Si Myung Byun

Effect on product specificity of cyclodextrin glycosyltransferase by site-directed mutagenesis

Cyclodextrin glycosyltransferase (CGTase; EC 2.4.1.19) catalyzes the degradation of starch into cyclodextrins through an intrarnolecular transglycosylation reaction. Tyr‐89, Asn‐94, and Tyr‐100 are located near the putative active center. To analyze their roles in product specificity, Tyr‐89, Asn‐94, and Tyr‐100 of CGTase from alkalophilic Bacillus sp. 1‐5 were replaced with different amino acids. Among the mutants, the N94S mutant protein produced about two times more α‐cyclodextrin than the wild‐type at all incubation times. The Y89F and Y100F mutant proteins were changed to more β‐specific enzymes. From these results it is suggested that the changing of the residues located at the near active site can change the product specificity of CGTase.

The pore size of the autotransporter domain is critical for the active translocation of the passenger domain.

The autotransporter mode of surface presentation in Gram-negative bacteria requires a hypothetical C-terminal beta-barrel which makes up an aqueous channel in the outer membrane. PalA is a Pseudomonas sp. autotransporter lipolytic protein. PalA is a 66 kDa protein that is composed of two parts, the N-terminal region (Ala(1)-Ala(296)) similar to the GDSL lipases and the C-terminal region (Leu(320)-Phe(612)) to the autotransporter. In this report, we provide biochemical and structural evidence demonstrating that the pore size of the beta-barrel conduit is important in delivering the N-terminal domain to the cell surface. Among all the autotransporter domains two strictly conserved residues (Pro(478) and Gly(576) in PalA) are converted to other various residues using site-directed mutagenesis. This investigation was made into the different pore-size mutants, affecting the folding of N-terminal domain. Wild beta-domain contains a cavity of approximately 2 nm diameter that is optimal for the active conformation of the N-terminal domains. However, deviation from the proper size of the pore, whether it is larger or smaller, is not suitable for the proper folding of the N-terminal catalytic domain.

C-Terminal Peptide of Streptokinase, Met369-Pro373, is Important in Plasminogen Activation

Streptokinase(SK), a plasminogen activator, is known to have multi‐domain structure. The function of the C‐terminal region of streptokinase was investigated with SK mutants constructed by truncating 26, 33, 37, 40, 41, 46, 47, 70 or 97 amino acid residues from the C‐terminus. The truncated SKs were expressed in E.coli and purified. The 41 residue deletion (SKP373) from the C‐terminus had not effect on the plasminogen activation activity. However, the deletion of 46 amino acid residues (SKP368) resulted in the dramatic reduction of the plasminogen activation efficiency. The result suggests that the C‐terminal peptide from Met369 to Pro373 of SK may play an important role on the plasminogen activation.

Purification and properties of ampicillin acylase from Pseudomonas melanogenum

Ampicillin acylase, which is known to have a novel substrate spectrum, was purified to homogeneity from Pseudomonas melanogenum by the crude extract preparation and chromatography with S-Sepharose, hydroxyapatite, CM-cellulose C-52, and CM-Sepharose. The molecular weight of the native enzyme was calculated to be 146,000 by Protein PAK-300 sw HPLC chromatography. SDS-polyacrylamide gel electrophoresis revealed that the enzyme consisted of two identical subunits with a molecular weight of 72,000. The enzyme was a glycoprotein containing 13% of total carbohydrate, and its isoelectric point was 7.2. The enzyme catalyzed both synthesis and hydrolysis of ampicillin and hydrolysis of the ester bond of phenylglycinemethylester hydrochloride substrate. The substrate specificity showed that the enzyme required a free amino group on the alpha-carbon of the acyl group. Chemical modification by diethylpyrocarbonate or N-bromosuccinimide resulted in time-dependent inactivation of the enzyme, and other results suggest the participation of essential histidine residue(s) in the catalytic activity of ampicillin acylase. Substrates of the enzyme, 6-aminopenicillanic acid and ampicillin, exhibited protective effects against N-bromosuccinimide inactivation, suggesting that the modification occurred near or at the active site.

Identification of H‐2Kb‐restricted T‐cell epitopes within the nucleocapsid protein of Hantaan virus and establishment of cytotoxic T‐cell clones

Although neutralizing antibodies against Hantaan virus (HTV) can protect hosts from viral infection, T‐cell responses to HTV are also important in host defense against HTV. However, much less is known about cytotoxic T lymphocyte (CTL) responses to HTV. To identify CTL epitopes in the HTV nucleocapsid protein (NP), we selected 7 H‐2Kb‐motif‐fitting peptides. Of these peptides, 3 peptides (NP3, NP4, and NP7) were recognized by CTL responses derived from HTV‐immunized mouse splenocytes. NP3 and NP4 peptides were also recognized by HTV‐immunized splenocytes after secondary in vitro stimulation with the relevant peptide, but NP7 could not be recognized after in vitro stimulation. These results agree well with peptide immunization studies showing that peptide‐specific CTL responses could be induced with NP3 and NP4 but not with NP7 peptide. Furthermore, CTL activity assay using targets, prepared to express the antigen (NP) endogenously, demonstrated that NP3 and NP4 peptides could be presented endogenously. CTL elicited with NP4 peptide retained some cross‐reactivity and was difficult to long‐term culture. However, NP3‐elicited CTL was very specific for NP3 peptide and was stable enough to be cloned. Among many CTL lines elicited with HTV or HTV NP peptides, 6 NP3‐specific CTL clones were established and have been maintained more than 2 years. All 6 CTL clones were characterized to be CD3+, CD4−, CD8+, CD25+, CD62L−, and NK1.1−, and to use TCR Vβ6. This preferential usage of TCR Vβ6 indicates that TCR Vβ6 regions are important for recognition of the HTV NP3 epitope (NP221–228, SVIGFLAL) on H‐2Kb molecule. Our data demonstrate the definition of mouse CTL epitopes in HTV and the generation of HTV‐specific mouse CTL clones. J. Med. Virol. 60:189–199, 2000.

