The key role of a non-active-site residue Met148 on the catalytic efficiency of meta-cleavage product hydrolase BphD

meta-Cleavage product (MCP) hydrolases (EC 3.7.1.9) can catalyze a specific C–C bond fission during the microbial aerobic degradation of aromatics. The previous studies on structure–function relationship of MCP hydrolases mainly focus on the active site residues by site-directed mutagenesis. However, the information about the role of the non-active-site residues is still unclear. In this study, a non-active-site residue Met148 of MCP hydrolase BphD was selected as the mutagenesis site according to the sequence alignments, structure superimpose and the tunnel analysis, which underwent the saturation mutagenesis resulting 19 mutants. The catalytic efficiencies of the mutants on 6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) were all decreased compared with the wild-type one except for the M148D mutant. Especially, the M148P mutant exhibited 290-fold lower k _cat/K _m than that of the wild-type BphD. Transient kinetic analyses of M148P showed the reciprocal relaxation time corresponded to C–C bond cleavage and product release steps (9.6 s^?1) was 4.08-fold lower than BphD WT (39.2 s^?1). Tunnel cluster analysis of BphD WT, M148P and M148W demonstrated that only the bulky Trp148 could block tunnel T2 in the BphD WT, but it exhibited slight effects on the catalytic efficiency (0.94-fold of BphD WT). Therefore, product release was not the main reason for the efficiency decrease of M148P. On the other hand, molecular dynamics simulations on the BphD WT and BphD M148P in complex with HOPDA indicated that the dramatic decrease of the catalytic efficiencies of BphD M148P should be due to the unproductive binding of HOPDA. The study demonstrated the catalytic efficiency of MCP hydrolase can be engineered by modification of non-active site residue.     

Characterization of a Carbon-Carbon Hydrolase from Mycobacterium tuberculosis Involved in Cholesterol Metabolism

In the recently identified cholesterol catabolic pathway of Mycobacterium tuberculosis, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (HsaD) is proposed to catalyze the hydrolysis of a carbon-carbon bond in 4,5–9,10-diseco-3-hydroxy-5,9,17-tri-oxoandrosta-1(10),2-diene-4-oic acid (DSHA), the cholesterol meta-cleavage product (MCP) and has been implicated in the intracellular survival of the pathogen. Herein, purified HsaD demonstrated 4–33 times higher specificity for DSHA (k_cat/K_m = 3.3 ± 0.3 × 10⁴ m⁻¹ s⁻¹) than for the biphenyl MCP 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) and the synthetic analogue 8-(2-chlorophenyl)-2-hydroxy-5-methyl-6-oxoocta-2,4-dienoic acid (HOPODA), respectively. The S114A variant of HsaD, in which the active site serine was substituted with alanine, was catalytically impaired and bound DSHA with a K_d of 51 ± 2 μm. The S114A·DSHA species absorbed maximally at 456 nm, 60 nm red-shifted versus the DSHA enolate. Crystal structures of the variant in complex with HOPDA, HOPODA, or DSHA to 1.8–1.9 Åindicate that this shift is due to the enzyme-induced strain of the enolate. These data indicate that the catalytic serine catalyzes tautomerization. A second role for this residue is suggested by a solvent molecule whose position in all structures is consistent with its activation by the serine for the nucleophilic attack of the substrate. Finally, the α-helical lid covering the active site displayed a ligand-dependent conformational change involving differences in side chain carbon positions of up to 6.7 Å, supporting a two-conformation enzymatic mechanism. Overall, these results provide novel insights into the determinants of specificity in a mycobacterial cholesterol-degrading enzyme as well as into the mechanism of MCP hydrolases.     

Crystal structures of two Bacillus carboxylesterases with different enantioselectivities

