Cloning and sequencing of an Agrobacterium tumefaciens beta-glucosidase gene involved in modifying a vir-inducing plant signal molecule.

Induction of Agrobacterium tumefaciens virulence genes by plant phenolic compounds is essential for successful T-DNA transfer to a host plant. In Douglas fir needles, the major virulence region inducer is the glycoside coniferin (J. W. Morris and R. O. Morris, Proc. Natl. Acad. Sci. USA 87:3612-3618, 1990). Agrobacterium strains with high beta-glucosidase activity respond to coniferin and infect Douglas fir seedlings, whereas most strains with low beta-glucosidase activity fail to respond to coniferin and are avirulent on this host. We have cloned two beta-glucosidase genes from A. tumefaciens B3/73 and sequenced one of them, cbg1. It appears to be part of a polycistronic unit and shows a high bias for GC-rich codons. When expressed in Escherichia coli, Cbg1 beta-glucosidase hydrolyzes coniferin but not cellobiose. The 88-kDa predicted product of cbg1 is highly similar to one other bacterial beta-glucosidase and several fungal beta-glucosidases. There is little homology between Cbg1 and other bacterial beta-glucosidases, including an Agrobacterium cellobiase.     

The multimerization of hantavirus nucleocapsid protein depends on type-specific epitopes

Multimerization of the Hantaan virus nucleocapsid protein (NP) in Hantaan virus-infected Vero E6 cells was observed in a competitive enzyme-linked immunosorbent assay (ELISA). Recombinant and truncated NPs of Hantaan, Seoul, and Dobrava viruses lacking the N-terminal 49 amino acids were also detected as multimers. Although truncated NPs of Hantaan virus lacking the N-terminal 154 amino acids existed as a monomer, those of Seoul and Dobrava formed multimers. The multimerized truncated NP antigens of Seoul and Dobrava viruses could detect serotype-specific antibodies, whereas the monomeric truncated NP antigen of Hantaan virus lacking the N-terminal 154 amino acids could not, suggesting that a hantavirus serotype-specific epitope on the NP results in multimerization. The NP-NP interaction was also detected by using a yeast two-hybrid assay. Two regions, amino acids 100 to 125 (region 1) and amino acids 404 to 429 (region 2), were essential for the NP-NP interaction in yeast. The NP of Seoul virus in which the tryptophan at amino acid number 119 was replaced by alanine (W119A mutation) did not multimerize in the yeast two-hybrid assay, indicating that tryptophan 119 in region 1 is important for the NP-NP interaction in yeast. However, W119A mutants expressed in mammalian cells were detected as the multimer by using competitive ELISA. Similarly, the truncated NP of Seoul virus expressing amino acids 155 to 429 showed a homologous interaction in a competitive ELISA but not in the yeast two-hybrid assay, indicating that the C-terminal region is important for the multimerization detected by competitive ELISA. Combined, the results indicate that several steps and regions are involved in multimerization of hantavirus NP.     

Mechanism of the family 1 beta-glucosidase from Streptomyces sp: catalytic residues and kinetic studies

