Mutational analysis of feedback inhibition and catalytic sites of prephenate dehydratase from Corynebacterium glutamicum

Prephenate dehydratase is a key regulatory enzyme in the phenylalanine-specific pathway of Corynebacterium glutamicum. PCR-based random mutagenesis and functional complementation were used to screen for m-fluorophenylalanine (mFP)-resistant mutants. Comparison of the amino acid sequence of the mutant prephenate dehydratases indicated that Ser-99 plays a role in the feedback regulation of the enzyme. When Ser-99 of the wild-type enzyme was replaced by Met, the specific activity of the mutant enzyme was 30% lower than that of the wild-type. The Ser99Met mutant was active in the presence of 50 muM phenylalanine, whereas the wild-type enzyme was not. The functional roles of the eight conserved residues of prephenate dehydratase were investigated by site-directed mutagenesis. Glu64Asp substitution reduced enzyme activity by 15%, with a 4.5- and 1.7-fold increase in K-m and k(cat) values, respectively. Replacement of Thr-183 by either Ala or Tyr resulted in a complete loss of enzyme activity. Substitution of Arg-184 with Leu resulted in a 50% decrease of enzyme activity. The specific activity for Phe185Tyr was more than 96% lower than that of the wild-type, and the K-m value was 26-fold higher. Alterations in the conserved Asp-76, Glu-89, His-115, and Arg-236 residues did not cause a significant change in the K-m and k(cat) values. These results indicated that Glu-64, Thr-183, Arg-184, and Phe-185 residues might be involved in substrate binding and/or catalytic activity

Expression of Pseudomonas amyloderamosa isoamylase gene in Saccharomyces cerevisiae

Isoamylase gene (iso) of Pseudomonas amyloderamosa was amplified by polymerase chain reaction and cloned into Saccharomyces cerevisiae vectors under the control of alcohol dehydrogenase gene and glyceraldehyde-3-phosphate dehydrogenase gene promoters. The signal sequence of iso gene was also replaced with that of Schwanniomyces occidentalis alpha-amylase gene. The extracellular isoamylase activity of transformed Sacc. cerevisiae could reach 86 U ml(-1) after a 4-days cultivation

Phylogenetic analysis and biochemical characterization of a thermostable dihydropyrimidinase from alkaliphilic Bacillus sp TS-23

Two degenerate primers established from the alignment of highly conserved amino acid sequences of bacterial dihydropyrimidinases (DHPs) were used to amplify a 330-bp gene fragment from the genomic DNA of Bacillus sp. TS-23 and the amplified DNA was successfully used as a probe to clone a dhp gene from the strain. The open reading frame of the gene consisted of 1422 bp and was deduced to contain 472 amino acids with a molecular mass of 52 kDa. The deduced amino acid sequence exhibited greater than 45% identity with that of prokaryotic D-hydantoinases and eukaryotic DHPs. Phylogenetic analysis showed that Bacillus sp. TS-23 DHP is grouped together with Bacillus stearothermophilus D-hydantoinase and related to dihydroorotases and allantoinases from various organisms. His(6)-tagged DHP was over-expressed in Escherichia coli and purified by immobilized metal affinity chromatography to a specific activity of 3.46 U mg(-1) protein. The optimal pH and temperature for the purified enzyme were 8.0 and 60 degrees C, respectively. The half-life of His(6)-tagged DHP was 25 days at 50 degrees C. The enzyme activity was stimulated by Co2+ and Mn2+ ions. His(6)-tagged DHP was most active toward dihydrouracil followed by hydantoin derivatives. The catalytic efficiencies (k(cat)/K-m) of the enzyme for dihydrouracil and hydantoin were 2.58 and 0.61 s(-1) mM(-1), respectively

The Host Galaxies of X-ray Quasars Are Not Strong Star Formers

We use ultradeep SCUBA-2 850 μm observations (~0.37 mJy rms) of the 2 Ms Chandra Deep Field-North (CDF-N) and 4 Ms Chandra Deep Field-South X-ray fields to examine the amount of dusty star formation taking place in the host galaxies of high-redshift X-ray active galactic nuclei (AGNs). Supplementing with COSMOS, we measure the submillimeter fluxes of the 4–8 keV sources at $z\gt 1$, finding little flux at the highest X-ray luminosities but significant flux at intermediate luminosities. We determine graybody and MIR luminosities by fitting spectral energy distributions to each X-ray source and to each radio source in an ultradeep Karl G. Jansky Very Large Array (VLA) 1.4 GHz (11.5 μJy at $5\sigma$) image of the CDF-N. We confirm the far-infrared (FIR)-radio and mid-infrared (MIR)-radio correlations to z = 4 using the non-X-ray detected radio sources. Both correlations are also obeyed by the X-ray less luminous AGNs but not by the X-ray quasars. We interpret the low FIR luminosities relative to the MIR for the X-ray quasars as being due to a lack of star formation, while the MIR stays high due to the AGN contribution. We find that the FIR luminosity distributions are highly skewed and the means are dominated by a small number of high-luminosity galaxies. Thus, stacking or averaging analyses will overestimate the level of star formation taking place in the bulk of the X-ray sample. We conclude that most of the host galaxies of X-ray quasars are not strong star formers, perhaps because their star formation is suppressed by AGN feedback