Naproxen esterase (NP) from Bacillus subtilis Thai I-8 is a carboxylesterase that catalyzes the enantioselective hydrolysis of naproxenmethylester to produce S-naproxen (E > 200). It is a homolog of CesA (98% sequence identity) and CesB (64% identity), both produced by B. subtilis strain 168. CesB can be used for the enantioselective hydrolysis of 1,2-O-isopropylideneglycerol (solketal) esters (E > 200 for IPG-caprylate). Crystal structures of NP and CesB, determined to a resolution of 1.75 Å and 2.04 Å, respectively, showed that both proteins have a canonical α/β hydrolase fold with an extra N-terminal helix stabilizing the cap subdomain. The active site in both enzymes is located in a deep hydrophobic groove and includes the catalytic triad residues Ser130, His274, and Glu245. A product analog, presumably 2-(2-hydroxyethoxy)acetic acid, was bound in the NP active site. The enzymes have different enantioselectivities, which previously were shown to result from only a few amino acid substitutions in the cap domain. Modeling of a substrate in the active site of NP allowed explaining the different enantioselectivities. In addition, Ala156 may be a determinant of enantioselectivity as well, since its side chain appears to interfere with the binding of certain R-enantiomers in the active site of NP. However, the exchange route for substrate and product between the active site and the solvent is not obvious from the structures. Flexibility of the cap domain might facilitate such exchange. Interestingly, both carboxylesterases show higher structural similarity to meta-cleavage compound (MCP) hydrolases than to other α/β hydrolase fold esterases.     

Characterization of a novel meta-fission product hydrolase from Dyella ginsengisoli LA-4

A novel meta-fission product hydrolase gene (mfphA) located in the bphX gene region of Dyella ginsengisoli LA-4 was successfully cloned and heterologously expressed in this study. The deduced amino acid sequence of MfphA showed 75% identity with the sequence of 2-hydroxymuconic semialdehyde hydrolase from Pseudomonas putida UCC22. The results suggested that MfphA belonged to the α/β hydrolase family, which could hydrolyze the meta-fission products (MFPs) during the biodegradation process of monocyclic compounds. The His-Tag MfphA was purified, and the subunit molecular mass of MfphA was about 35.3 kDa by SDS-PAGE analysis. According to the apparent kinetic parameters, the specificity of MfphA was determined in the following order: 6-methyl-HODA > HODA > 5-methyl-HODA > 6-phenyl-HODA > 5-chloro-HODA (HODA, 2-hydroxy-6-oxohexa-2,4-dienoate). The activity of MfphA for HODA was higher than that for 6-methyl-HODA. It was shown that the maximum activity was at 70 °C, and MfphA was stable for storage at low temperature for 30 days.     

Characterization of a C-C bond hydrolase from Sphingomonas wittichii RW1 with novel specificities towards polychlorinated biphenyl metabolites

Sphingomonas wittichii RW1 degrades chlorinated dibenzofurans and dibenzo-p-dioxins via meta cleavage. We used inverse PCR to amplify dxnB2, a gene encoding one of three meta-cleavage product (MCP) hydrolases identified in the organism that are homologues of BphD involved in biphenyl catabolism. Purified DxnB2 catalyzed the hydrolysis of 8-OH 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) approximately six times faster than for HOPDA at saturating substrate concentrations. Moreover, the specificity of DxnB2 for HOPDA (k(cat)/K(m) = 1.2 x 10(7) M(-1) s(-1)) was about half that of the BphDs of Burkholderia xenovorans LB400 and Rhodococcus globerulus P6, two potent polychlorinated biphenyl (PCB)-degrading strains. Interestingly, DxnB2 transformed 3-Cl and 4-OH HOPDAs, compounds that inhibit the BphDs and limit PCB degradation. DxnB2 had a higher specificity for 9-Cl HOPDA than for HOPDA but a lower specificity for 8-Cl HOPDA (k(cat)/K(m) = 1.7 x 10(6) M(-1) s(-1)), the chlorinated analog of 8-OH HOPDA produced during dibenzofuran catabolism. Phylogenetic analyses based on structure-guided sequence alignment revealed that DxnB2 belongs to a previously unrecognized class of MCP hydrolases, evolutionarily divergent from the BphDs although the physiological substrates of both enzyme types are HOPDAs. However, both classes of enzymes have mainly small hydrophobic residues lining the subsite that binds the C-6 phenyl of HOPDA, in contrast to the bulky hydrophobic residues (Phe106, Phe135, Trp150, and Phe197) found in the class II enzymes that prefer substrates possessing a C-6 alkyl. Thr196 and/or Asn203 appears to be an important determinant of specificity for DxnB2, potentially forming hydrogen bonds with the 8-OH substituent. This study demonstrates that the substrate specificities of evolutionarily divergent hydrolases may be useful for degrading mixtures of pollutants, such as PCBs.     