The Streptomyces sp. beta-glucosidase (Bgl3) is a retaining glycosidase that belongs to family 1 glycosyl hydrolases. Steady-state kinetics with p-nitrophenyl beta-D-glycosides revealed that the highest k(cat)/K(M) values are obtained with glucoside (with strong substrate inhibition) and fucoside (with no substrate inhibition) substrates and that Bgl3 has 10-fold glucosidase over galactosidase activity. Reactivity studies by means of a Hammett analysis using a series of substituted aryl beta-glucosides gave a biphasic plot log k(cat) vs pK(a) of the phenol aglycon: a linear region with a slope of beta(lg) = -0.8 for the less reactive substrates (pK(a) > 8) and no significant dependence for activated substrates (pK(a) < 8). Thus, according to the two-step mechanism of retaining glycosidases, formation of the glycosyl-enzyme intermediate is rate limiting for the former substrates, while hydrolysis of the intermediate is for the latter. To identify key catalytic residues and on the basis of sequence similarity to other family 1 beta-glucosidases, glutamic acids 178 and 383 were changed to glutamine and alanine by site-directed mutagenesis. Mutation of Glu178 to Gln and Ala yielded enzymes with 250- and 3500-fold reduction in their catalytic efficiencies, whereas larger reduction (10(5)-10(6)-fold) were obtained for mutants at Glu383. The functional role of both residues was probed by a chemical rescue methodology based on activation of the inactive Ala mutants by azide as exogenous nucleophile. The E178A mutant yielded the beta-glucosyl azide adduct (by (1)H NMR) with a 200-fold increase on k(cat) for the 2,4-dinitrophenyl glucoside but constant k(cat)/K(M) on azide concentration. On the other hand, the E383A mutant with the same substrate gave the alpha-glucosyl azide product and a 100-fold increase in k(cat) at 1 M azide. In conclusion, Glu178 is the general acid/base catalyst and Glu383 the catalytic nucleophile. The results presented here indicate that Bgl3 beta-glucosidase displays kinetic and mechanistic properties similar to other family 1 enzymes analyzed so far. Subtle differences in behavior would lie in the fine and specific architecture of their respective active sites.     

Identification of the lipoprotein initiating domain of apolipoprotein B

We have explored the minimum sequence requirement for the initiation of apolipoprotein B (apoB)-mediated triglyceride-rich lipoprotein assembly. A series of apoB COOH-terminal truncation mutants, spanning a range from apoB34 (amino acid residues 1-1544 of apoB100) to apoB19 (residues 1-862) were transfected into COS cells with and without coexpression of the microsomal triglyceride transfer protein (MTP). ApoB34, -25, -23, -21, -20.5, and -20.1 underwent efficient conversion to buoyant lipoproteins when coexpressed with MTP. ApoB19.5 (amino acids 1-884) also directed MTP-dependent particle assembly, although at reduced efficiency. When apoB19.5 was truncated by another 22 amino acids to form apoB19, MTP-dependent lipoprotein assembly was abolished. Analysis of the lipid stoichiometry of secreted lipoproteins revealed that all apoB truncation mutants formed spherical particles containing a hydrophobic core. Even highly truncated assembly-competent forms of apoB, such as apoB19.5 and 20.1, formed lipoproteins with surface:core lipid ratios of <1. We conclude that the translation of the first approximately 884 amino acids of apoB completes a domain capable of initiating nascent lipoprotein assembly. The composition of lipids recruited into lipoproteins by this initiating domain is consistent with formation of small emulsion particles, perhaps by simultaneous desorption of both polar and neutral lipids from a saturated bilayer.     

Mutants of the membrane-binding region of Semliki Forest virus E2 protein. I. Cell surface transport and fusogenic activity

Three mutations of the membrane-binding region of the Semliki Forest virus (SFV) p62 polypeptide (the precursor for virion E3 and E2) have been made by oligonucleotide-directed mutagenesis of a cDNA clone encoding the SFV structural proteins. One of the mutations (A2) substitutes a Glu for an Ala in the middle of the hydrophobic stretch which spans the bilayer. A1 and A3 alter the two basic charged amino acids in the cytoplasmic domain next to the hydrophobic region. The wild-type charge cluster of Arg-Ser-Lys (+2) has been changed to Gly-Ser-Met (0;A3) or to Gly-Ser-Glu (-1;A1). The mutant p62 proteins have been analyzed both in the presence and the absence of E1, the other half of the heterodimer spike complex of SFV. The mutant proteins expressed in COS-7 cells are glycosylated and are of the expected sizes. When co-expressed with E1, all three mutants are cleaved to yield the E2 protein and transported to the surface of COS-7 cells. When expressed in the absence of E1, the mutant p62 proteins remain uncleaved but still reach the cell surface. Once at the cell surface, all three mutants, when co-expressed with E1, can promote low pH-triggered cell-cell fusion. These results show that the three mutant p62/E2 proteins are still membrane associated in a functionally unaltered way.     

