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.     

Identification of Residues Essential for the Activity and Substrate Affinity of l-Carnitine Dehydrogenase

Recently, two l-carnitine dehydrogenases from soil isolates Rhizobium sp. (Rs-CDH) and Xanthomonas translucens (Xt-CDH) have demonstrated to exhibit mutually differing affinities toward l-carnitine. To identify residues important for affinity to the substrate, we compared the primary structure of Xt-CDH and Rs-CDH with the recognized 3D structure of 3-hydroxyacyl-CoA dehydrogenase (PDB code: 1F0Y). Then, six residues of Xt-CDH (Phe143, Gly188, Ile190, Ala191, Gly223, and Ala224) and the corresponding residues of Rs-CDH (Tyr140, Ala185, Val187, Gly188, Ser220, and Phe221) were selected for further mutagenesis. The residues of Xt-CDH were replaced with that of Rs-CDH at the corresponding position and vice versa. All Rs-CDH mutants exhibited slight effects on substrate affinity, except for the double mutants Rs-V187I/G188A, which was devoid of enzyme activity. All Xt-CDH mutants showed different K _m values. Xt-F143Y caused a higher increase in the K _m value. Furthermore, the kinetic parameters of 10 mutants at Xt-F143 and Rs-Y140 were investigated. All Rs-Y140 mutants, except aromatic residues (Phe, Trp), produced proteins that were almost entirely devoid of enzyme activity and with disrupted affinity to l-carnitine. All Xt-F143 variants showed a marked reduction (P ≤ 0.05) in enzyme activity. Overall, our results suggest that the aromatic rings of Tyr140 in Rs-CDH and Phe143 of Xt-CDH are essential for substrate recognition.     

Molecular analysis of the role of tyrosine 224 in the active site of Streptomyces coelicolor RppA, a bacterial type III polyketide synthase

Streptomyces coelicolor RppA (Sc-RppA), a bacterial type III polyketide synthase, utilizes malonyl-CoA as both starter and extender unit substrate to form 1,3,6,8-tetrahydroxynaphthalene (THN) (therefore RppA is also known as THN synthase (THNS)). The significance of the active site Tyr(224) for substrate specificity has been established previously, and its aromatic ring is believed to be essential for RppA to select malonyl-CoA as starter unit. Herein, we describe a series of Tyr(224) mutants of Sc-RppA including Y224F, Y224L, Y224C, Y224M, and Y224A that were able to catalyze a physiological assembly of THN, albeit with lower efficiency, challenging the necessity for the Tyr(224) aromatic ring. Steady-state kinetics and radioactive substrate binding analysis of the mutant enzymes corroborated these unexpected results. Functional examination of the Tyr(224) series of RppA mutants using diverse unnatural acyl-CoA substrates revealed the unique role of malonyl-CoA as starter unit substrate for RppA, leading to the development of a novel stericelectronic constraint model.     

Structural Basis of Substrate Specificity of Plant 12-Oxophytodienoate Reductases

12-Oxophytodienoate reductase 3 (OPR3) is a FMN-dependent oxidoreductase that catalyzes the reduction of the cyclopentenone (9S,13S)-12-oxophytodienoate [(9S,13S)-OPDA] to the corresponding cyclopentanone in the biosynthesis of the plant hormone jasmonic acid. In vitro, however, OPR3 reduces the jasmonic acid precursor (9S,13S)-OPDA as well as the enantiomeric (9R,13R)-OPDA, while its isozyme OPR1 is highly selective, accepting only (9R,13R)-OPDA as a substrate. To uncover the molecular determinants of this remarkable enantioselectivity, we determined the crystal structures of OPR1 and OPR3 in complex with the ligand p-hydroxybenzaldehyde. Structural comparison with the OPR1:(9R,13R)-OPDA complex and further biochemical and mutational analyses revealed that two active-site residues, Tyr78 and Tyr246 in OPR1 and Phe74 and His244 in OPR3, are critical for substrate filtering. The relatively smaller OPR3 residues allow formation of a wider substrate binding pocket that is less enantio-restrictive. Substitution of Phe74 and His244 by the corresponding OPR1 tyrosines resulted in an OPR3 mutant showing enhanced, OPR1-like substrate selectivity. Moreover, sequence analysis of the OPR family supports the filtering function of Tyr78 and Tyr246 and allows predictions with respect to substrate specificity and biological function of thus far uncharacterized OPR isozymes. The discovered structural features may also be relevant for other stereoselective proteins and guide the rational design of stereospecific enzymes for biotechnological applications.     

