Structure, specificity and function of cyclomaltodextrinase, a multispecific enzyme of the alpha-amylase family

Cyclomaltodextrinase (CDase, EC 3.2.1.54), maltogenic amylase (EC 3. 2.1.133), and neopullulanase (EC 3.2.1.135) are reported to be capable of hydrolyzing all or two of the following three types of substrates: cyclomaltodextrins (CDs); pullulan; and starch. These enzymes hydrolyze CDs and starch to maltose and pullulan to panose by cleavage of alpha-1,4 glycosidic bonds whereas alpha-amylases essentially lack activity on CDs and pullulan. They also catalyze transglycosylation of oligosaccharides to the C3-, C4- or C6-hydroxyl groups of various acceptor sugar molecules. The present review surveys the biochemical, enzymatic, and structural properties of three types of such enzymes as defined based on the substrate specificity toward the CDs: type I, cyclomaltodextrinase and maltogenic amylase that hydrolyze CDs much faster than pullulan and starch; type II, Thermoactinomyces vulgaris amylase II (TVA II) that hydrolyzes CDs much less efficiently than pullulan; and type III, neopullulanase that hydrolyzes pullulan efficiently, but remains to be reported to hydrolyze CDs. These three types of enzymes exhibit 40-60% amino acid sequence identity. They occur in the cytoplasm of bacteria and have molecular masses from 62 to 90 kDa which are slightly larger than those of most alpha-amylases. Multiple amino acid sequence alignment and crystal structures of maltogenic amylase and TVA II reveal the presence of an N-terminal extension of approximately 130 residues not found in alpha-amylases. This unique N-terminal domain as seen in the crystal structures apparently contributes to the active site structure leading to the distinct substrate specificity through a dimer formation. In aqueous solution, most of these enzymes show a monomer-dimer equilibrium. The present review discusses the multiple specificity in the light of the oligomerization and the molecular structures arriving at a clarified enzyme classification. Finally, a physiological role of the enzymes is proposed.     

Identification of Tyr(290(6.58)) of the human gonadotropin-releasing hormone (GnRH) receptor as a contact residue for both GnRH I and GnRH II: importance for high-affinity binding and receptor activation

Molecular modeling showed interactions of Tyr (290(6.58)) in transmembrane domain 6 of the GnRH receptor with Tyr (5) of GnRH I, and His (5) of GnRH II. The wild-type receptor exhibited high affinity for [Phe (5)]GnRH I and [Tyr (5)]GnRH II, but 127- and 177-fold decreased affinity for [Ala (5)]GnRH I and [Ala (5)]GnRH II, indicating that the aromatic ring in position 5 is crucial for receptor binding. The receptor mutation Y290F decreased affinity for GnRH I, [Phe (5)]GnRH I, GnRH II and [Tyr (5)]GnRH II, while Y290A and Y290L caused larger decreases, suggesting that both the para-OH and aromatic ring of Tyr (290(6.58)) are important for binding of ligands with aromatic residues in position 5. Mutating Tyr (290(6.58)) to Gln increased affinity for Tyr (5)-containing GnRH analogues 3-12-fold compared with the Y290A and Y290L mutants, suggesting a hydrogen-bond between Gln of the Y290Q mutant and Tyr (5) of GnRH analogues. All mutations had small effects on affinity of GnRH analogues that lack an aromatic residue in position 5. These results support direct interactions of the Tyr (290(6.58)) side chain with Tyr (5) of GnRH I and His (5) of GnRH II. Tyr (290(6.58)) mutations, except for Y290F, caused larger decreases in GnRH potency than affinity, indicating that an aromatic ring is important for the agonist-induced receptor conformational switch.     