Catalytic role for arginine 188 in the C-C hydrolase catalytic mechanism for Escherichia coli MhpC and Burkholderia xenovorans LB400 BphD

The alpha/beta-hydrolase superfamily, comprised mainly of esterase and lipase enzymes, contains a family of bacterial C-C hydrolases, including MhpC and BphD which catalyze the hydrolytic C-C cleavage of meta-ring fission intermediates on the Escherichia coli phenylpropionic acid pathway and Burkholderia xenovorans LB400 biphenyl degradation pathway, respectively. Five active site amino acid residues (Arg-188, Asn-109, Phe-173, Cys-261, and Trp-264) were identified from sequence alignments that are conserved in C-C hydrolases, but not in enzymes of different function. Replacement of Arg-188 in MhpC with Gln and Lys led to 200- and 40-fold decreases, respectively, in k(cat); the same replacements for Arg-190 of BphD led to 400- and 700-fold decreases, respectively, in k(cat). Pre-steady-state kinetic analysis of the R188Q MhpC mutant revealed that the first step of the reaction, keto-enol tautomerization, had become rate-limiting, indicating that Arg-188 has a catalytic role in ketonization of the dienol substrate, which we propose is via substrate destabilization. Mutation of nearby residues Phe-173 and Trp-264 to Gly gave 4-10-fold reductions in k(cat) but 10-20-fold increases in K(m), indicating that these residues are primarily involved in substrate binding. The X-ray structure of a succinate-H263A MhpC complex shows concerted movements in the positions of both Phe-173 and Trp-264 that line the approach to Arg-188. Mutation of Asn-109 to Ala and His yielded 200- and 350-fold reductions, respectively, in k(cat) and pre-steady-state kinetic behavior similar to that of a previous S110A mutant, indicating a role for Asn-109 is positioning the active site loop containing Ser-110. The catalytic role of Arg-188 is rationalized by a hydrogen bond network close to the C-1 carboxylate of the substrate, which positions the substrate and promotes substrate ketonization, probably via destabilization of the bound substrate.     

Site Directed Mutagenesis of Schizosaccharomyces pombe Glutathione Synthetase Produces an Enzyme with Homoglutathione Synthetase Activity

Three different His-tagged, mutant forms of the fission yeast glutathione synthetase (GSH2) were derived by site-directed mutagenesis. The mutant and wild-type enzymes were expressed in E. coli DH5α and affinity purified in a two-step procedure. Analysis of enzyme activity showed that it was possible to shift the substrate specificity of GSH2 from Gly (k_m 0,19; wild-type) to β-Ala or Ser. One mutation (substitution of Ile471, Cy472 to Met and Val and Ala 485 and Thr486 to Leu and Pro) increased the affinity of GSH2 for β-Ala (k_m 0,07) and lowered the affinity for Gly (k_m 0,83), which is a characteristic of the enzyme homoglutathione synthetase found in plants. Substitution of Ala485 and Thr486 to Leu and Pro only, increased instead the affinity of GSH2 for Ser (k_m 0,23) as a substrate, while affinity to Gly was preserved (k_m 0,12). This provides a new biosynthetic pathway for hydroxymethyl glutathione, which is known to be synthesized from glutathione and Ser in a reaction catalysed by carboxypeptidase Y. The reported findings provide further insight into how specific amino acids positioned in the GSH2 active site facilitate the recognition of different amino acid substrates, furthermore they support the evolutionary theory that homoglutathione synthetase evolved from glutathione synthetase by a single gene duplication event.     

The conserved glycine/alanine residue of the active-site loop containing the putative acetylCoA-binding motif is essential for the overall structural integrity of Mesorhizobium loti arylamine N-acetyltransferase 1

The arylamine N-acetyltransferases are important xenobiotic-metabolizing enzymes that catalyze an acetyl group transfer from acetylCoA to arylamine substrates. NAT enzymes possess an active-site loop (the active-site P-loop) involved in substrate binding and selectivity. The Gly/Ala residue present at the start of the active-site P-loop, although conserved in all NAT enzymes, is not involved in the catalytic mechanism or substrate binding. Here we show that a small amino acid (such as Gly or Ala) at this position is important not only for maintaining the functions of the active-site P-loop but, more surprisingly, also important for maintaining the overall structural integrity of NAT enzymes. Our data thus suggest that in addition to its role in substrate binding and selectivity, the active-site P-loop could play a wider structural role in NAT enzymes.     