Insensitivity of perturbed carboxyl pK(a) values in the ovomucoid third domain to charge replacement at a neighboring residue

A number of carboxyl groups in turkey ovomucoid third domain (OMTKY3) have low pK(a) values. A previous study suggested that neighboring amino groups were primarily responsible for the low carboxyl pK(a) values. However, the expected elevation in pK(a) values for these amino groups was not observed. In the present study, site-directed mutagenesis is used to investigate the origins of perturbed carboxyl pK(a) values in OMTKY3. Electrostatic calculations suggest that Lys 34 has large effects, 0.4-0.6 unit, on Asp 7, Glu 10, and Glu 19 which are 5-11 A away from Lys 34. Two-dimensional (1)H NMR techniques were used to determine pK(a) values of the acidic residues in OMTKY3 mutants in which Lys 34 has been replaced with threonine and glutamine. Surprisingly, the pK(a) values in the mutants are very close to those of the wild-type protein. The insensitivity of the acidic residues to replacement of Lys 34 suggests that long-range electrostatic interactions play less of a role in perturbing carboxyl pK(a) values than originally thought. We hypothesize that hydrogen bonds play a key role in perturbing some of the carboxyl ionization equilibria in OMTKY3.     

Mutational analysis of Thermus caldophilus GK24 beta-glycosidase: role of His119 in substrate binding and enzyme activity

Three amino acid residues (His119, Glu164, and Glu338) in the active site of Thermus caldophilus GK24 beta- glycosidase (Tca beta-glycosidase), a family 1 glycosyl hydrolase, were mutated by site-directed mutagenesis. To verify the key catalytic residues, Glu164 and Glu338 were changed to Gly and Gln, respectively. The E164G mutation resulted in drastic reductions of both beta-galactosidase and beta-glucosidase activities, and the E338Q mutation caused complete loss of activity, confirming that the two residues are essential for the reaction process of glycosidic linkage hydrolysis. To investigate the role of His119 in substrate binding and enzyme activity, the residue was substituted with Gly. The H119G mutant showed 53-fold reduced activity on 5 mM p-nitrophenyl beta-Dgalactopyranoside, when compared with the wild type; however, both the wild-type and mutant enzymes showed similar activity on 5 mM p-nitrophenyl beta-D-glucopyranoside at 75degreeC. Kinetic analysis with p-nitrophenyl beta-D-galactopyranoside revealed that the kcat value of the H119G mutant was 76.3-fold lower than that of the wild type, but the Km of the mutant was 15.3-fold higher than that of the wild type owing to the much lower affinity of the mutant. Thus, the catalytic efficiency (kcat/Km) of the mutant decreased to 0.08% to that of the wild type. The kcat value of the H119G mutant for p-nitrophenyl beta- D-glucopyranoside was 5.1-fold higher than that of the wild type, but the catalytic efficiency of the mutant was 2.5% of that of the wild type. The H119G mutation gave rise to changes in optima pH (from 5.5-6.5 to 5.5) and temperature (from 90 degrees C to 80-85 degrees C). This difference of temperature optima originated in the decrease of H119G's thermostability. These results indicate that His119 is a crucial residue in beta- galactosidase and beta-glucosidase activities and also influences the enzyme's substrate binding affinity and thermostability.     

Reorganization in apo- and holo-beta-lactoglobulin upon protonation of Glu89: molecular dynamics and pKa calculations

Molecular dynamics (MD) simulations starting from crystallographic data allowed us to directly account for the effects of the protonation state of Glu89 on the conformational stability of apo- and holo-beta-lactoglobulin (BLG). In apo-BLG simulations starting from the protonated crystal structure, we observe a long-lived H-bond interaction between the protonated Glu89 and Ser116. This interaction, sequestering the proton from the aqueous medium, explains a pK(half) value evaluated at pH 7.3 by continuum electrostatics/Monte Carlo computation on MD data, using linear response approximation. A very large root-mean-square deviation (RMSD; 5.11 A) is observed for the EF loop between protonated and unprotonated apo-BLG. This results from a quite different orientation of the EF loop that acts either as a closed or as an open lid above the protein calyx. Proton exchange by Glu89 in apo- but not in holo-BLG is associated with a reorganization energy of 4.7 kcal/mol. A 3-ns MD simulation starting from the crystal structure of protonated apo-BLG, but considering the Glu89 as unprotonated, shows the progressive opening of the lid giving rise to the Tanford transition. In both holo-BLG forms, the lid is most probably held in place by hydrophobic interactions of amino acid side-chains of the EF loop with the palmitate hydrocarbon tail.     