Region and amino acid residues required for Rad51C binding in the human Xrcc3 protein

The Xrcc3 protein, which is required for the homologous recombinational repair of damaged DNA, forms a complex with the Rad51C protein in human cells. Mutations in either the Xrcc3 or Rad51C gene cause extreme sensitivity to DNA-damaging agents and generate the genomic instability frequently found in tumors. In the present study, we found that the Xrcc3 segment containing amino acid residues 63–346, Xrcc3_63–346, is the Rad51C-binding region. Biochemical analyses revealed that Xrcc3_63–346 forms a complex with Rad51C, and the Xrcc3_63–346– Rad51C complex possesses ssDNA and dsDNA binding abilities comparable to those of the full-length Xrcc3–Rad51C complex. Based on the structure of RecA, which is thought to be the ancestor of Xrcc3, six Xrcc3 point mutants were designed. Two-hybrid and biochemical analyses of the Xrcc3 point mutants revealed that Tyr139 and Phe249 are essential amino acid residues for Rad51C binding. Superposition of the Xrcc3 Tyr139 and Phe249 residues on the RecA structure suggested that Tyr139 may function to ensure proper folding and Phe249 may be important to constitute the Rad51C-binding interface in Xrcc3.     

Role of Tryptophan 95 in substrate specificity and structural stability of Sulfolobus solfataricus alcohol dehydrogenase

A mutant of the thermostable NAD⁺-dependent (S)-stereospecific alcohol dehydrogenase from Sulfolobus solfataricus (SsADH) which has a single substitution, Trp95Leu, located at the substrate binding pocket, was fully characterized to ascertain the role of Trp95 in discriminating between chiral secondary alcohols suggested by the wild-type SsADH crystallographic structure. The Trp95Leu mutant displays no apparent activity with short-chain primary and secondary alcohols and poor activity with aromatic substrates and coenzyme. Moreover, the Trp → Leu substitution affects the structural stability of the archaeal ADH, decreasing its thermal stability without relevant changes in secondary structure. The double mutant Trp95Leu/Asn249Tyr was also purified to assist in crystallographic analysis. This mutant exhibits higher activity but decreased affinity toward aliphatic alcohols, aldehydes as well as NAD⁺ and NADH compared to the wild-type enzyme. The crystal structure of the Trp95Leu/Asn249Tyr mutant apo form, determined at 2.0 Å resolution, reveals a large local rearrangement of the substrate site with dramatic consequences. The Leu95 side-chain conformation points away from the catalytic metal center and the widening of the substrate site is partially counteracted by a concomitant change of Trp117 side chain conformation. Structural changes at the active site are consistent with the reduced activity on substrates and decreased coenzyme binding.     

Structural Basis for Substrate Recognition and Specificity in Aklavinone-11-Hydroxylase from Rhodomycin Biosynthesis

In the biosynthesis of several anthracyclines, aromatic polyketides produced by many Streptomyces species, the aglycone core is modified by a specific flavin adenine dinucleotide (FAD)- and NAD(P)H-dependent aklavinone-11-hydroxylase. Here, we report the crystal structure of a ternary complex of this enzyme from Streptomyces purpurascens, RdmE, with FAD and the substrate aklavinone. The enzyme is built up of three domains, a FAD-binding domain, a domain involved in substrate binding, and a C-terminal thioredoxin-like domain of unknown function. RdmE exhibits structural similarity to aromatic hydroxylases from the p-hydroxybenzoate hydroxylase family, but unlike most other related enzymes, RdmE is a monomer. The substrate is bound in a hydrophobic pocket in the interior of the enzyme, and access to this pocket is provided through a different route than for the isoalloxazine ring of FAD—the backside of the ligand binding cleft. The architecture of the substrate binding pocket and the observed enzyme-aklavinone interactions provide a structural explanation for the specificity of the enzyme for non-glycosylated substrates with C9-R stereochemistry. The isoalloxazine ring of the flavin cofactor is bound in the “out” conformation but can be modeled in the “in” conformation without invoking large conformational changes of the enzyme. This model places the flavin ring in a position suitable for catalysis, almost perpendicular to the tetracyclic ring system of the substrate and with a distance of the C4a carbon atom of the isoalloxazine ring to the C-11 carbon atom of the substrate of 4.8 Å. The structure suggested that a Tyr224-Arg373 pair might be involved in proton abstraction at the C-6 hydroxyl group, thereby increasing the nucleophilicity of the aromatic ring system and facilitating electrophilic attack by the perhydroxy-flavin intermediate. Replacement of Tyr224 by phenylalanine results in inactive enzyme, whereas mutants at position Arg373 retain catalytic activity close to wild-type level. These data establish an essential role of residue Tyr224 in catalysis, possibly in aligning the substrate in a position suitable for catalysis.     

