit of the dimer unit, and binds one PLP with its re-face directed toward the protein side. Upon binding of a substrate, Arg40 changes its side-chain direction to interact with the interdomain loop, and the loop, which is disordered in the unliganded form, shows its ordered structure on the active-site cavity, interacts with the hydrophobic side chain of the substrate, and shields it from the solvent region. The substrate binds to the active-site pocket with its alpha-hydrogen toward the protein side, its side-chain on the side of O3 of PLP, and its alpha-carboxylate on the side of the phosphate group of PLP. The hydrophobic side-chain of the substrate is recognized by Phe36, Trp126, Tyr129, Tyr164, Tyr31*, and Val109*. The alpha-carboxylate of the substrate binds to the unique site constructed by three polar groups (two main-chain NH groups of the beta-turn at Thr257 and Ala258 and the hydroxy group of Tyr95) which are activated by the access of Arg40 to the main-chain C=O group of the beta-turn and the coordination of Arg97 to the hydroxy group. Since Arg40 is the only residue that significantly changes its side-chain conformation and directly interacts with the interdomain loop and the beta-turn, the residue plays important roles in the induced fit of the interdomain loop and the alpha-carboxylate recognition of the substrate.     

Crystal Structure of Three Consecutive Laminin-type Epidermal Growth Factor-like (LE) Modules of Laminin γ1 Chain Harboring the Nidogen Binding Site

The structure of three consecutive laminin-type EGF-like (LE) modules of mouse laminin γ1 chain, γ1III3-5 (positions 738 to 899), has been determined by multiple isomorphous replacement in a crystal of space groupP6₄22 (a=b=74.57 Å,c=185.11 Å and γ=120°). The crystal structure was refined using restrained crystallographic refinement to an R-factor of 19.72 % for 14,983 independent reflections with intensitiesF_obs> 0 at 2.1 Å resolution, with root mean square deviations of 0.012 Å and 1.690° from ideal bond lengths and bond angles, respectively. The final model consisted of 1179 (non-hydrogen) protein atoms within 162 residues and 119 water molecules. The molecule showed a rod-like structure of about 76 Å length with individual modules twisted relative to each other by about 70°. Each module had the same disulfide bond connections Cys1-Cys3 (loop a), Cys2-Cys4 (loop b), Cys5-Cys6 (loop c) and Cys7-Cys8 (loop d), the first three being identical to epidermal growth factor (EGF). All three LE modules showed little secondary structure which was mainly restricted to loop d, but they differed in several other details of their structure. The interface contacts between the LE modules are based on hydrogen bonds and hydrophobic interactions between the hydrophobic core of loop d of the preceding module and the first cysteine and an exposed residue in loop b of the following module. Module 4 was previously shown to contribute the major nidogen binding site of laminins and site-directed mutagenesis demonstrated a specific binding role for Asp800, Asn802, Val804 and Tyr819 in loops a and c. The side-chains of these four residues are all located on the surface in a linear array and separated by a distance of 17 Å between Tyr819 and Val804. The entire nidogen binding site is stabilizedviamain-chain hydrogen bonds which are in part derived from the link between loops b and c (residues Leu815 and Lys816). The data demonstrate the unique nature of the LE modules and only a remote similarity to EGF. They also indicate that the crucial residues in the binding loops provide direct contacts with nidogen and explain the synergism between loops a and c which is essential for binding.     

Mutagenesis of folylpolyglutamate synthetase indicates that dihydropteroate and tetrahydrofolate bind to the same site

The folylpolyglutamate synthetase (FPGS) enzyme of Escherichia coli differs from that of Lactobacillus casei in having dihydrofolate synthetase activity, which catalyzes the production of dihydrofolate from dihydropteroate. The present study undertook mutagenesis to identify structural elements that are directly responsible for the functional differences between the two enzymes. The amino terminal domain (residues 1-287) of the E. coli FPGS was found to bind tetrahydrofolate and dihydropteroate with the same affinity as the intact enzyme. The domain-swap chimera proteins between the E. coli and the L. casei enzymes possess both folate or pteroate binding properties and enzymatic activities of their amino terminal portion, suggesting that the N-terminal domain determines the folate substrate specificity. Recent structural studies have identified two unique folate binding sites, the omega loop in L. casei FPGS and the dihydropteroate binding loop in the E. coli enzyme. Mutants with swapped omega loops retained the activities and folate or pteroate binding properties of the rest of the enzyme. Mutating L. casei FPGS to contain an E. coli FPGS dihydropteroate binding loop did not alter its substrate specificity to using dihydropteroate as a substrate. The mutant D154A, a residue specific for the dihydropteroate binding site in E. coli FPGS, and D151A, the corresponding mutant in the L. casei enzyme, were both defective in using tetrahydrofolate as their substrate, suggesting that the binding site corresponding to the E. coli pteroate binding site is also the tetrahydrofolate binding site for both enzymes. Tetrahydrofolate diglutamate was a slightly less effective substrate than the monoglutamate with the wild-type enzyme but was a 40-fold more effective substrate with the D151A mutant. This suggests that the 5,10-methylenetetrahydrofolate binding site identified in the L. casei ternary structure may bind diglutamate and polyglutamate folate derivatives.