SCH28080, a K+-competitive inhibitor of the gastric H,K-ATPase,binds near the M5-6 luminal loop,preventing K+ access to the ion binding domain

Inhibition of the gastric H,K-ATPase by the imidazo[1,2-alpha]pyridine, SCH28080, is strictly competitive with respect to K+ or its surrogate, NH4+. The inhibitory kinetics [V(max), K(m,app)(NH4+), K(i)(SCH28080), and competitive, mixed, or noncompetitive] of mutants can define the inhibitor binding domain and the route to the ion binding region within M4-6. While mutations Y799F, Y802F, I803L, S806N, V807I (M5), L811V (M5-6), Y928H (M8), and Q905N (M7-8) had no effect on inhibitor kinetics, mutations P798C, Y802L, P810A, P810G, C813A or -S, I814V or -F, F818C, T823V (M5, M5-6, and M6), E914Q, F917Y, G918E, T929L, and F932L (M7-8 and M8) reduced the affinity for SCH28080 up to 10-fold without affecting the nature of the kinetics. In contrast, the L809F substitution in the loop between M5 and M6 resulted in an approximately 100-fold decrease in inhibitor affinity, and substitutions L809V, I816L, Y925F, and M937V (M5-6, M6, and M8) reduced the inhibitor affinity by 10-fold, all resulting in noncompetitive kinetics. The mutants L811F, Y922I, and I940A also reduced the inhibitor affinity up to 10-fold but resulted in mixed inhibition. The mutations I819L, Q923V, and Y925A also gave mixed inhibition but without a change in inhibitor affinity. These data, and the 9-fold loss of SCH28080 affinity in the C813T mutant, suggest that the binding domain for SCH28080 contains the surface between L809 in the M5-6 loop and C813 at the luminal end of M6, approximately two helical turns down from the ion binding region, where it blocks the normal ion access pathway. On the basis of a model of the Ca-ATPase in the E2 conformation (PDB entry 1kju), the mutants that change the nature of the kinetics are arranged on one side of M8 and on the adjacent side of the M5-6 loop and M6 itself. This suggests that mutations in this region modify the enzyme structure so that K+ can access the ion binding domain even with SCH28080 bound.     

Analysis of catalytic residues of Thermoactinomyces vulgaris R-47 alpha-amylase II (TVA II) by site-directed mutagenesis

To confirm that the catalytic residues (Asp325, Glu354, and Asp421) are necessary for the hydrolysis of starch, pullulan, and cyclodextrins, we constructed TVA II mutated by site-directed mutagenesis. The mutated enzymes (D325N, E354Q, and D421N) had markedly reduced levels of activity, less than 0.006% of the wild type, indicating that these three residues are the catalytic sites for these substrates. Even E354D had reduced levels of activity, less than 0.05% of wild type. These four mutated enzymes retained a trace of activity. From the result of hydrolysis patterns for maltohexaose, in particular, D421N, unlike D325N and E354Q, catalyzed transglycosylation rather than hydrolysis. The results suggest that Asp421 could function to capture water molecules.     

The 'pair of sugar tongs' site on the non-catalytic domain C of barley alpha-amylase participates in substrate binding and activity

Some starch-degrading enzymes accommodate carbohydrates at sites situated at a certain distance from the active site. In the crystal structure of barley alpha-amylase 1, oligosaccharide is thus bound to the 'sugar tongs' site. This site on the non-catalytic domain C in the C-terminal part of the molecule contains a key residue, Tyr380, which has numerous contacts with the oligosaccharide. The mutant enzymes Y380A and Y380M failed to bind to beta-cyclodextrin-Sepharose, a starch-mimic resin used for alpha-amylase affinity purification. The K(d) for beta-cyclodextrin binding to Y380A and Y380M was 1.4 mm compared to 0.20-0.25 mm for the wild-type, S378P and S378T enzymes. The substitution in the S378P enzyme mimics Pro376 in the barley alpha-amylase 2 isozyme, which in spite of its conserved Tyr378 did not bind oligosaccharide at the 'sugar tongs' in the structure. Crystal structures of both wild-type and S378P enzymes, but not the Y380A enzyme, showed binding of the pseudotetrasaccharide acarbose at the 'sugar tongs' site. The 'sugar tongs' site also contributed importantly to the adsorption to starch granules, as Kd = 0.47 mg.mL(-1) for the wild-type enzyme increased to 5.9 mg.mL(-1) for Y380A, which moreover catalyzed the release of soluble oligosaccharides from starch granules with only 10% of the wild-type activity. beta-cyclodextrin both inhibited binding to and suppressed activity on starch granules for wild-type and S378P enzymes, but did not affect these properties of Y380A, reflecting the functional role of Tyr380. In addition, the Y380A enzyme hydrolyzed amylose with reduced multiple attack, emphasizing that the 'sugar tongs' participates in multivalent binding of polysaccharide substrates.     