Flavonoids biotransformation by bacterial non-heme dioxygenases,biphenyl and naphthalene dioxygenase

This review details recent progresses in the flavonoid biotransformation by bacterial non-heme dioxygenases, biphenyl dioxygenase (BDO), and naphthalene dioxygenase (NDO), which can initially activate biphenyl and naphthalene with insertion of dioxygen in stereospecfic and regiospecific manners. Flavone, isoflavone, flavanone, and isoflavanol were biotransformed by BDO from Pseudomonas pseudoalcaligenes KF707 and NDO from Pseudomonas sp. strain NCIB9816-4, respectively. In general, BDO showed wide range of substrate spectrum and produced the oxidized products, whereas NDO only metabolized flat two-dimensional substrates of flavone and isoflavone. Furthermore, biotransformation of B-ring skewed substrates, flavanone and isoflavanol, by BDO produced the epoxide products, instead of dihydrodiols. These results support the idea that substrate-driven reactivity alteration of the Fe-oxo active species may occur in the active site of non-heme dioxygenases. The study of flavonoid biotransformation by structurally-well defined BDO and NDO will provide the substrate structure and reactivity relationships and eventually establish the production of non-plant-originated flavonoids by means of microbial biotechnology.     

Engineering of substrate specificity of D-amino acid oxidase from the yeast Trigonopsis variabilis: Directed mutagenesis of Phe258 residue

Natural D-amino acid oxidases (DAAO) are not suitable for selective determination of D-amino acids due to their broad substrate specificity profiles. Analysis of the 3D-structure of the DAAO enzyme from the yeast Trigonopsis variabilis (TvDAAO) revealed the Phe258 residue located at the surface of the protein globule to be in the entrance to the active site. The Phe258 residue was mutated to Ala, Ser, and Tyr residues. The mutant TvDAAOs with amino acid substitutions Phe258Ala, Phe258Ser, and Phe258Tyr were purified to homogeneity and their thermal stability and substrate specificity were studied. These substitutions resulted in either slight stabilization (Phe258Tyr) or destabilization (Phe258Ser) of the enzyme. The change in half-inactivation periods was less than twofold. However, these substitutions caused dramatic changes in substrate specificity. Increasing the side chain size with the Phe258Tyr substitution decreased the kinetic parameters with all the D-amino acids studied. For the two other substitutions, the substrate specificity profiles narrowed. The catalytic efficiency increased only for D-Tyr, D-Phe, and D-Leu, and for all other D-amino acids this parameter dramatically decreased. The improvement of catalytic efficiency with D-Tyr, D-Phe, and D-Leu for TvDAAO Phe258Ala was 3.66-, 11.7-, and 1.5-fold, and for TvDAAO Phe258Ser it was 1.7-, 4.75-, and 6.61-fold, respectively.     

Aromatic stacking in the sugar binding site of the lactose permease

Major determinants for substrate recognition by the lactose permease of Escherichia coli are at the interface between helices IV (Glu126, Ala122), V (Arg144, Cys148), and VIII (Glu269). We demonstrate here that Trp151, one turn of helix V removed from Cys148, also plays an important role in substrate binding probably by aromatic stacking with the galactopyranosyl ring. Mutants with Phe or Tyr in place of Trp151 catalyze active lactose transport with time courses nearly the same as wild type. In addition, apparent K(m) values for lactose transport in the Phe or Tyr mutants are only 6- or 3-fold higher than wild type, respectively, with a comparable V(max). Surprisingly, however, binding of high-affinity galactoside analogues is severely compromised in the mutants; the affinity of mutant Trp151-->Phe or Trp151-->Tyr is diminished by factors of at least 50 or 20, respectively. The results demonstrate that Trp151 is an important component of the binding site, probably orienting the galactopyranosyl ring so that important H-bond interactions with side chains in helices IV, V, and VIII can be realized. The results are discussed in the context of a current model for the binding site.     