Identification of regions of leukotriene C4 synthase which direct the enzyme to its nuclear envelope localization

Leukotrienes (LTs) are fatty acid derivatives formed by oxygenation of arachidonic acid via the 5-lipoxygenase (5-LO) pathway. Upon activation of inflammatory cells 5-LO is translocated to the nuclear envelope (NE) where it converts arachidonic acid to the unstable epoxide LTA4. LTA4 is further converted to LTC4 by conjugation with glutathione, a reaction catalyzed by the integral membrane protein LTC4 synthase (LTC4S), which is localized on the NE and endoplasmic reticulum (ER). We now report the mapping of regions of LTC4S that are important for its subcellular localization. Multiple constructs encoding fusion proteins of green fluorescent protein (GFP) as the N-terminal part and various truncated variants of human LTC4S as C-terminal part were prepared and transfected into HEK 293/T or COS-7 cells. Constructs encoding hydrophobic region 1 of LTC4S (amino acids 6-27) did not give distinct membrane localized fluorescence. In contrast hydrophobic region 2 (amino acids 60-89) gave a localization pattern similar to that of full length LTC4S. Hydrophobic region 3 (amino acids 114-135) directed GFP to a localization indistinguishable from that of full length LTC4S. A minimal directing sequence, amino acids 117-132, was identified by further truncation. The involvement of the hydrophobic regions in the homo-oligomerization of LTC4S was investigated using bioluminescence resonance energy transfer (BRET) analysis in living cells. BRET data showed that hydrophobic regions 1 and 3 each allowed oligomerization to occur. These regions most likely form transmembrane helices, suggesting that homo-oligomerization of LTC4S is due to helix-helix interactions in the membrane.     

Completely buried, non-ion-paired glutamic acid contributes favorably to the conformational stability of pyrrolidone carboxyl peptidases from hyperthermophiles

Pyrrolidone carboxyl peptidases (PCPs) from hyperthermophiles have a structurally conserved and completely buried Glu192 in the hydrophobic core; in contrast, the corresponding residue in the mesophile protein is a hydrophobic residue, Ile. Does the buried ionizable residue contribute to stabilization or destabilization of hyperthermophile PCPs? To elucidate the role of the buried glutamic acid in stabilizing PCP from hyperthermophiles, we constructed five Glu192 mutants of PCP-0SH (C142S/C188S, Cys-free double mutant of PCP) from Pyrococcus furiosus and examined their thermal and pH-induced unfolding and crystal structures and compared them with those of PCP-0SH. The stabilities of apolar (E192A/I/V) and polar (E192D/Q) mutants were less than PCP-0SH at acidic pH values. In the alkaline region, the mutant proteins, except for E192D, were more stable than PCP-0SH. The thermal stability data and theoretical calculations indicated an apparent pKa value > or = 7.3 for Glu192. Present results confirmed that the protonated Glu192 in PCP-0SH forms strong hydrogen bonds with the carbonyl oxygen and peptide nitrogen of Pro168. New intermolecular hydrogen bonds in the E --> A/D mutants were formed by a water molecule introduced into the cavity created around position 192, whereas the hydrogen bonds disappeared in the E --> I/V mutants. Structure-based empirical stability of mutant proteins was in good agreement with the experimental results. The results indicated that (1) completely buried Glu192 contributes to the stabilization of PCP-0SH because of the formation of strong intramolecular hydrogen bonds and (2) the hydrogen bonds by the nonionized and buried Glu can contribute more than the burial of hydrophobic groups to the conformational stability of proteins.     

Analysis of the mechanism of the Serratia nuclease using site-directed mutagenesis.