Analysis of Catalytic Residues of Thermoactinomyces vulgaris R-47 α-Amylase II (TVA II) by Site-directed Mutagenesis

To confirm that the catalytic residues (Asp325, Glu354, and Asp421) are necessary for the hydrolysis of starch, pullulan, and cyclodextrins, we constructed TVA II mutated by site-directed mutagenesis. The mutated enzymes (D325N, E354Q, and D421N) had markedly reduced levels of activity, less than 0.006% of the wild type, indicating that these three residues are the catalytic sites for these substrates. Even E354D had reduced levels of activity, less than 0.05% of wild type. These four mutated enzymes retained a trace of activity. From the result of hydrolysis patterns for maltohexaose, in particular, D421N, unlike D325N and E354Q, catalyzed transglycosylation rather than hydrolysis. The results suggest that Asp421 could function to capture water molecules.     

Crystallographic structure of ChitA,a glycoside hydrolase family 19, plant class IV chitinase from Zea mays

Maize ChitA chitinase is composed of a small, hevein‐like domain attached to a carboxy‐terminal chitinase domain. During fungal ear rot, the hevein‐like domain is cleaved by secreted fungal proteases to produce truncated forms of ChitA. Here, we report a structural and biochemical characterization of truncated ChitA (ChitA ΔN), which lacks the hevein‐like domain. ChitA ΔN and a mutant form (ChitA ΔN‐EQ) were expressed and purified; enzyme assays showed that ChitA ΔN activity was comparable to the full‐length enzyme. Mutation of Glu62 to Gln (ChitA ΔN‐EQ) abolished chitinase activity without disrupting substrate binding, demonstrating that Glu62 is directly involved in catalysis. A crystal structure of ChitA ΔN‐EQ provided strong support for key roles for Glu62, Arg177, and Glu165 in hydrolysis, and for Ser103 and Tyr106 in substrate binding. These findings demonstrate that the hevein‐like domain is not needed for enzyme activity. Moreover, comparison of the crystal structure of this plant class IV chitinase with structures from larger class I and II enzymes suggest that class IV chitinases have evolved to accommodate shorter substrates.     

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.     

Cytomegalovirus capsid protease: biological substrates are cleaved more efficiently by full-length enzyme (pUL80a) than by the catalytic domain (assemblin)

We compared the full-length capsid maturational protease (pPR, pUL80a) of human cytomegalovirus with its proteolytic domain (assemblin) for the ability to cleave two biological substrates, and we found that pPR is more efficient with both. Affinity-purified, refolded enzymes and substrates were combined under defined reaction conditions, and cleavage was monitored and quantified following staining of the resulting electrophoretically separated fragments. The enzymes were stabilized against self-cleavage by a single point mutation in each cleavage site (ICRMT-pPR and IC-assemblin). The substrates were pPR itself, inactivated by replacing its catalytic nucleophile (S132A-pPR), and the sequence-related assembly protein precursor (pAP, pUL80.5). Our results showed that (i) ICRMT-pPR is 5- to 10-fold more efficient than assemblin for all cleavages measured (i.e., the M site of pAP and the M, R, and I sites of S132A-pPR). (ii) Cleavage of substrate S132A-pPR proceeded M>R>I for both enzymes. (iii) Na(2)SO(4) reduced M- and R-site cleavage efficiency by ICRMT-pPR, in contrast to its enhancing effect for both enzymes on I site and small peptide cleavage. (iv) Disrupting oligomerization of either the pPR enzyme or substrate by mutating Leu382 in the amino-conserved domain reduced cleavage efficiency two- to fourfold. (v) Finally, ICRMT-pPR mutants that include the amino-conserved domain, but terminate with Pro481 or Tyr469, retain the enzymatic characteristics that distinguish pPR from assemblin. These findings show that the scaffolding portion of pPR increases its enzymatic activity on biologically relevant protein substrates and provide an additional link between the structure of this essential viral enzyme and its biological mechanism.     

