Site-directed mutagenesis of cysteine residues of large neutral amino acid transporter LAT1

The large neutral amino acid transporter type 1, LAT1, is the principal neutral amino acid transporter expressed at the blood-brain barrier (BBB). Owing to the high affinity (low K_m) of the LAT1 isoform, BBB amino acid transport in vivo is very sensitive to transport competition effects induced by hyperaminoacidemias, such as phenylketonuria. The low K_m of LAT1 is a function of specific amino acid residues, and the transporter is comprised of 12 phylogenetically conserved cysteine (Cys) residues. LAT1 is highly sensitive to inhibition by inorganic mercury, but the specific cysteine residue(s) of LAT1 that account for the mercury sensitivity is not known. LAT1 forms a heterodimer with the 4F2hc heavy chain, which are joined by a disulfide bond between Cys¹⁶⁰ of LAT1 and Cys¹¹⁰ of 4F2hc. The present studies use site-directed mutagenesis to convert each of the 12 cysteines of LAT1 and each of the 2 cysteines of 4F2hc into serine residues. Mutation of the cysteine residues of the 4F2hc heavy chain of the hetero-dimeric transporter did not affect transporter activity. The wild type LAT1 was inhibited by HgCl₂ with a K_i of 0.56 ± 0.11 μM. The inhibitory effect of HgCl₂ for all 12 LAT1 Cys mutants was examined. However, except for the C439S mutant, the inhibition by HgCl₂ for 11 of the 12 Cys mutants was comparable to the wild type transporter. Mutation of only 2 of the 12 cysteine residues of the LAT1 light chain, Cys⁸⁸ and Cys⁴³⁹, altered amino acid transport. The V_max was decreased 50% for the C88S mutant. A kinetic analysis of the C439S mutant could not be performed because transporter activity was not significantly above background. Confocal microscopy showed the C439S LAT1 mutant was not effectively transferred to the oocyte plasma membrane. These studies show that the Cys⁴³⁹ residue of LAT1 plays a significant role in either folding or insertion of the transporter protein in the plasma membrane.     

Mechanism of chitosanase-oligosaccharide interaction: subsite structure of Streptomyces sp. N174 chitosanase and the role of Asp57 carboxylate.

We have investigated the mechanism of the interaction of Streptomyces sp. N174 chitosanase with glucosamine hexasaccharide [(GlcN)(6)] by site-directed mutagenesis, thermal unfolding, and (GlcN)(6) digestion experiments, followed by theoretical calculations. From the energy-minimized model of the chitosanase-(GlcN)(6) complex structure (Marcotte et al., 1996), Asp57, which is present in all known chitosanases, was proposed to be one of the amino acid residues that interacts with the oligosaccharide substrate. The chitosanase gene was mutated at Asp57 to Asn (D57N) and Ala (D57A), and the relative activities of the mutated chitosanases were found to be 72 and 0.5% of that of the wild type, respectively. The increase in the transition temperature of thermal unfolding (T(m)), usually observed upon the addition of (GlcN)(n) to chitosanase mutants unaffected in terms of substrate binding, was considerably suppressed in the D57A mutant. These data suggest that Asp57 is important for substrate binding. The experimental time-courses of [(GlcN)(6)] degradation were analyzed by a theoretical model in order to obtain the binding free energy values of the individual subsites of the chitosanases. A (-3, -2, -1, +1, +2, +3) subsite model agreed best with the experimental data. This analysis also indicated that the mutation of Asp57 affects substrate affinity at subsite (-2), suggesting that Asp57 most likely participates in the substrate binding at this subsite.     