Yeast sterol C24-methyltransferase: role of highly conserved tyrosine-81 in catalytic competence studied by site-directed mutagenesis and thermodynamic analysis

The role of Try-81 in the reaction catalyzed by Saccharomyces cerevisiae sterol 24-C-methyltransferase (Erg6p) was investigated kinetically and for product differences against a panel of position-81 mutants in which Tyr was substituted with Trp, Phe, Ile, Leu, Val and Ala. The residue chosen for mutation is one that was reported previously to accept fecosterol and yield a set 24-ethyl (idene) sterol products typical of plants, showing the amino acid residue is located close to the transient C25 carbocation intermediate in the active site. One group of mutants (aromatic) tested with the natural substrate zymosterol accelerated the C-methylation reaction (k_cat/K_m) whereas the other group of mutants (aliphatics) decreased catalytic competence as the amino acid side chain was downsized. Mutating to aromatic and assaying with the substrate analog designed as a suicide substrate 26,27-dehydrozymosterol favored C26-monol formation, whereas mutating to the aliphatic of smaller size favored C26-diol formation (a measure of enzyme alkylation). In no case was zymosterol converted to an intermediate that formed a C25-diol. Thermodynamic analysis (determination of E_a, ΔG^‡, ΔH^‡ and TΔS^‡) for the C-methylation reaction performed by these enzymes assayed with the substrate and its analog or zymosterol paired with the “charged’ high energy intermediate (HEI) analogs 24(R,S)25,epiminolanosterol and 25-azalanosterol or “neutral” membrane insert ergosterol showed that mutation to aromatics can reduce inhibitor potency (measured as K_m/K_i), yet catalysis can improve in Trp81 by the introduction of a gain in free energy associated with stabilization of the transition state of a rate-controlling step directed toward turnover. Alternatively, mutation to the smaller aliphatic amino acid side chains led to a destabilization in the active site structure which was accompanied by increases in the partition ratios associated with abortive complex formation. The results are explained by consideration of the functional differences attributed to Tyr81 substitution to aromatics and aliphatics of different size involved with cation-π or hydrogen bonding interactions and in the activation barriers required of differing side chain conformations to orient the reactants in the direction of turnover versus enzyme inactivation.     

Crystal structure and characterization of esterase Est25 mutants reveal improved enantioselectivity toward (S)-ketoprofen ethyl ester

Esterases comprise a group of enzymes that catalyze the cleavage and synthesis of ester bonds. They are important in biotechnological applications owing to their enantioselectivity, regioselectivity, broad substrate specificity, and the fact that they do not require cofactors. In a previous study, we isolated the esterase Est25 from a metagenomic library. Est25 showed catalytic activity toward the (R,S)-ketoprofen ethyl ester but had low enantioselectivity toward the (S)-ketoprofen ethyl ester. Because (S)-ketoprofen has stronger anti-inflammatory effects and fewer side effects than (R)-ketoprofen, enantioselectivity of this esterase is important. In this study, we generated Est25 mutants with improved enantioselectivity toward the (S)-ketoprofen ethyl ester; improved enantioselectivity of mutants was established by analysis of their crystal structures. The enantioselectivity of mutants was influenced by substitution of Phe72 and Leu255. Substituting these residues changed the size of the binding pocket and the entrance hole that leads to the active site. The enantioselectivity of Est25 (E = 1.1 ± 0.0) was improved in the mutants F72G (E = 1.9 ± 0.2), L255W (E = 16.1 ± 1.1), and F72G/L255W (E = 60.1 ± 0.5). Finally, characterization of Est25 mutants was performed by determining the optimum reaction conditions, thermostability, effect of additives, and substrate specificity after substituting Phe72 and Leu255.

