Yoshinori Ishii

Chemical synthesis, cloning, and expression of genes for human somatomedin C (insulin-like growth factor I) and 59val-somatomedin C

In 1957, it was found that the action of growth hormone was mediated by a sulfation factor that promotes the incorporation of sulfate into the bone.’ This substance was renamed somatomedin as the mediator of growth hormone (somatotropin) in 1972; and many somatomedins have been reported since then. Somatomedins are produced mainly in the liver and kidney by the stimulation of growth hormone. Most of the biological activity of growth hormone is mediated by somatomedins. Somatomedins promote both the incorporation of sulfate into the bone and DNA synthesis in all kinds of cells. They also have insulin-like activity. Among them somatomedin C (SM-C) and insulin-like growth factor I, I1 (IGF-1,II) have been purified to homogeneity from human plasma.’*‘ The structures of IGF-I and I1 have been very well elucidated. Various bioassay and radioimmunoassay (RIA) data indicate that IGF-I and SM-C are identical. The primary structure of SM-C was proposed by Humbel el al.’ in 1978 as shown in FIGURE 1. SM-C was found to consist of 70 amino-acid residues with three disulfide bridges and to have high homology to the structure of proinsulin in both A and B domains. Although it is an interesting substance, it is very difficult to get a sufficient amount of it for study because several tons of serum yield only a few milligrams of SM-C; thus, biological studies with it have been insufficient. In order to study the biological and pharmacological properties and to supply enough samples for further evaluation, the mass production of SM-C using recombinant DNA technique is now under study.’ One of our strategies was to synthesize chemically the SM-C gene on the basis of the amino-acid sequence

A novel 7-β-(4-carboxybutanamido)-cephalosporanic acid acylase isolated from Pseudomonas strain C427 and its high-level production in Escherichia coli

Abstract We cloned the gene for 7-β-(4-carboxybutanamido)-cephalosporanic acid (GL-7ACA) acylase from Pseudomonas strain C427. The DNA sequence revealed an open reading frame of 2154 bp coding for 718 amino acid residues. The deduced amino acid sequence consists of 4 structural domains: (i) a signal peptide (positions 1–27), (ii) a small subunit of the acylase (positions 28–190), designated as α, (iii) a spacer peptide (positions 191–198), (iv) a large subunit (positions 199–718), designated as β. Plasmids were constructed to direct the synthesis of the acylase in Escherichia coli and the following results were obtained. The active acylase consists of two subunits which are processed from a single precursor protein, removing the spacer peptide during processing. A proportion of active acylase is secreted into the periplasm and the remainder is retained in the cytoplasm. The amount of precursor protein accumulated in the cytoplasm is greatly reduced when plasmids for the acylase lacking the signal sequence are expressed. Therefore, processing is independent of the translocation of the gene product through the cytoplasmic membrane, in contrast to the situation for penicillin G acylase. A high level of active enzyme production was achieved with a plasmid coding for an acylase in which the amino terminal sequence (positions 1–32) of native acylase is replaced by MFPTT.

Chemical modification and site-directed mutagenesis of tyrosine residues in cephalosporin C acylase from Pseudomonas strain N176

Cephalosporin C (CC) acylase from Pseudomonas strain N176 was chemically modified by tetranitromethane (TNM), causing complete loss of activity. Modification using molar excesses of TNM up to 10 resulted in complete inactivation when 1.4 mol tyrosines/mol enzyme were modified. Digestion of native and TNM-modified acylase with Achromobacter protease I (API), separation by high performance liquid phase chromatography (HPLC) and amino terminal sequencing of the resultant peptides were used to identify the modified tyrosine residues. The major difference in HPLC profile between these API digests was shown to be the peak corresponding to the peptide Ser239-Lys301 of native acylase. A portion of the peak for the peptide Ala45-Lys73 was also shifted in HPLC analysis of TNM-modified acylase. The peptides isolated from the modified acylase were shown to contain nitrated tyrosines (3-nitrotyrosine) at positions 270 and 52, respectively. These findings indicate that Tyr270 is completely modified, and Tyr52 is partially modified in the inactivated acylase. Each of the fifteen tyrosines in the acylase was altered to leucine by site-directed mutagenesis to complement the chemical modification with TNM. At pH 8.7, the mutant acylase in which tyrosine at position 270 is changed to leucine showed GL-7ACA and CC acylase activities reduced to 28.0 and 32.2% of native acylase, respectively. The results correspond to those obtained from TNM-modification. A similar reduction in activity was also obtained in the case of Tyr491 mutant, although nitration of this residue was not confirmed by chemical modification. Therefore Tyr270 and Tyr491 are important for exerting the maximum activity of the enzyme, but are not essential for catalysis. However, mutation of Tyr52 to Leu produced little change in acylase activity. The mutant acylase in which Tyr705 is changed to leucine has a lowered pH optimum for GL-7ACA, which may be useful for further improvement of the acylase.