Functional significance of some particular amino acid residues in Bombyx mori pyridoxal kinase

Pyridoxal kinase (PLK; EC 2.7.1.35) is a key enzyme for vitamin B₆ metabolism in animals. It catalyzes the ATP-dependent phosphorylation of pyridoxal, generating pyridoxal 5′-phosphate, an important cofactor for many enzymatic reactions. Bombyx mori PLK (BmPLK) is 10 or more residues shorter than mammalian PLKs, and some amino acid residues conserved in the PLKs from mammals are not maintained in the protein. Multiple sequence alignment suggested that amino acid residues Thr⁴⁷, Ile⁵⁴, Arg⁸⁸, Asn¹²¹ and Glu²³⁰ might play important roles in BmPLK. In this study, we used a site-directed specific mutagenesis approach to determine the functional significance of these particular amino acid residues in BmPLK. Our results demonstrated that the mutation of Asn¹²¹ to Glu did not affect the catalytic function of BmPLK. The corresponding site-directed mutants of Thr⁴⁷ to Asn, Ile⁵⁴ to Phe, and Arg⁸⁸ to Ile displayed a decreased catalytic efficiency and an elevated Km value for substrate relative to the wild-type value, and no enzyme activity could be detected in mutant of Trp²³⁰ to Glu. Circular dichroism analysis revealed that the mutation of Trp²³⁰ to Glu resulted in mis-folding of the protein. Our results provided direct evidence that residue Trp²³⁰ is crucial to maintain the structural and functional integrity of BmPLK. This study will add to the existing understanding of the characteristic of structure and function of BmPLK.     

Role of the amino acid invariants in the active site of MurG as evaluated by site-directed mutagenesis

To evaluate their role in the active site of the MurG enzyme from Escherichia coli, 13 residues conserved in the sequences of 73 MurG orthologues were submitted to site-directed mutagenesis. All these residues lay within, or close to, the active site of MurG as defined by its tridimensional structure [Ha et al., Prot. Sci. 9 (2000) 1045-1052, and Hu et al., Proc. Natl. Acad. Sci. USA 100 (2003) 845-849]. Thirteen mutants proteins, in which residues T15, H18, Y105, H124, E125, N127, N134, S191, N198, R260, E268, Q288 or N291 have been replaced by alanine, were obtained as the C-terminal His-tagged forms. The effects of the mutations on the activity were checked: (i) by functional complementation of an E. coli murG mutant strain by the mutated genes; and (ii) by the determination of the steady-state kinetic parameters of the purified proteins. Most mutations resulted in an important loss of activity and, in the case of N134A, in the production of a highly unstable protein. The results correlated with the assigned or putative functions of the residues based on the tridimensional structure.     

Theoretical calculation of pKa reveals an important role of Arg205 in the activity and stability of Streptomyces sp. N174 chitosanase

Based on the crystal structure of chitosanase from Streptomyces sp. N174, we have calculated theoretical pK(a) values of the ionizable groups of this protein using a combination of the boundary element method and continuum electrostatics. The pK(a) value obtained for Arg(205), which is located in the catalytic cleft, was abnormally high (>20.0), indicating that the guanidyl group may interact strongly with nearby charges. Chitosanases possessing mutations in this position (R205A, R205H, and R205Y), produced by Streptomyces lividans expression system, were found to have less than 0.3% of the activity of the wild type enzyme and to possess thermal stabilities 4-5 kcal/mol lower than that of the wild type protein. In the crystal structure, the Arg(205) side chain is in close proximity to the Asp(145) side chain (theoretical pK(a), -1.6), which is in turn close to the Arg(190) side chain (theoretical pK(a), 17.7). These theoretical pK(a) values are abnormal, suggesting that both of these residues may participate in the Arg(205) interaction network. Activity and stability experiments using Asp(145)- and Arg(190)-mutated chitosanases (D145A and R190A) provide experimental data supporting the hypothesis derived from the theoretical pK(a) data and prompt the conclusion that Arg(205) forms a strong interaction network with Asp(145) and Arg(190) that stabilizes the catalytic cleft.     

Energetic contributions of amino acid residues and its cross-talk to delineate ligand-binding mechanism