     

Roles of the N-terminal domain and remote substrate binding subsites in activity of the debranching barley limit dextrinase

Barley limit dextrinase (HvLD) of glycoside hydrolase family 13 is the sole enzyme hydrolysing α-1,6-glucosidic linkages from starch in the germinating seed. Surprisingly, HvLD shows 150- and 7-fold higher activity towards pullulan and β-limit dextrin, respectively, than amylopectin. This is investigated by mutational analysis of residues in the N-terminal CBM-21-like domain (Ser14Arg, His108Arg, Ser14Arg/His108Arg) and at the outer subsites +2 (Phe553Gly) and +3 (Phe620Ala, Asp621Ala, Phe620Ala/Asp621Ala) of the active site. The Ser14 and His108 mutants mimic natural LD variants from sorghum and rice with elevated enzymatic activity. Although situated about 40 Å from the active site, the single mutants had 15–40% catalytic efficiency compared to wild type for the three polysaccharides and the double mutant retained 27% activity for β-limit dextrin and 64% for pullulan and amylopectin. These three mutants hydrolysed 4,6-O-benzylidene-4-nitrophenyl-6³-α-d-maltotriosyl-maltotriose (BPNPG3G3) with 51–109% of wild-type activity. The results highlight that the N-terminal CBM21-like domain plays a role in activity. Phe553 and the highly conserved Trp512 sandwich a substrate main chain glucosyl residue at subsite +2 of the active site, while substrate contacts of Phe620 and Asp621 at subsite +3 are less prominent. Phe553Gly showed 47% and 25% activity on pullulan and BPNPG3G3, respectively having a main role at subsite +2. By contrast at subsite +3, Asp621Ala increased activity on pullulan by 2.4-fold, while Phe620Ala/Asp621Ala retained only 7% activity on pullulan albeit showed 25% activity towards BPNPG3G3. This outcome supports that the outer substrate binding area harbours preference determinants for the branched substrates amylopectin and β-limit dextrin.     

Single-site mutations on the catalase–peroxidase from Sinorhizobium meliloti: role of the distal Gly and the three amino acids of the putative intrinsic cofactor

KatB is the only catalase–peroxidase identified so far in Sinorhizobium meliloti. It plays a housekeeping role, as it is expressed throughout all the growth phases of the free-living bacterium and also during symbiosis. This paper describes the functional and structural characterization of the KatB mutants Gly303Ser, Trp95Ala, Trp95Phe, Tyr217Leu, Tyr217Phe and Met243Val carried out by optical and electron spin resonance spectroscopy. The aim of this work was to investigate the involvement of these residues in the catalatic and/or peroxidatic reaction and falls in the frame of the open dispute around the factors that influence the balance between catalatic and peroxidatic activity in heme enzymes. The Gly303 residue is not conserved in any other protein of this family, whereas the Trp95, Tyr217 and Met243 residues are thought to form an intrinsic cofactor that is likely to play a role in intramolecular electron transfer. Spectroscopic investigations show that the Gly303Ser mutant is almost similar to the wild-type KatB and should not be involved in substrate binding. Mutations on Trp95, Tyr217 and Met243 clear out the catalatic activity completely, whereas the peroxidatic activity is maintained or even increased with respect to that of the wild-type enzyme. The k _cat values obtained for these mutants suggest that Trp95 and Tyr217 form a huge delocalized system that provides a pathway for electron transfer to the heme. Conversely, Met243 is likely to be placed close to the binding site of the organic molecules and plays a crucial role in substrate docking.     

Role of the conserved phenylalanine 181 of NADPH-cytochrome P450 oxidoreductase in FMN binding and catalytic activity.

NADPH-cytochrome P450 oxidoreductase (P450 reductase, EC 1.6.2.4) is an essential component of the P450 monooxygenase complex and binds FMN, FAD, and NADPH cofactors. Residues Tyr140 and Tyr178 are known to be involved in FMN binding. A third aromatic side chain, Phe181, is also located in the proximity of the FMN ring and is highly conserved in FMN-binding proteins, suggesting an important functional role. This role has been investigated by site-directed mutagenesis. Substitution of Phe181 with leucine or glutamine decreased the cytochrome c reductase activity of the enzyme by approximately 50%. Ferricyanide reductase activity was unaffected, indicating that the FAD domain was unperturbed. The mutant FMN domains were expressed in Escherichia coli, and the redox potentials and binding energies of their complexes with FMN were determined. The affinity for FMN was decreased approximately 50-fold in the Leu181 and Gln181 mutants. Comparison of the binding energies of the wild-type and mutant enzymes in the three redox states of FMN suggests that Phe181 stabilizes the FMN-apoprotein complex. The amide 1H and 15N resonances of the Phe181Leu FMN domain were assigned; comparison of their chemical shifts with those of the wild-type domain indicated that the effect of the substitution on FMN affinity results from perturbation of two loops which form part of the FMN binding site. The results indicate that Phe181 cooperates with Tyr140 and Tyr178 to play a major role in the binding and stability of FMN.     