The crystal structure of Pseudomonas aeruginosa lipoxygenase Ala420Gly mutant explains the improved oxygen affinity and the altered reaction specificity

Secreted LOX from Pseudomonas aeruginosa (PA-LOX) has previously been identified as arachidonic acid 15S-lipoxygenating enzyme. Here we report that the substitution of Ala420Gly in PA-LOX leads to an enzyme variant with pronounced dual specificity favoring arachidonic acid 11R-oxygenation. When compared with other LOX-isoforms the molecular oxygen affinity of wild-type PA-LOX is 1–2 orders of magnitude lower (Km O₂ of 0.4 mM) but Ala420Gly exchange improved the molecular oxygen affinity (Km O₂ of 0.2 mM). Experiments with stereo-specifically deuterated linoleic acid indicated that the formation of both 13S- and 9R-HpODE involves abstraction of the proS-hydrogen from C11 of the fatty acid backbone. To explore the structural basis for the observed functional changes (altered specificity, improved molecular oxygen affinity) we solved the crystal structure of the Ala420Gly mutant of PA-LOX at 1.8 Å resolution and compared it with the wild-type enzyme. Modeling of fatty acid alignment at the catalytic center suggested that in the wild-type enzyme dioxygen is directed to C15 of arachidonic acid by a protein tunnel, which interconnects the catalytic center with the protein surface. Ala420Gly exchange redirects intra-enzyme O₂ diffusion by bifurcating this tunnel so that C11 of arachidonic acid also becomes accessible for O₂ insertion.     

Conversion of a Rhizopus chinensis lipase into an esterase by lid swapping

In an effort to explore the feasibility of converting a lipase into an esterase by modifying the lid region, we designed and characterized two novel Rhizopus chinensis lipase variants by lid swapping. The substrate specificity of an R. chinensis lipase was successfully modified toward water-soluble substrates, that is, turned into an esterase, by replacing the hydrophobic lid with a hydrophilic lid from ferulic acid esterase from Aspergillus niger. Meanwhile, as a comparison, the lid of R. chinensis lipase was replaced by a hydrophobic lid from Rhizomucor miehei lipase, which did not alter its substrate specificity but led to a 5.4-fold higher catalytic efficiency (k*_cat/K*_m) toward p-nitrophenyl laurate. Based on the analysis of structure-function relationships, it suggests that the amphipathic nature of the lid is very important for the substrate specificity. This study provides new insight into the structural basis of lipase specificities and a way to tune the substrate preference of lipases.     

Engineering of Yarrowia lipolytica lipase Lip8p by circular permutation to alter substrate and temperature characteristics

Applications of lipases are mainly based on their catalytic efficiency and substrate specificity. In this study, circular permutation (CP), an unconventional protein engineering technique, was employed to acquire active mutants of Yarrowia lipolytica lipase Lip8p. A total of 21 mutant lipases exhibited significant shifts in substrate specificity. Cp128, the most active enzyme mutant, showed higher catalytic activity (14.5-fold) and higher affinity (4.6-fold) (decreased K _m) to p-nitrophenyl-myristate (pNP-C₁₄) than wild type (WT). Based on the three-dimensional (3D) structure model of the Lip8p, we found that most of the functional mutation occurred in the surface-exposed loop region in close proximity to the lid domain (S112–F122), which implies the steric effect of the lid on lipase activity and substrate specificity. The temperature properties of Cp128 were also investigated. In contrast to the optimal temperature of 45 °C for the WT enzyme, Cp128 exhibited the maximal activity at 37 °C. But it is noteworthy that there is no change in thermostability.     

Thermodynamic profiles of penicillin G hydrolysis catalyzed by wild-type and Met→Ala168 mutant penicillin acylases from Kluyvera citrophila

The Met-168 residue in penicillin acylase from Kluyvera citrophila was changed to Ala by oligonucleotide site-directed mutagenesis. The Ala-168 mutant exhibited different substrate specificity than wild-type and enhanced thermal stability. The thermodynamic profiles for penicillin G hydrolysis catalyzed by both enzymes were obtained from the temperature dependence of the steady-state kinetic parameters K_m and k_cat. The high values of enthalpy and entropy of activation determined for the binding of substrate suggest that an induced-fit-like mechanism takes place. The Met→Ala168 mutation unstabilizes the first transition-state (E··S^≠) and the enzyme-substrate complex (ES) causing a decrease in association equilibrium and specificity constants in the enzyme. However, no change is observed in the acyl-enzyme formation. It is concluded that residue 168 is involved in the enzyme conformational rearrangements caused by the interaction of the acid moiety of the substrate at the active site.     