Receptor-based QSAR approaches can enumerate the energetic contributions of amino acid residues toward ligand binding only when experimental binding affinity is associated. The structural data of protein-ligand complexes are witnessing a tremendous growth in the Protein Data Bank deposited with a few entries on binding affinity. We present here a new approach to compute the E nergetic CONT ributions of A mino acid residues and its possible C ross-T alk (ECONTACT) to study ligand binding using per-residue energy decomposition, molecular dynamics simulations and rescoring method without the need for experimental binding affinity. This approach recognizes potential cross-talks among amino acid residues imparting a nonadditive effect to the binding affinity with evidence of correlative motions in the dynamics simulations. The protein-ligand interaction energies deduced from multiple structures are decomposed into per-residue energy terms, which are employed as variables to principal component analysis and generated cross-terms. Out of 16 cross-talks derived from eight datasets of protein-ligand systems, the ECONTACT approach is able to associate 10 potential cross-talks with site-directed mutagenesis, free energy, and dynamics simulations data strongly. We modeled these key determinants of ligand binding using joint probability density function (jPDF) to identify cross-talks in protein structures. The top two cross-talks identified by ECONTACT approach corroborated with the experimental findings. Furthermore, virtual screening exercise using ECONTACT models better discriminated known inhibitors from decoy molecules. This approach proposes the jPDF metric to estimate the probability of observing cross-talks in any protein-ligand complex. The source code and related resources to perform ECONTACT modeling is available freely at https://www.gujaratuniversity.ac.in/econtact /.     

Studies on the key amino acid residues responsible for the alkali-tolerance of the xylanase by site-directed or random mutagenesis

Asparagine (Asn)-71 of the xylanase (XYN) from Bacillus pumilus A-30 was found highly conserved in alkaline xylanases of family G/11. The mutated gene fragments containing different substitutions of Asn-71 was obtained by site-directed mutagenesis to study its role in the alkali-tolerant mechanism of xylanase. The xylanase activity was completely lost if Asn-71 residue was replaced by alkaline arginine (Arg) or lysine (Lys) residues, but obviously depressed with a shift in the pH optimum of the enzyme from 6.7 to 6.3 if substituted by serine (Ser) or aspartate (Asp) residues. No mutant with a shift of the pH optimum to a more basic value was found. Furthermore, N71D lost its activity in the alkaline pH range completely, while N71S did not lose as much as that of N71D. Except for Asn-71, the random mutagenesis to other residues of the xylanase was also studied. The alkali-tolerant mechanism of the xylanase was analyzed by their charged character, ionized state, and the hydrogen bond network of the residues surrounding the two catalytic residues on the basis of homology modeling of the mutated xylanases.     

Competition between citrate and heptapeptide DDSDEEN binding to DNA in presence of divalent cations

The binding of citrate and acidic peptide DDSDEEN with DNA in the presence of divalent cations is compared. Citric acid shows a higher number of binding sites on the DNA compared to the peptide; this is probably due to the bigger sitric hindrance of the peptide compared to the citric acid for the binding in the DNA grooves. Moreover, DNA preincubated with saturating amounts of citric acid is not available for the binding with successively added peptide. Therefore the peptide and citrate binding sites to some extent overlap.     

Modulation of NO binding to cytochrome <Emphasis Type="Italic">c</Emphasis>′ by distal and proximal haem pocket residues

We have cloned and expressed the cycP gene encoding cytochrome c′ from Alcaligenes xylosoxidans and generated mutations in Arg-124 and Phe-59, residues close to the haem, to probe their involvement in modulating the unusual spin-state equilibrium of the haem Fe and the unique proximal mode of binding of NO to form a stable five-coordinate adduct. Arg-124 is located in the proximal pocket of the haem and forms a hydrogen bond to the stable five-coordinated bound NO. Phe-59 provides steric hindrance at the distal face where NO binds initially to form a six-coordinate adduct. Optical spectroscopy showed altered electronic properties of the oxidised haem centre resulting from the mutations of both residues. The high affinity of the ferrous proteins for NO remained unchanged and all of the mutational variants formed a stable five-coordinate NO species (λ _Soret 395 nm) in the presence of stoichiometric concentrations of NO. However, the kinetics of the reactivity towards NO were altered, with mutation of the distal Phe-59 residue resulting in the transient six-coordinate distally bound NO adduct (λ _Soret 415 nm) not being detected. Surprisingly, substitution of the proximal residue Arg-124 with Phe, Ala, Gln or Glu also resulted in the six-coordinate adduct not being detected, showing that this proximal residue also modulates reactivity towards NO on the opposite haem face. In contrast, the R124L substitution retained the property of the native protein in the initial formation of a six-coordinate NO adduct, a finding of functional importance since a Lys or an Arg residue is invariant in these proteins.     