S1 site residues of Lactococcus lactis prolidase affect substrate specificity and allosteric behaviour

Lactococcus lactis prolidase preferably hydrolyzes Xaa-Pro dipeptides where Xaa is a hydrophobic amino acid. Anionic Glu-Pro and Asp-Pro dipeptides cannot be hydrolyzed at any observable rates and the hydrolysis of cationic Arg-Pro and Lys-Pro dipeptides is at about one tenth of the rate of Leu-Pro. It was hypothesized that the hydrophobic residues in the S₁ site were responsible for this substrate specificity, thus the residues in the S₁ site were substituted with hydrophilic residues. The substitution of Leu193 and Val302 revealed that these residues influenced the substrate specificity. The introduction of a cationic residue, L193R, allowed Asp-Pro to be utilized as a substrate at 37.0% of the rate of Leu-Pro, and the anionic mutation, V302D, yielded mutants that could hydrolyze Asp-Pro, Arg-Pro and Lys-Pro at 25.9 to 57.4% rates. Interestingly, these mutants of S₁ site residues eliminated the allosteric behaviour of L. lactis prolidase that makes this enzyme unique among known prolidases. Results of pH dependency, thermal dependency, and molecular modelling suggested that these observed changes were due to the alteration of the interactions among catalytic zinc cations, Arg293, His296, and the mutated residues.     

Energetic and mechanistic studies of glucoamylase using molecular recognition of maltose OH groups coupled with site-directed mutagenesis

Molecular recognition using a series of deoxygenated maltose analogues was used to determine the substrate transition-state binding energy profiles of 10 single-residue mutants at the active site of glucoamylase from Aspergillus niger. The individual contribution of each substrate hydroxyl group to transition-state stabilization with the wild type and each mutant GA was determined from the relation Delta(DeltaG()) = -RT ln[(k(cat)/K(M))(x)/(k(cat)/K(M))(y)], where x represents either a mutant enzyme or substrate analogue and y the wild-type enzyme or parent substrate. The resulting binding energy profiles indicate that disrupting an active site hydrogen bond between enzyme and substrate, as identified in crystal structures, not only sharply reduces or eliminates the energy contributed from that particular hydrogen bond but also perturbs binding contributions from other substrate hydroxyl groups. Replacing the active site acidic groups, Asp55, Glu180, or Asp309, with the corresponding amides, and the neutral Trp178 with the basic Arg, all substantially reduced the binding energy contribution of the 4'- and 6'-OH groups of maltose at subsite -1, even though both Glu180 and Asp309 are localized at subsite 1. In contrast, the substitution, Asp176 --> Asn, located near subsites -1 and 1, did not substantially perturb any of the individual hydroxyl group binding energies. Similarly, the substitutions Tyr116 --> Ala, Ser119 --> Tyr, or Trp120 --> Phe also did not substantially alter the energy profiles even though Trp120 has a critical role in directing conformational changes necessary for activity. Since the mutations at Trp120 and Asp176 reduced k(cat) values by 50- and 12-fold, respectively, a large effect on k(cat) is not necessarily accompanied by changes in hydroxyl group binding energy contributions. Two substitutions, Asn182 --> Ala and Tyr306 --> Phe, had significant though small effects on interactions with 3- and 4'-OH, respectively. Binding interactions between the enzyme and the glucosyl group in subsite -1, particularly with the 4'- and 6'-OH groups, play an important role in substrate binding, while subsite 1 interactions may play a more important role in product release.     

Structure and Haem-Distal Site Plasticity in Methanosarcina acetivorans Protoglobin