Binding site analysis of cellulose binding domain CBD(N1) from endoglucanse C of Cellulomonas fimi by site-directed mutagenesis

Endoglucanase C (CenC), a beta1,4 glucanase from the soil bacterium Cellulomonas fimi, binds to amorphous cellulose via two homologous cellulose binding domains, termed CBD(N1) and CBD(N2). In this work, the contributions of 10 amino acids within the binding cleft of CBD(N1) were evaluated by single site-directed mutations to alanine residues. Each isolated domain containing a single mutation was analyzed for binding to an insoluble amorphous preparation of cellulose, phosphoric acid swollen Avicel (PASA), and to a soluble glucopyranoside polymer, barley beta-glucan. The effect of any given mutation on CBD binding was similar for both substrates, suggesting that the mechanism of binding to soluble and insoluble substrates is the same. Tyrosines 19 and 85 were essential for tight binding by CBD(N1) as their replacement by alanine results in affinity decrements of approximately 100-fold on PASA, barley beta-glucan, and soluble cellooligosaccharides. The tertiary structures of unbound Y19A and Y85A were assessed by heteronuclear single quantum coherence (HSQC) spectroscopy. These studies indicated that the structures of both mutants were perturbed but that all perturbations are very near to the site of mutation.     

Identification of amino acids involved in catalytic process of M. tuberculosis GlmU acetyltransferase

M. tuberculosis GlmU is a bifunctional enzyme with acetyltransferase activity in C-terminus and uridyltransferase activity in N-terminus, and it is involved in the biosynthesis of glycosyl donor UDP-N-acetylglucosamine (UDP-GlcNAc). The crystal structure of M. tuberculosis GlmU clearly determines the active site and catalytic mechanism of GlmU uridyltransferase domain but not succeed in GlmU acetyltransferase domain. Sequence comparison analysis revealed highly conserved amino acid residues in the C-terminus between M. tuberculosis GlmU and GlmU enzymes from other bacteria. To find the essential amino acids related to M. tuberculosis GlmU acetyltransferase activity, we substituted 10 conserved amino acids in the acetyltransferase domain of M. tuberculosis GlmU by site-directed mutagenesis. All the mutant GlmU proteins were largely expressed in soluble and purified by affinity chromatography. Enzyme assays showed that K362A, H374A, Y398A and W460A mutants abolished more than 90?% activity of M. tuberculosis GlmU acetyltransferase and totally lost the affinity with two substrates, suggesting the potential substrate-binding functions. However, K403A, S416A, N456A and E458A mutants exhibited decreased GlmU acetyltransferase activity and lower kinetic parameters, probably responsible for substrate releasing by conformation shifting.     

Tryptophan scanning mutagenesis in the TM3 domain of the Torpedo californica acetylcholine receptor beta subunit reveals an alpha-helical structure

We used tryptophan substitutions to characterize the beta M3 transmembrane domain (betaTM3) of the acetylcholine receptor (AChR). We generated 15 mutants with tryptophan substitutions within the betaTM3 domain, between residues R282W and I296W. The various mutants were injected into Xenopus oocytes, and expression levels were measured by [125I]-alpha-bungarotoxin binding. Expression levels of the M288W, I289W, L290W, and F293W mutants were similar to that of wild type, whereas the other mutants (R282W, Y283W, L284W, F286W, I287W, V291W, A292W, S294W, V295W, and I296W) were expressed at much lower levels than that of wild type. None of these tryptophan mutants produced peak currents larger than that of wild type. Five of the mutants, L284W, F286W, I287W, V295W, and I296W, were expressed at levels <15% of the wild type. I296W had the lowest expression levels and did not display any significant ACh-induced current, suggesting that this position is important for the function and assembly of the AChR. Tryptophan substitution at three positions, L284, V291, and A292, dramatically inhibited AChR assembly and function. A periodicity analysis of the alterations in AChR expression at positions 282-296 of the betaTM3 domain was consistent with an alpha-helical structure. Residues known to be exposed to the membrane lipids, including R282, M285, I289, and F293, were all found in all the upper phases of the oscillatory pattern. Mutants that were expressed at lower levels are clustered on one side of a proposed alpha-helical structure. These results were incorporated into a structural model for the spatial orientation of the TM3 of the Torpedo californica beta subunit.     