Based on crystal structure analysis of the Serratia nuclease and a sequence alignment of six related nucleases, conserved amino acid residues that are located in proximity to the previously identified catalytic site residue His89 were selected for a mutagenesis study. Five out of 12 amino acid residues analyzed turned out to be of particular importance for the catalytic activity of the enzyme: Arg57, Arg87, His89, Asn119 and Glu127. Their replacement by alanine, for example, resulted in mutant proteins of very low activity, < 1% of the activity of the wild-type enzyme. Steady-state kinetic analysis of the mutant proteins demonstrates that some of these mutants are predominantly affected in their kcat, others in their Km. These results and the determination of the pH and metal ion dependence of selected mutant proteins were used for a tentative assignment for the function of these amino acid residues in the mechanism of phosphodiester bond cleavage by the Serratia nuclease.     

Interaction of wild-type and mutant adeno-associated virus (AAV) Rep proteins on AAV hairpin DNA.

Both the Rep68 and Rep78 proteins of adeno-associated virus type 2 (AAV) bind to AAV terminal repeat hairpin DNA and can mediate site-specific nicking in vitro at the terminal resolution site (trs) within the terminal repeats. To define the regions of the Rep proteins required for these functions, a series of truncated Rep78 derivatives was created. Wild-type and mutant proteins were synthesized by in vitro translation and analyzed for AAV hairpin DNA binding, trs endonuclease activity, and interaction on hairpin DNA. Amino-terminal deletion mutants which lacked the first 29 or 79 amino acid residues of Rep78 did not bind hairpin DNA, which is consistent with our previous identification of a DNA-binding domain in this region. Progressive truncation of the carboxyl-terminal region of Rep78 did not eliminate hairpin DNA binding until the deletion reached amino acid 443. The electrophoretic mobility of the Rep-specific protein-DNA complexes was inversely related to the molecular weight of the Rep derivative. Analysis of the C-terminal deletion mutants by the trs endonuclease assay identified a region (amino acids 467 to 476) that is essential for nicking but is not necessary for DNA binding. When endonuclease-positive, truncated Rep proteins that bound hairpin DNA were mixed with full-length Rep78 or Rep68 protein in electrophoretic mobility shift assays, a smear of protein-DNA complexes was observed. This smear migrated at an intermediate position with respect to the bands generated by the proteins individually. An antibody recognizing only the full-length protein produced a novel supershift band when included in a mixed binding assay containing Rep68 and a truncated Rep mutant. These experiments suggest that the Rep proteins can form hetero-oligomers on the AAV hairpin DNA.     

Carboxyl-terminal hydrophilic tail of a NhaP type Na+/H+ antiporter from cyanobacteria is involved in the apparent affinity for Na+ and pH sensitivity

Little information is available on the C-terminal hydrophilic tails of prokaryotic Na(+)/H(+) antiporters. To address functional properties of the C-terminal tail, truncation mutants in this domain were constructed. Truncation of C-terminal amino acid residues of NhaP1 type antiporter from Synechocystis PCC6803 (SynNhaP1) did not change the V(max) values, but increased the K(m) values for Na(+) and Li(+) about 3 to 15-fold. Truncation of C-terminal tail of a halotolerant cyanobacterium Aphanothece halophytica (ApNhaP1) significantly decreased the V(max) although it did not alter the K(m) values for Na(+). The C-terminal part of SynNhaP1 was expressed in E. coli and purified as a 16kDa soluble protein. Addition of purified polypeptide to the membrane vesicles expressing the C-terminal truncated SynNhaP1 increased the exchange activities. Change of Glu519 and Glu521 to Lys in C-terminal tail altered the pH dependence of Na(+)/H(+) and Li(+)/H(+) exchange activities. These results indicate that the specific acidic amino acid residues at C-terminal domain play important roles for the K(m) and the pH dependence of the exchange activity.     

De novo design of a pentameric coiled-coil: decoding the motif for tetramer versus pentamer formation in water-soluble phospholamban.