Affinity labelling of cephalosporin C acylase from Pseudomonas sp. N176 with a substrate analogue, 7β-(6-bromohexanoylamido)cephalosporanic acid

Abstract We synthesized 7β-(6-bromohexanoylamido)cephalosporanic acid (6-BH-7ACA), a substrate analogue of an acylase from Pseudomonas N176 (N176 acylase) to determine the substrate binding site of the acylase. The enzyme was inactivated by incubation with 6-BH-7ACA in a time-dependent manner. A double reciprocal plot of the pseudo-first-order rate constant ( k obs ) against the 6-BH-7ACA concentration gave a straight line ( k max =0.113 min −1 , K I =0.51 mM). A plot of log k obs against log [6-BH-7ACA] was linear with a slope of 0.87. Inactivation of the enzyme with 6-BH-7ACA was inhibited by addition of 7-aminocephalosporanic acid and glutaric acid. These data indicate that 6-BH-7ACA functions as an affinity label reagent and the inactivation by 6-BH-7ACA is due to the modification of a single residue located in the neighborhood of the substrate binding region of the acylase. The digest of the inactivated enzyme with lysylendopeptidase was fractionated by reversed phase high-performance liquid chromatography (HPLC). One fragment was eluted with a different retention time from the corresponding fragment of the intact enzyme. From additional α-chymotryptic digestion followed by amino acid sequence analysis, Tyr 270 was determined as the site labelled by 6-BH-7ACA. Replacing Tyr 270 with a Phe residue by site-directed mutagenesis caused a decrease in the enzyme capability ( k cat / K m ). While the K m of the mutant acylase increased slightly, the k cat decreased to about 50% of that of the wild-type. These results indicate that although labelled Tyr 270 is not essential, it does play an important role in the enzymatic activity.

Purification and molecular characterization of a quinoprotein alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes IFO 14464

We have cloned and verified a gene for a novel quinoprotein alcohol dehydrogenase (ADH) from Pseudogluconobacter saccharoketogenes IFO 14464 that has the ability to oxidize L-sorbose to 2-keto-L-gulonic acid (2-KLGA). The enzyme was purified from the soluble fraction of the bacterium and was estimated to be a monomeric protein with a molecular weight of 65 kDa from the analyses of SDS-PAGE and gel-filtration chromatography. An open reading frame of 1824 bp for 608 amino acid residues was estimated as the gene for ADH because of the consistency of the calculated molecular mass and the elucidated partial amino acid sequences of the native enzyme. Homology search revealed that the enzyme showed close similarity to quinoprotein alcohol dehydrogenases isolated from Methylobacterium extorquens and Acetobacter aceti, particularly in the tryptophan docking motifs in the alpha-subunits of those dehydrogenases. The ability to convert L-sorbose to 2-KLGA was found when the lysate of recombinant Escherichia coli DH10B transformed with the gene for ADH was mixed with CaCl2and pyrroloquinoline quinone (PQQ). These data indicate that the cloned DNA is the desired gene for the ADH in which CaCl2 and PQQ are essential for enzymatic activity.

An Approach to High-Level Production of Cephalosporin Acylase from Pseudomonas strain N176 in Escherichia coli

Abstract The cultivation conditions for N176 cephalosporin acylase production were optimized using Escherichia coli JM109 pCK305S , which encodes a highly expressed mutant, C305S, in which Cys305 is altered to Ser, under the control of the trp promoter. Processing of the precursor protein to a two-chain active form in vivo was a key factor for high-level production of the enzyme. The active acylase was produced in a high amount under the following conditions: (i) addition of Trp in the second seed medium, (ii) the use of glycerol as a carbon source, (iii) induction with IAA at 9 h, and (iv) a shift down in the cultivation temperature from 25 to 22.5°C after induction. Under these high-level production conditions, the OD at 600 nm was found to increase in proportion to the number of colony forming units (CFUs), indicating that the CFU is an important index for the expression of the active enzyme.

Cloning, expression, purification, crystallization and preliminary diffraction analysis of the C-terminal catalytic domain of human poly(ADP-ribose) polymerase.

Two fragments of the C-terminal catalytic domain of human poly(ADP-ribose) polymerase (catPARP), Met-catPARP and Gly-Ser-catPARP, were purified and crystallized. Both catPARP crystals belong to space group C2, with almost the same unit-cell parameters. However, the shapes and harvest periods of both crystals were quite different owing to the slight mutation at the N-terminal position. Gly-Ser-catPARP was found to be more suitable for X-ray crystallography and crystals showed diffraction to at least 3.5 A resolution.

Crystallization and preliminary X-ray analysis of cephalosporin C acylase from Pseudomonas sp. strain N176

Cephalosporin C acylase from Pseudomonas sp. strain N176, a heterodimer of 25 and 58 kDa, has been crystallized using polyethylene glycol 6000 as precipitant. The crystals are orthorhombic and have unit-cell parameters a = 141.41, b = 192.10, c = 80.75 A. They belong to space group P2(1)2(1)2(1) and diffract to at least 2.7 A resolution. Calculations indicate that there are two heterodimers in the asymmetric unit. The structure is being solved by molecular replacement using penicillin G acylase from Escherichia coli as a search model and by multiple isomorphous replacement.