Regulation of glycan processing by Golgi enzymes from red kidney bean (Phaseolus vulgaris) seedlings.

The effect of a protein matrix on the processing of glycoprotein glycans by Golgi enzymes from plant seedlings has been determined with an artificial glycoprotein system, comparing the processing rates of glycan-(biotinyl)Asn (or glycan-(biotinamidohexanoyl)Asn) substrates either free or bound to avidin. An analysis of the pooled glycoproteins from the seedlings suggested that the most common glycan structure is a complex one (GlcNAc-Man3GlycNAc2-protein), and consistent with this processing end-product, mannosidases I and II and GlcNAc transferases I and II were all found to be present in the seedling Golgi membrane preparations. The effect of the avidin matrix either in a proximal (biotinyl substrates) or distal (N-(biotinamido)hexonoyl substrates) association with the appropriate glycan substrate for these four enzymes was assessed from the direct comparison of the apparent first-order rate constants for the free and avidin-bound substrate-product conversions. All four plant enzymes were inhibited by the association of the glycan substrates with avidin, but the inhibition was much less pronounced than that observed with the corresponding enzymes from rat liver and hen oviduct. The rate effect shows a progression from 3- to 10-fold rate decreases in the proximal complexes and 2- to 3-fold in the distal complexes in going from the first (mannosidase I) to the fourth (GlcNAc transferase II) enzyme; with the mammalian and avian enzymes the largest effects were for the first ones and much larger absolute rate effects were observed. The results suggest that the nature of the processing enzymes in terms of this response to the avidin glycan substrates may differ in different organisms.     

The crystal structure of Thermoactinomyces vulgaris R-47 alpha-amylase II (TVA II) complexed with transglycosylated product.

Alphan alpha-amylase (TVA II) from Thermoactinomyces vulgaris R-47 efficiently hydrolyzes alpha-1,4-glucosidic linkages of pullulan to produce panose in addition to hydrolyzing starch. TVA II also hydrolyzes alpha-1,4-glucosidic linkages of cyclodextrins and alpha-1,6-glucosidic linkages of isopanose. To clarify the basis for this wide substrate specificity of TVA II, we soaked 4(3)-alpha-panosylpanose (4(3)-P2) (a pullulan hydrolysate composed of two panosyl units) into crystals of D325N inactive mutated TVA II. We then determined the crystal structure of TVA II complexed with 4(2)-alpha-panosylpanose (4(2)-P2), which was produced by transglycosylation from 4(3)-P2, at 2.2-A resolution. The shape of the active cleft of TVA II is unique among those of alpha-amylase family enzymes due to a loop (residues 193-218) that is located at the end of the cleft around the nonreducing region and forms a 'dam'-like bank. Because this loop is short in TVA II, the active cleft is wide and shallow around the nonreducing region. It is assumed that this short loop is one of the reasons for the wide substrate specificity of TVA II. While Trp356 is involved in the binding of Glc +2 of the substrate, it appears that Tyr374 in proximity to Trp356 plays two roles: one is fixing the orientation of Trp356 in the substrate-liganded state and the other is supplying the water that is necessary for substrate hydrolysis.     