Amino acid residues adjacent to the catalytic cavity of tetramer l-asparaginase II contribute significantly to its catalytic efficiency and thermostability

l-Asparaginase (l-asparagine amidohydrolase, EC 3.5.1.1) catalyzes the hydrolysis of l-asparagine to l-aspartic acid and ammonia. It can be used to reduce the formation of acrylamide, which is carcinogenic to humans in foods, via removal of the precursor, asparagine, from the primary ingredients. However, low activity and poor thermostability of l-asparaginase restrict its application in food industry. In this study, we successfully improved thermostability and catalytic efficiency of l-asparaginase II (BsAII) from Bacillus subtilis B11-06 by site-directed mutagenesis. According to sequences alignment and homologous modeling, residues G107, T109 and S166 which were adjacent to the catalytic cavity were selected and substituted by Asp, Gln/Ser and Ala, respectively, to construct mutants G107D, T109Q, T109S and S166A. The BsAII mutant of G107D (G107D_ansz) displayed superior performance in thermal tolerance and higher activity than the wild-type enzyme (towards l-asparagine). Comparative analysis of hydrogen bond interactions, surface electrostatic potential and structure of substrate binding pocket between G107D_anszand BsAII indicated that the substitution of G107, which was adjacent to catalytic cavity with Asp, resulted in small conformational changes and surface electrostatic potential redistribution and contributed to the improved protein stability and catalytic efficiency.     

Identification of N-acetylglucosamine binding residues in Griffonia simplicifolia lectin II

Primary structure and crystallographic data of several legume lectins were used to predict the involvement in carbohydrate binding of six amino acid residues (Asp⁸⁸, Glu¹⁰⁸, Tyr¹³⁴, Asn¹³⁶, Leu²²⁶ and Gln²²⁷) in Griffonia simplicifolia lectin II (GS-II). The functional involvement of these residues was evaluated by assessing GlcNAc binding of modified forms of GS-II in which these residues were eliminated in truncated peptides or systematically substituted with other amino acids by site-specific mutations. Mutations at (Asp⁸⁸, Tyr¹³⁴ or Asn¹³⁶ eliminated GlcNAc binding activity by GS-II, while those at Glut¹⁰⁸, Leu²²⁶ or Gln²²⁷ did not alter the activity. The former three amino acids were functionally essential for carbohydrate binding by GS-II presumably through hydrogen bonding to and hydrophobic interactions with GlcNAc. Although an Asp or Gly substitution for Tyr¹³⁴ eliminated GlcNAc affinity, substitution with Phe did not appreciably affect binding. Despite the fact that mutations to Leu²²⁶ and Gln²²⁷ did not alter carbohydrate binding, a truncated form of GS-II lacking these residues no longer exhibited carbohydrate binding affinity.     

Identification of amino acid residues involved in the binding of Huperzine A to cholinesterases.

Huperzine A, a potential agent for therapy in Alzheimer's disease and for prophylaxis of organophosphate toxicity, has recently been characterized as a reversible inhibitor of cholinesterases. To examine the specificity of this novel compound in more detail, we have examined the interaction of the 2 stereoisomers of Huperzine A with cholinesterases and site-specific mutants that detail the involvement of specific amino acid residues. Inhibition of fetal bovine serum acetylcholinesterase by (-)-Huperzine A was 35-fold more potent than (+)-Huperzine A, with KI values of 6.2 nM and 210 nM, respectively. In addition, (-)-Huperzine A was 88-fold more potent in inhibiting Torpedo acetylcholinesterase than (+)-Huperzine A, with KI values of 0.25 microM and 22 microM, respectively. Far larger KI values that did not differ between the 2 stereoisomers were observed with horse and human serum butyrylcholinesterases. Mammalian acetylcholinesterase, Torpedo acetylcholinesterase, and mammalian butyrylcholinesterase can be distinguished by the amino acid Tyr, Phe, or Ala in the 330 position, respectively. Studies with mouse acetylcholinesterase mutants, Tyr 337 (330) Phe and Tyr 337 (330) Ala yielded a difference in reactivity that closely mimicked the native enzymes. In contrast, mutation of the conserved Glu 199 residue to Gln in Torpedo acetylcholinesterase produced only a 3-fold increase in KI value for the binding of Huperzine A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid residues involved in organic solvent-stability of the LST-03 lipase