Water-soluble phospholamban (WSPLB) is a designed, water-soluble analogue of the pentameric membrane protein phospholamban (PLB), which contains the same core and interhelical residues as PLB, with only the solvent-exposed positions mutated. WSPLB contains the same secondary and quaternary structure as PLB. The hydrophobic cores of PLB and WSPLB contain Leu and Ile at the a- and d-positions of a heptad repeat (abcdefg) from residues 31-52, while residues 21-30 are rich in polar amino acids at these positions. While the full-length WSPLB forms pentamers in solution, truncated peptides lacking residues 21-30 are largely tetrameric. Thus, truncation of residues 1-20 promotes a switch from pentamer to tetramer formation. Here, the motifs for WSPLB pentamerization were elucidated by characterizing a series of peptides, which were progressively truncated in this polar 'switch' region. When fully present, the 'switch' region promotes pentamer formation in WSPLB, by destabilizing a more stable tetrameric species which exists in its absence. We find that the burial of hydrogen bonding residues from 21 to 30 drives WSPLB from a tetramer to a pentamer, with direct implications for coiled-coil design.     

AdoMet-dependent methyl-transfer: Glu119 is essential for DNA C5-cytosine methyltransferase M.HhaI

The role of Glu119 in S-adenosyl-L-methionine-dependent DNA methyltransferase M.HhaI-catalyzed DNA methylation was studied. Glu119 belongs to the highly conserved Glu/Asn/Val motif found in all DNA C5-cytosine methyltransferases, and its importance for M.HhaI function remains untested. We show that formation of the covalent intermediate between Cys81 and the target cytosine requires Glu119, since conversion to Ala, Asp or Gln lowers the rate of methyl transfer 10(2)-10(6) fold. Further, unlike the wild-type M.HhaI, these mutants are not trapped by the substrate in which the target cytosine is replaced with the mechanism-based inhibitor 5-fluorocytosine. The DNA binding affinity for the Glu119Asp mutant is decreased 10(3)-fold. Thus, the ability of the enzyme to stabilize the extrahelical cytosine is coupled directly to tight DNA binding. The structures of the ternary protein/DNA/AdoHcy complexes for both the Glu119Ala and Glu119Gln mutants (2.70 A and 2.75 A, respectively) show that the flipped base is positioned nearly identically with that observed in the wild-type M.HhaI complex. A single water molecule in the Glu119Ala structure between Ala119 and the extrahelical cytosine N3 is lacking in the Glu119Gln and wild-type M.HhaI structures, and most likely accounts for this mutant's partial activity. Glu119 has essential roles in activating the target cytosine for nucleophilic attack and contributes to tight DNA binding.     

Isolation of a series of single missense mutants of a dna gene of phage psi29, gene 1, utilizing their inhibitory effect on E. coli growth

Gene 1 product (gp1) of Bacillus subtilis phage psi29 is known to promote DNA replication of the phage. Although its role in the DNA replication is not clear, gp1 is reported to exhibit multiple characteristics, including RNA binding, cell membrane localization, and self-association. To investigate these characteristics, we undertook the isolation of a series of missense mutants of gene 1 bearing substitutions at various regions. During cloning of gene 1, we found that its expression severely inhibited the growth of its host Escherichia coli cells. In this study, we utilized this growth-inhibition phenomenon to screen a random library muta-genized by error-prone PCR, expecting that mutants which could not inhibit cell growth would be affected in the authentic functions of gp1. Using this approach, we obtained 31 different mutants bearing single amino acid substitutions at 26 positions along the entire length of gp1. As a preliminary analysis of these mutants, we compared the deduced amino acid sequences of gp1s from psi29 and its related phages PZA, B103 and M2. Alignment of these sequences revealed three conserved regions, i.e. a hydrophobic region near the carboxyl terminus (assumed to be involved in the membrane localization and self-association of gp1), coiled-coil motif (essential for self-association), and a region of unknown function near the amino terminus. Interestingly, many of the substitutions in the isolated mutants occurred at strongly conserved residues in these regions and affected characteristic features of the regions (e.g. hydrophobicity of the hydrophobic region). These substitutions are expected to affect authentic functions of gp1, and the mutants will be useful for studies of the structure and functions of gp1.     