The peptide-substrate-binding domain of human collagen prolyl 4-hydroxylases. Backbone assignments,secondary structure,and binding of proline-rich peptides

The collagen prolyl 4-hydroxylases (C-P4Hs) catalyze the formation of 4-hydroxyproline by the hydroxylation of proline residues in -Xaa-Pro-Gly-sequences. The vertebrate enzymes are alpha 2 beta 2 tetramers in which protein-disulfide isomerase serves as the beta subunit. Two isoforms of the catalytic alpha subunit have been identified and shown to form [alpha(I)]2 beta 2 and [alpha(II)]2 beta 2 tetramers, the type I and type II C-P4Hs, respectively. The peptide-substrate-binding domain of type I C-P4H has been shown to be located between residues 138 and 244 in the 517-residue alpha(I) subunit and to be distinct from the catalytic domain that is located in the C-terminal region. We report here that a recombinant human C-P4H alpha(I) polypeptide Phe144-Ser244 forms a folded domain consisting of five alpha helices and one short beta strand. This structure is quite different from those of other proline-rich peptide-binding modules, which consist mainly of beta strands. Binding of the peptide (Pro-Pro-Gly)2 to this domain caused major chemical shifts in many backbone amide resonances, the residues showing the largest shifts being mainly hydrophobic, including three tyrosines. The Kd values determined by surface plasmon resonance and isothermal titration calorimetry for the binding of several synthetic peptides to the alpha(I) and the corresponding alpha(II) domain were very similar to the Km and Ki values for these peptides as substrates and inhibitors of the type I and type II C-P4H tetramers. The Kd values of the alpha(I) and alpha(II) domains for (Gly-Pro-4Hyp)5 were much higher than those for (Pro-Pro-Gly)5, indicating a marked decrease in the affinity of hydroxylated peptides for the domain. Many characteristic features of the binding of peptides to the type I and type II C-P4H tetramers can thus be explained by the properties of binding to this domain rather than the catalytic domain.     

Site-directed mutagenesis studies of the NADPH-binding domain of rat steroid 5alpha-reductase (isozyme-1) I: analysis of aromatic and hydroxylated amino acid residues.

Previous studies have shown that the reduced nicotinamide adenine dinucleotide phosphate (NADPH)- binding domain of rat liver microsomal steroid 5alpha-reductase isozyme-1 (r5alphaR-1) is in a highly conserved region of the polypeptide sequence (residues 160-190). In this study, we investigated, by site-directed mutagenesis, the role of hydroxylated and aromatic amino acids within the NADPH-binding domain. The r5alphaR-1 cDNA was cloned into a pCMV vector, and the double strand site-directed mutagenesis method was used to create mutants Y179F, Y179S, Y189F, Y189S, S164A, S164T, and Y187F, which were subsequently expressed in COS-1 cells. Kinetic studies of the expressed enzymes showed that the mutation Y179F resulted in an approximately 40-fold increase in the Km for NADPH versus wild-type, with only a 2-fold increase in the Km for testosterone. The mutants Y189F and S164A showed smaller increases (4 and 6-fold) in Kms for NADPH and no significant change in the Km for testosterone, whereas Y189S had kinetic properties similar to the wild-type r5alphaR-1. Mutants Y179S and S164T both resulted in inactive enzymes, whereas F187Y showed an approximately 5-fold decrease in Km for NADPH and a significant increase (approximately 18-fold) in the Km for testosterone. The results suggest that the -OH functionality of Y179 is involved in cofactor binding, but is not essential for the activity of the enzyme, whereas the -OH functionalities of Y189 and S164 play lesser roles in cofactor binding to r5alphaR-1 and may not be required for enzyme activity. On the other hand, the residue F187 may be important for the binding of both NADPH and testosterone.     

Substitution of asparagine 76 by a tyrosine residue induces domain swapping in Helicobacter pylori phosphopantetheine adenylyltransferase

Phosphopantetheine adenylyltransferase (PPAT) catalyses the penultimate step in coenzyme A biosynthesis in bacteria and is therefore a candidate target for antibacterial drug development. We randomly mutated the residues in the Helicobacter pylori PPAT sequence to identify those that govern protein folding and ligand binding, and we describe the crystal structure of one of these mutants (I4V/N76Y) that contains the mutations I4?→?V and N76?→?Y. Unlike other PPATs, which are homohexamers, I4V/N76Y is a domain-swapped homotetramer. The protomer structure of this mutant is an open conformation in which the 65 C-terminal residues are intertwined with those of a neighbouring protomer. Despite structural differences between wild-type PPAT and IV4/N76Y, they had similar ligand-binding properties. ATP binding to these two proteins was enthalpically driven, whereas that for Escherichia coli PPAT is entropically driven. The structural packing of the subunits may affect the thermal denaturation of wild-type PPAT and I4V/N76Y. Mutations in hinge regions often induce domain swapping, i.e. the spatial exchange of portions of adjacent protomers, but residues 4 and 76 of H. pylori PPAT are not located in or near to the hinge region. However, one or both of these residues is responsible for the large conformational change in the C-terminal region of each protomer. To identify the residue(s) responsible, we constructed the single-site mutant, N76Y, and found a large displacement of α-helix 4, which indicated that its flexibility allowed the domain swap to occur.     