Protoglobin from Methanosarcina acetivorans C2A (MaPgb), a strictly anaerobic methanogenic Archaea, is a dimeric haem-protein whose biological role is still unknown. As other globins, protoglobin can bind O₂, CO and NO reversibly in vitro, but it displays specific functional and structural properties within members of the hemoglobin superfamily. CO binding to and dissociation from the haem occurs through biphasic kinetics, which arise from binding to (and dissociation from) two distinct tertiary states in a ligation-dependent equilibrium. From the structural viewpoint, protoglobin-specific loops and a N-terminal extension of 20 residues completely bury the haem within the protein matrix. Thus, access of small ligand molecules to the haem is granted by two apolar tunnels, not common to other globins, which reach the haem distal site from locations at the B/G and B/E helix interfaces. Here, the roles played by residues Trp(60)B9, Tyr(61)B10 and Phe(93)E11 in ligand recognition and stabilization are analyzed, through crystallographic investigations on the ferric protein and on selected mutants. Specifically, protein structures are reported for protoglobin complexes with cyanide, with azide (also in the presence of Xenon), and with more bulky ligands, such as imidazole and nicotinamide. Values of the rate constant for cyanide dissociation from ferric MaPgb-cyanide complexes have been correlated to hydrogen bonds provided by Trp(60)B9 and Tyr(61)B10 that stabilize the haem-Fe(III)-bound cyanide. We show that protoglobin can strikingly reshape, in a ligand-dependent way, the haem distal site, where Phe(93)E11 acts as ligand sensor and controls accessibility to the haem through the tunnel system by modifying the conformation of Trp(60)B9.     

Role of active‐site residues Tyr55 and Tyr114 in catalysis and substrate specificity of Corynebacterium diphtheriae C‐S lyase

In recent years, there has been increased interest in bacterial methionine biosynthesis enzymes as antimicrobial targets because of their pivotal role in cell metabolism. C‐S lyase from Corynebacterium diphtheriae is a pyridoxal 5′‐phosphate‐dependent enzyme in the transsulfuration pathway that catalyzes the α,β‐elimination of sulfur‐containing amino acids, such as l ‐cystathionine, to generate ammonia, pyruvate, and homocysteine, the immediate precursor of L ‐methionine. In order to gain deeper insight into the functional and dynamic properties of the enzyme, mutants of two highly conserved active‐site residues, Y55F and Y114F, were characterized by UV‐visible absorbance, fluorescence, and CD spectroscopy in the absence and presence of substrates and substrate analogs, as well as by steady‐state kinetic studies. Substitution of Tyr55 with Phe apparently causes a 130‐fold decrease in at pH 8.5 providing evidence that Tyr55 plays a role in cofactor binding. Moreover, spectral data show that the mutant accumulates the external aldimine intermediate suggesting that the absence of interaction between the hydroxyl moiety and PLP‐binding residue Lys222 causes a decrease in the rate of substrate deprotonation. Mutation of Tyr114 with Phe slightly influences hydrolysis of l ‐cystathionine, and causes a change in substrate specificity towards l ‐serine and O‐acetyl‐l ‐serine compared to the wild type enzyme. These findings, together with computational data, provide useful insights in the substrate specificity of C‐S lyase, which seems to be regulated by active‐site architecture and by the specific conformation in which substrates are bound, and will aid in development of inhibitors. Proteins 2015; 83:78–90. © 2014 Wiley Periodicals, Inc.     

The Critical Role of N- and C-Terminal Contact in Protein Stability and Folding of a Family 10 Xylanase under Extreme Conditions

Background

Stabilization strategies adopted by proteins under extreme conditions are very complex and involve various kinds of interactions. Recent studies have shown that a large proportion of proteins have their N- and C-terminal elements in close contact and suggested they play a role in protein folding and stability. However, the biological significance of this contact remains elusive.

Methodology

In the present study, we investigate the role of N- and C-terminal residue interaction using a family 10 xylanase (BSX) with a TIM-barrel structure that shows stability under high temperature, alkali pH, and protease and SDS treatment. Based on crystal structure, an aromatic cluster was identified that involves Phe4, Trp6 and Tyr343 holding the N- and C-terminus together; this is a unique and important feature of this protein that might be crucial for folding and stability under poly-extreme conditions.

Conclusion

A series of mutants was created to disrupt this aromatic cluster formation and study the loss of stability and function under given conditions. While the deletions of Phe4 resulted in loss of stability, removal of Trp6 and Tyr343 affected in vivo folding and activity. Alanine substitution with Phe4, Trp6 and Tyr343 drastically decreased stability under all parameters studied. Importantly, substitution of Phe4 with Trp increased stability in SDS treatment. Mass spectrometry results of limited proteolysis further demonstrated that the Arg344 residue is highly susceptible to trypsin digestion in sensitive mutants such as ΔF4, W6A and Y343A, suggesting again that disruption of the Phe4-Trp6-Tyr343 (F-W-Y) cluster destabilizes the N- and C-terminal interaction. Our results underscore the importance of N- and C-terminal contact through aromatic interactions in protein folding and stability under extreme conditions, and these results may be useful to improve the stability of other proteins under suboptimal conditions.