Stabilization of plant formate dehydrogenase by rational design

Recombinant formate dehydrogenase (FDH, EC 1.2.1.2) from soy Glycine max (SoyFDH) has the lowest values of Michaelis constants for formate and NAD⁺ among all studied formate dehydrogenases from different sources. Nevertheless, it also has the lower thermal stability compared to enzymes from bacteria and yeasts. The alignment of full sequences of FDHs from different sources as well as structure of apo- and holo-forms of SoyFDH has been analyzed. Ten mutant forms of SoyFDH were obtained by site-directed mutagenesis. All of them were purified to homogeneity and their thermal stability and substrate specificity were studied. Thermal stability was investigated by studying the inactivation kinetics at different temperatures and by differential scanning calorimetry (DSC). As a result, single-point (Ala267Met) and double mutants (Ala267Met/Ile272Val) were found to be more stable than the wild-type enzyme at high temperatures. The stabilization effect depends on temperature, and at 52°C it was 3.6- and 11-fold, respectively. These mutants also showed higher melting temperatures in DSC experiments — the differences in maxima of the melting curves (T _m) for the single and double mutants were 2.7 and 4.6°C, respectively. For mutations Leu24Asp and Val127Arg, the thermal stability at 52°C decreased 5- and 2.5-fold, respectively, and the T _m decreased by 3.5 and 1.7°C, respectively. There were no differences in thermal stability of six mutant forms of SoyFDH — Gly18Ala, Lys23Thr, Lys109Pro, Asn247Glu, Val281Ile, and Ser354Pro. Analysis of kinetic data showed that for the enzymes with mutations Val127Arg and Ala267Met the catalytic efficiency increased 1.7- and 2.3-fold, respectively.     

Evidence for a gem-diol reaction intermediate in bacterial C-C hydrolase enzymes BphD and MhpC from 13C NMR spectroscopy

C-C hydrolase enzymes MhpC and BphD catalyze the hydrolytic C-C cleavage of meta-ring fission intermediates on the Escherichia coli phenylpropionic acid and Burkholderia xenovorans LB400 biphenyl degradation pathways and are both members of the alpha/beta-hydrolase family containing a Ser-His-Asp catalytic triad. The catalytic mechanism of this family of enzymes is thought to proceed via a gem-diol reaction intermediate, which has not been observed directly. Site-directed single mutants of BphD in which catalytic residues His-265 and Ser-112 were replaced with Ala were found to possess 10(4)-fold reduced k(cat) values, and in each case, the C-C cleavage step was shown by pre-steady-state kinetic analysis to be rate-limiting. The processing of a 6-(13)C-labeled aryl-containing substrate by these H265A or S112A mutant BphD enzymes was monitored directly by (13)C NMR spectroscopy. A new line-broadened signal was observed at 128 ppm for each enzyme, corresponding to the proposed gem-diol reaction intermediate, over a time scale of 1-24 h. A similar signal was observed upon incubation of the (13)C-labeled substrate with an H114A MhpC mutant, which is able to accept the 6-phenyl-containing substrate, on a shorter time scale. The direct observation of a gem-diol intermediate provides further evidence that supports a general base mechanism for this family of enzymes.     

Frequent Missense and Insertion/Deletion Polymorphisms in the Ovine Shadoo Gene Parallel Species-Specific Variation in PrP

Background

The cellular prion protein PrP^C is encoded by the Prnp gene. This protein is expressed in the central nervous system (CNS) and serves as a precursor to the misfolded PrP^Sc isoform in prion diseases. The prototype prion disease is scrapie in sheep, and whereas Prnp exhibits common missense polymorphisms for V136A, R154H and Q171R in ovine populations, genetic variation in mouse Prnp is limited. Recently the CNS glycoprotein Shadoo (Sho) has been shown to resemble PrP^C both in a central hydrophobic domain and in activity in a toxicity assay performed in cerebellar neurons. Sho protein levels are reduced in prion infections in rodents. Prompted by these properties of the Sho protein we investigated the extent of natural variation in SPRN.