Effect of alanine-293 replacement on the activity, ATP binding, and editing of Escherichia coli leucyl-tRNA synthetase

Leucyl-tRNA synthetase (LeuRS) is a class I aminoacyl-tRNA synthetase that catalyzes leucylation of tRNA(Leu). Several mutants in the CP1 domain of Escherichia coli LeuRS were obtained by introduction of restriction endonuclease sites into its gene, leuS. Of these mutants, only LeuRS-A293F had decreased activity (46%) compared to the native enzyme. To investigate the effect of A293 on enzyme function, A293 was mutated to Y, G, I, R, or D. The mutants were impaired in activity and editing function to varying extents. The decrease in K(m) values for three substrates showed that the binding of ATP to these mutants became much stronger. The inhibition of ATP binding to most of the mutants was also stronger. In particular, LeuRS-A293D had the lowest activity, the strongest ATP binding, and the most impaired editing function. A red shift of the fluorescence emission maximum of LeuRS-A293D indicated a less hydrophobic chromophore environment and a relatively more flexible dynamic conformation. The change in T(m) of LeuRS-A293D was higher than that of all other substitutions. Evidence from sequence alignment and crystal structure of LeuRS from Thermus thermophilus shows that A293 was conserved as R (K) or A and is located at a small helix in the editing domain of the enzyme facing the active site. Hence, any amino acid substitution of A293 may affect the stability of the helix, which may lead to impaired editing function and aminoacylation activity and may be indirectly involved in ATP binding.     

Role of aromatic transmembrane residues of the organic anion transporter,rOAT3, in substrate recognition

Organic anion transporters (OATs, SLC21) are important in the excretion of endogenous and exogenous compounds in the kidney. The rat organic anion transporter, rOAT3, mediates the transport of organic anions such as p-aminohippurate (PAH) and estrone sulfate as well as the basic compound, cimetidine. In the present study, we examined the role of conserved transmembrane aromatic amino acid residues of rOAT3 in substrate recognition and transport. Alanine scanning followed by amino acid replacements was used to construct mutants of rOAT3. The uptake of model compounds was studied in Xenopus laevis oocytes expressing the mutant transporters. We observed that four mutants in transmembrane domain 7 (TMD 7), W334A, F335A, Y341A, and Y342Q, and one mutant in transmembrane domain 8 (TMD 8), F362S, exhibited a less than 2-fold enhanced uptake of PAH and cimetidine in comparison to wild-type rOAT3, which exhibited a 16-fold enhanced uptake of PAH and an 8-fold enhanced uptake of cimetidine. Estrone sulfate uptake in oocytes expressing any one of these five mutants remained at least 8-fold enhanced. The data suggest that the five residues, W334, F335, Y341, Y342, and F362, contribute differently to the transport of the small hydrophilic organic substrates PAH and cimetidine in comparison to the large hydrophobic organic substrate estrone sulfate. The effects of side chains of these five residues on transporter functions were also evaluated by constructing conservative mutations. We observed that the residues contribute to PAH and cimetidine transport in different ways: the -OH group of Y342, the indole ring of W334, and the aromatic rings of F335, Y341, and F362 are important for PAH and cimetidine transport by rOAT3. These data suggest that there is an aromatic pocket composed mainly of residues in TMD 7 in the translocation pathway of rOAT3, which is important for the transport of PAH and cimetidine. Aromatic residues in this pocket may interact directly with substrates of rOAT3 through hydrogen bonds and pi-pi interactions.