Site-specific alteration of Gly-24 in streptokinase: Its effect on plasminogen activation

Oligonucleotide-directed mutagenesis was carried out to replace glycine-24 of streptokinase with histidine, glutamic acid, or alanine. Substitutions with either histidine or glutamic acid resulted in almost complete loss of streptokinase activity but streptokinase replaced with alanine retained its activity. Although streptokinases with histidine-24 or glutamic acid-24 bound normally to human plasminogen, they were not able to generate active plasmin, whereas those with alanine-24 or glycine-24 (wild-type) could generate active plasmin. The results indicate that the small, uncharged alkyl group side-chain on the 24th amino acid residue of streptokinase is indispensable for the activity of the human plasminogen-streptokinase complex.

Elongation factor-2 in chick embryo is phosphorylated on tyrosine as well as serine and threonine.

An endogenous 95 kDa chick embryo cytosolic protein (p95) was phosphorylated in the presence of [gamma-32P]ATP and the kinase activity for p95 was mostly associated with particulate fraction. Phosphorylation of p95 was prominent in embryos of early developmental stage. Hydrolysis of p95 phosphoprotein yielded phosphotyrosine in addition to phosphothreonine and phosphoserine. Native p95 was also tyrosine-phosphorylated. p95 phosphoprotein was purified by DEAE-Sephacel chromatography and immunoprecipitation with anti-phosphotyrosine antibody and the amino acid sequence was determined. The N-terminal sequence, Val-Asn-Phe-Thr-Val-Asp-Gln-Ile-Arg-Ala-Ile-Met-Asp- Lys-Lys-Ala-Asn-Ile-Arg-Asn-Met-, was found to be identical to those of elongation factor-2 (EF-2) of both rat and hamster. Our results suggest the presence of other EF-2 kinase in chick embryo cell than the previously reported Ca2+/calmodulin-dependent protein kinase III.

Effect of the weak Ca2+-binding site of subtilisin J by site-directed mutagenesis on heat stability

The functional role of the negatively charged amino acid residue in subtilisin J from Bacillus stearothermophilus has been investigated by site-directed mutagenesis. Glu-195 located at the weak Ca2+-binding site was replaced with Gln to examine the role of Glu-195 in the heat stability of subtilisin J. Mutant enzyme was expressed in Bacillus subtilis and was purified from the culture supernatant. When the mutant enzyme was expressed at 37 degrees C in the presence of 2mM calcium chloride, the pattern of enzyme production was quite different from that of wild-type. The purified Gln-195 mutant enzyme was analyzed with respect to optimal temperature, optimal pH, and heat stability. The mutation was found to decrease the heat stability but not catalytic efficiency (kcat/Km) and optimal pH. These results demonstrate the important role of the negatively charged side chains at the weak Ca(2+)-binding site in the heat stability of subtilisin.

The leader sequence of streptokinase is responsible for its post-translational carboxyl-terminal cleavage.

When the expression of streptokinase from two tac promoter-controlled expression vectors, one of these deleted a putative leader sequence of streptokinase and the other not, was compared, both normal and degraded streptokinase were detected in proteins expressed from the leader-contained vector, but only normal streptokinase was detected from the leader-deleted vector. These findings indicate that the characteristic carboxyl-terminal cleavage of streptokinase is correlated with its leader sequence and occurs during the defective secretion. The homogeneous preparation of streptokinase was facilitated by expressing from this leader-deleted vector.

Secretion of Bacillus α‐amylase from yeast directed by glucoamylase I signal sequence of Saccharomyces diastaticus

For the secretion of Bacillus stearothermophilus α ‐amylase from yeast, a recombinant plasmid pGAT17 was constructed by fusing B. stearothermophilus α‐amylase structural gene in frame to the promoter and signal sequence of Saccharomyces diastaticus glucoamylase I gene (STA1). The secretion of the heterologous α‐amylase from S. diastaticus transformed with pGAT17 was confirmed by the halo formation around colonies on selective starch agar medium. About 80% of the total α‐amylase activity was detected in the extracellular culture medium. The secreted α‐amylase was glycosylated and its molecular weight increased from 61 kDa to 75 kDa. The thermostability of the glycosylated α‐amylase was markedly enhanced, compared with that of the non‐glycosylated enzyme from E. coli.