Principal Findings

Paralleling the case for ovine versus human and murine PRNP, we failed to detect significant coding polymorphisms that alter the mature Sho protein in a sample of neurologically normal humans, or in diverse strains of mice. However, ovine SPRN exhibited 4 missense mutations and expansion/contraction in a series of 5 tandem Ala/Gly-containing repeats R1-R5 encoding Sho''s hydrophobic domain. A Val71Ala polymorphism and polymorphic expansion of wt 67(Ala)₃Gly70 to 67(Ala)₅Gly72 reached frequencies of 20%, with other alleles including Δ67–70 and a 67(Ala)₆Gly73 expansion. Sheep V71, A71, Δ67–70 and 67(Ala)₆Gly73 SPRN alleles encoded proteins with similar stability and posttranslational processing in transfected neuroblastoma cells.

Significance

Frequent coding polymorphisms are a hallmark of the sheep PRNP gene and our data indicate a similar situation applies to ovine SPRN. Whether a common selection pressure balances diversity at both loci remains to be established.     

Substrate Specificity of Cucumisin on Synthetic Peptides

The substrate specificity of cucumisin [EC 3.4.21.25] was identified by the use of the synthetic peptide substrates Leu_m-Pro-Glu-Ala-Leu_n (m=0-4, n=0-3). Neither Pro-Glu-Ala-Leu (m=0) nor Leu-Pro-Glu-Ala (n=0) was cleaved by cucumisin, however other analogus peptides were cleaved between Glu-Ala. The hydrolysis rates of Leu_m-Pro-Glu-Ala-Leu increased with the increase of m=1 to 2 and 3, but was however, essentially same with the increase of m=3 to 4. Similarly, the hydrolysis rates of Leu-Leu-Pro-Glu-Ala-Leu_n increased with the increase of n=0 to 1 and 2, but was essentially same with the increase of n=2 to 3. Then, it was concluded that cucumisin has a S₅-S₃′ subsite length. In order to identify the substrate specificity at P₁ position, Leu-Leu-Pro-X-Ala-Leu (X; Gly, Ala, Val, Leu, Ile, Pro, Asp, Glu, Lys, Arg, Asn, Gln, Phe, Tyr, Ser, Thr, Met, Trp, His) were synthesized and digested by cucumisin. Cucumisin showed broad specificity at the P₁ position. However, cucumisin did not cleave the C-terminal side of Gly, Ile, Pro, and preferred Leu, Asn, Gln, Thr, and Met, especially Met. Moreover, the substrates, Leu-Leu-Pro-Glu-Y-Leu (Y; Gly, Ala, Ser, Leu, Val, Glu, Lys, Phe) were synthesized and digested by cucumisin. Cucumisin did not cleave the N-terminal side of Val but preferred Gly, Ser, Ala, and Lys especially Ser. The specificity of cucumisin for naturally occurring peptides does not agree strictly with the specificity obtained by synthetic peptides at the P₁ or P₁′ position alone, but it becomes clear that the most of the cleavage sites on naturally occurring peptides by cucumisin contain suitable amino acid residues at P₁ and (or) P₁′ positions. Moreover, cucumisin prefers Pro than Leu at P₂ position, indicating that the specificity at P₂ position differs from that of papain.     

Enhanced antibacterial activity of an attacin-coleoptericin hybrid protein fused with a helical linker

Previously, we isolated and characterized attacin from Spodoptera exigua and a coleoptericin-like protein from Protaetia brevitarsis seulensis. In this study, we fused these two genes encoding antimicrobial proteins to obtain a hybrid protein with enhanced antimicrobial activity. To fuse the two antimicrobial proteins, we employed helical and non-helical linker sequences that function as inter-domain linkers in proteins. We used the Gly–Gly–Gly–Gly–Ser peptide as a non-helical linker. The hybrid protein produced using this linker showed less antimicrobial activity against Escherichia coli, Bacillus subtilis, Burkholderia glumae, Pseudomonas corrugate, and Erwinia rhapontici than either of the two parental antimicrobial proteins. In addition, the MIC value of the hybrid protein was 23.1 μM, which indicates poor activity against E. coli. When we used three Glu–Ala–Ala–Ala–Lys (EAAAK) peptide sequences as a helical linker to fuse the two proteins, the resultant hybrid protein had much higher antimicrobial activity than the parental antimicrobial proteins. In particular, this hybrid protein had strong antimicrobial activity against P. corrugate. These results indicate that the EAAAK motif can be used to effectively separate two antimicrobial proteins and produce a hybrid protein with more antimicrobial activity than either of the parent proteins.     

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.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (M_{r ca.} 29,000), and the S-peptide (M_r 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH₂-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH₂-terminal 60-amino acid peptide.