LST-03 lipase from Pseudomonas aeruginosa LST-03 is highly active and stable in the presence of various organic solvents. To further characterize and improve the organic solvent-stability of the LST-03 lipase, residues that potentially provide this stability were identified and mutated to other amino acids in an effort to increase the organic solvent-stability of the protein. S155L, G157R, S164K, S194R, and D209N mutations were found to improve the organic solvent-stability of the wild-type LST-03 lipase. Such mutations were found to induce structural changes, including the formation of a salt bridge, hydrogen bonds, lead to an improved packing of the hydrophobic core, and pI shift of side chain. These changes increased the stability of the protein, thereby improving the organic solvent-stability of the wild-type LST-03 lipase. In addition, a single mutation was found to stabilize the lipase by single or multiple factors.     

Site-directed mutagenesis of the conserved Ala348 and Gly350 residues at the putative active site of Bacillus kaustophilus leucine aminopeptidase

Leucine aminopeptidase (LAP) is an exopeptidase that catalyzes the hydrolysis of amino acid residues from the amino terminus of proteins and peptides. Sequence alignment shows that the conserved Ala348 and Gly350 residues of Bacillus kaustophilus LAP (BkLAP) are located right next to a coordinated ligand. We further investigated the roles of these two residues by performing computer modeling and site-directed mutagenesis. Based on the modeling, the carbonyl group of Ala348 interacts with Asn345 and Asn435, and that of Gly350 with Ile353 and Leu354, where these interactions might maintain the zinc-coordinated residues at their correct positions. Replacement of Ala348 with Arg resulted in a dramatic reduction in LAP activity. A complete loss of the activity was also observed in A348E, A348V, and the Gly350 variants. Measurement of intrinsic tryptophan fluorescence revealed alteration of the microenvironment of aromatic amino acid residues, while circular dichroism spectra were nearly identical for wild-type and all mutant enzymes. Protein modeling and site-directed mutagenesis suggest that residues Ala348 and Gly350 are essential for BkLAP in maintaining a stable active-site environment for the catalytic reaction.     

Site-directed mutagenesis of arginine-89 supports the role of its guanidino side-chain in substrate binding by Cephalosporium acremonium isopenicillin N synthase

Isopenicillin N synthase (IPNS) catalyses a key step in the penicillin and cephalosporin biosynthetic pathway which involves the oxidative cyclisation of the acyclic peptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N. Based on crystallographic evidence from the Aspergillus nidulans IPNS crystal structure complexed with the substrate ACV (Roach et al. (1997) Nature 387, 827-830), we were able to provide mutational evidence for the critical involvement of the conserved R-X-S motif in ACV binding in IPNS. The crystal structure further implicated arginine-87 in the binding of the aminoadipyl portion of ACV. Thus, in this study, the site-directed mutagenesis of the corresponding arginine-89 in Cephalosporium acremonium IPNS (cIPNS) was performed to ascertain its role in cIPNS. Alteration of arginine-89 to five amino acids from different amino acid groups, namely lysine, serine, alanine, aspartate and leucine, was performed and no activity was detected in all the mutants obtained when enzyme bioassays were performed. Furthermore, the solubility of the mutants was considerably lower than the wild-type cIPNS after expression at 37 degrees C, but could be recovered when the expression temperature was lowered to 25 degrees C. This suggests that arginine-89 could be critical for the activity of cIPNS due to its involvement in ACV binding and the solubility of wild-type enzyme.     

Histidine-272 of isopenicillin N synthase of Cephalosporium acremonium, which is possibly involved in iron binding, is essential for its catalytic activity

Abstract Amino acid sequence alignment of the Cephalosporium acremonium isopenicillin N synthase (cIPNS) to similar non-heme Fe²⁺-containing enzymes from 28 different sources (bacterial, fungal, plant and animals) revealed a homologous region of high sequence conservation containing an invariant histidine residue at position 272 in cIPNS. The importance of this histidine residue in cIPNS was investigated through site-directed mutagenesis by replacing the histidine residue with leucine. The mutated gene was verified by DNA sequence analysis and expressed in Escherichia coli . When analyzed by denaturing gel electrophoresis and immunoblotting, the mutant cIPNS had identical mobility as that of the wild-type enzyme. Enzyme studies on the mutant enzyme showed loss of enzymatic activity indicating that His272 is essential for the catalytic function of cIPNS, possibly as a ligand for iron binding.     