Contribution of extracellular Glu residues to the structure and function of bacteriorhodopsin. Presence of specific cation-binding sites

Single and multiple mutants of extracellular Glu side chains of bacteriorhodopsin were analyzed by acid and calcium titration, differential scanning calorimetry, and thermal difference spectrophotometry. Acid titration spectra show that the second group protonating with Asp(85) is revealed in E204Q in the absence of Cl(-) but is not observed in the triple mutant E9Q/E194Q/E204Q or in the quadruple mutant E9Q/E74Q/E194Q/E204Q. The results point to Glu(9) as the second group protonating cooperatively with Asp(85). Comparison of the apparent pK(a) of Asp(85) protonation in water and in the deionized forms and results of calcium titration suggest that cation-binding sites are of low affinity in the multiple Glu mutants. Like for deionized wild type bacteriorhodopsin, differential scanning calorimetry reveals a lack of the pretransition in the multiple mutants, whereas in E9Q it appears at lower temperature and with lower cooperativity. Additionally, at neutral pH the band at 630 nm arising from cation release upon temperature increase is absent for the multiple mutants. Based on these results, we propose the presence of two cation-binding sites in the extracellular region of bacteriorhodopsin having as ligands Glu(9), Glu(194), Glu(204), and water molecules.     

Structural properties of the truncated and wild types of Taka-amylase: A molecular dynamics simulation and docking study

In this work, structures of the native (Amyl-C) and truncated Taka amylase were compared by molecular modeling methods. Using in silico enzyme engineering approach, 50 (Amyl-S1) and 100 (Amyl-S2) amino acids were eliminated from Amyl-C to produce the truncated forms. Analysis of the tertiary structures showed that three essential domains of the enzyme including super secondary structure (αβ)₈, the barrel region, and the large cleft remained native in Amyl-S1 and Amyl-S2. Secondary structures of Met112-Val118, Gly202-His211, Gln230-Asp233, Phe292-Asp297 residues in Amyl-C, Amyl-S1, and Amyl-S2 remained unchanged. These domains are necessary for catalytic function of alpha-amylase superfamily. Flexibility analysis of the three forms was examined and it is obtained that by truncation, the flexibility of the C-terminal domain was increased. This shows that C-terminal domain is essential for the stability of the structure which is in agreement with experimental observations. However, Glu156, Gln 162, Gly234, Val 245, Asn260, Ser264, Asp 297 of Amyl-C had higher flexibility than those in truncated enzymes. Maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose as five substrates were docked to the three enzyme forms. Binding affinity of maltoheptaose was higher in Amyl-C and Amyl-S1and lower in Amyl-S2 than that of maltotriose. In all forms the substrates were associated with three residues of the catalytic triad.     

Protein kinase C contains two phorbol ester binding domains

A series of deletion and truncation mutants of protein kinase C (PKC) were expressed in the baculovirus-insect cell expression system in order to elucidate the ability of various domains of the enzyme to bind phorbol dibutyrate (PDBu). A PKC truncation mutant consisting of only the catalytic domain of the enzyme did not bind [3H]PDBu, whereas a PKC truncation mutant consisting of the regulatory domain (containing the tandem cysteine-rich putative zinc finger regions) bound [3H]PDBu. Deletion of the second conserved region (C2) of PKC did not abolish [3H]PDBu binding, whereas a deletion of the first conserved region (C1) of PKC, containing the two cysteine-rich sequences, completely abolished [3H]PDBu binding. Additional truncation and deletion mutants helped to localize the region necessary for [3H]PDBu binding; all PKC mutants that contained either one of the cysteine-rich zinc finger-like regions possessed phorbol ester binding activity. Scatchard analyses of these mutants indicated that each bound [3H]PDBu with equivalent affinity (21-41 nM); approximately 10-20-fold less than the native enzyme. In addition, a peptide of 146 amino acid residues from the first cysteine-rich region, as well as a peptide of only 86 amino acids residues from the second cysteine-rich region, both bound [3H]PDBu with high affinity (31 +/- 4 and 59 +/- 13 nM, respectively). These data establish that PKC contains two phorbol ester binding domains which may function in its regulation.