Cloning and high-level production of a chitinase from <Emphasis Type="Italic">Chromobacterium</Emphasis> sp. and the role of conserved or nonconserved residues on its catalytic activity

A gene encoding an alkaline (pI of 8.67) chitinase was cloned and sequenced from Chromobacterium sp. strain C-61. The gene was composed of 1,611 nucleotides and encoded a signal sequence of 26 N-terminal amino acids and a mature protein of 510 amino acids. Two chitinases of 54 and 52 kDa from both recombinant Escherichia coli and C-61 were detected on SDS-PAGE. Maximum chitinase activity was obtained in the culture supernatant of recombinant E. coli when cultivated in TB medium for 6 days at 37°C and was about fourfold higher than that from C-61. Chi54 from the culture supernatants could be purified by a single step based on isoelectric point. The purified Chi54 had about twofold higher binding affinity to chitin than to cellulose. The chi54 encoded a protein that included a type 3 chitin-binding domain belonging to group A and a family 18 catalytic domain belonging to subfamily A. In the catalytic domain, mutation of perfectly conserved residues and highly conserved residues resulted in loss of nearly all activity, while mutation of nonconserved residues resulted in enzymes that retained activity. In this process, a mutant (T218S) was obtained that had about 133% of the activity of the wild type, based on comparison of K _cat values.     

The role of conserved tryptophan and acidic residues in the human dopamine transporter as characterized by site-directed mutagenesis

The human dopamine (DA) transporter (hDAT) contains multiple tryptophans and acidic residues that are completely or highly conserved among Na(+)/Cl(-)-dependent transporters. We have explored the roles of these residues using non-conservative substitution. Four of 17 mutants (E117Q, W132L, W177L and W184L) lacked plasma membrane immunostaining and were not functional. Both DA uptake and cocaine analog (i.e. 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane, CFT) binding were abolished in W63L and severely damaged in W311L. Four of five aspartate mutations (D68N, D313N, D345N and D436N) shifted the relative selectivity of the hDAT for cocaine analogs and DA by 10-24-fold. In particular, mutation of D345 in the third intracellular loop still allowed considerable [(3)H]DA uptake, but caused undetectable [(3)H]CFT binding. Upon anti-C-terminal-hDAT immunoblotting, D345N appeared as broad bands of 66-97 kDa, but this band could not be photoaffinity labeled with cocaine analog [(125)I]-3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid ([(125)I]RTI-82). Unexpectedly, in this mutant, cocaine-like drugs remained potent inhibitors of [(3)H]DA uptake. CFT solely raised the K(m) of [(3)H]DA uptake in wild-type hDAT, but increased K(m) and decreased V(max) in D345N, suggesting different mechanisms of inhibition. The data taken together indicate that mutation of conserved tryptophans or acidic residues in the hDAT greatly impacts ligand recognition and substrate transport. Additionally, binding of cocaine to the transporter may not be the only way by which cocaine analogs inhibit DA uptake.     

Targeted mutagenesis of a fatty acid Delta6-desaturase from Mucor rouxii: role of amino acid residues adjacent to histidine-rich motif II

The amino acid residues serine at position 213 (S213) and lysine at position 218 (K218), which are present in close proximity to the histidine-rich motif II of Mucor rouxii fatty acid Delta(6)-desaturase isoform II, were targeted for studying structure-function relationships using site-directed mutagenesis. The mutants were functionally characterized in a heterologous host, Saccharomyces cerevisiae. Substrate specificity and preference studies revealed that S213 and K218 are involved in substrate recognition. K218 plays a role in substrate preference by involvement in the binding of substrates, particularly C15-C18 monoene fatty acids. Modification of the M. rouxii Delta(6)-desaturase therefore has potential in specifically altering substrate utilization for production of desired fatty acids.