Substitution of the critical methionine residues in Trigonopsis variabilis D-amino acid oxidase with leucine enhances its resistance to hydrogen peroxide

Each of the six oxidative-sensitive methionine residues in Trigonopsis variabilis D-amino acid oxidase (DAAO) was changed to leucine by site-directed mutagenesis. The wild-type and mutant enzymes with an apparent molecular mass of about 39.3 kDa were expressed in Escherichia coli. The specific activity of four mutant DAAOs (Met(104)Leu, Met(226)Leu, Met(245)Leu, and Met(339)Leu) was decreased by more than 96% while Met(156)Leu and Met(209)Leu showed about 23% and 96% higher activity, respectively, than the wild-type enzyme. The kinetic parameters of the two mure active enzymes were determined and a 2.2-fold increase in K(m) was observed for Met(209)Leu. Comparison of Met(156)Leu and wild-type DAAO revealed a 95% increase in k(cat)/K(m). Met(156)Leu, Met(209)Leu, and Met(226)Leu were resistant to inactivation by 50 mM H(2)O(2) The other three mutant DAAOs were also slightly more resistant than the wild-type enzyme to chemical oxidation. These observations indicate that the oxidative stability in T. variabilis DAAO can be improved by substitution of methionine residues with leucine. (C) 2000 Published by Elsevier Science B.V. All rights reserved

High-level expression of Trigonopsis variabilis D-amino acid oxidase in Escherichia coli using lactose as inducer

The use of lactose as inducer for the expression of Trigonopsis variabilis D-amino acid oxidase gene (daao) was investigated in Escherichia coli regulated by T7 or T5 promoter. The daao gene was prepared by reverse transcriptase-polymerase chain reaction and cloned into pET21b and pQE-30 to yield pET-DAAO and pQE-DAAO, respectively. The His(6)-tagged DAAO was expressed in E. coli and had a M-r value of approximately 39.3 kDa. In lactose-induced E. coli BL21 (DE3) (pET-DAAO), the expressed DAAO could comprise up to 15% of total soluble proteins and a productivity of 23.4 U ml(-1) was obtained

Overexpression, one-step purification, and biochemical characterization of a recombinant gamma-glutamyltranspeptidase from Bacillus licheniformis

A truncated gene from Bacillus lichenifromis ATCC 27811 encoding a recombinant gamma-glutamyltranspeptidase (BLrGGT) was cloned into pQE-30 to generate pQE-BLGGT, and the overexpressed enzyme was purified from the crude extract of IPTG-induced E. coli M15 (pQE-BLGGT) to homogeneity by nickel-chelate chromatography. This protocol yielded over 25 mg of purified BLrGGT per liter of growth culture under optimum conditions. The molecular masses of the subunits of the purified enzyme were determined to be 41 and 22 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature for the recombinant enzyme were 6-8 and 40 degrees C, respectively. The chloride salt of metal ions Mg2+, K+, and Na+ can activate BLrGGT, whereas that of Pb2+ dramatically inhibited it. The substrate specificity study showed that L-gamma-glutamyl-p-nitroanilide (L-gamma-Glu-p-NA) is a preference for the enzyme. Steady-state kinetic study revealed that BLrGGT has a k (cat) of 105 s(-1) and a K (m) of 21 mu M when using L-gamma-Glu-p-NA as the substrate. With this overexpression and purification system, BLrGGT can now be obtained in quantities necessary for structural characterization and synthesis of commercially important gamma-glutamyl compounds

Identification of essential cysteine residues in 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Corynebacterium glutamicum

To ascertain the functional role of cysteine residue in 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase from Corynebacterium glutamicum, site-directed mutagenesis was performed to change each of the three residues to serine. Plasmids were constructed for high-level overproduction and one-step purification of histidine-tagged DAHP synthase. Analysis of the purified wild-type and mutant enzymes by SDS-polyacrylamide gel electrophoresis showed an apparent protein band with a molecular mass of approximately 45 kDa. Cys(145)Ser mutant retained about 16% of the enzyme activity, while DAHP synthase activity was abolished in Cys(67)Ser mutant. Kinetic analysis of Cys(145)Ser mutant with PEP as a substrate revealed a marked increase in K-m with significant change in k(cat), resulting in a 13.6-fold decrease in k(cat)/K-m(PEP). Cys(334) was found to be nonessential for catalytic activity, although it is highly conserved in DAHP synthases. From these studies, Cys(67) appears important for synthase activity, while Cys(145) plays a crucial role in the catalytic efficiency through affecting the mode of substrate binding

Serine 187 is a crucial residue for allosteric regulation of Corynebacterium glutamicum 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase

Corynebacterium glutamicum 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase is sensitive to feedback inhibition by tyrosine. One feedback-insensitive mutant was obtained by in vitro chemical mutagenesis and the mutation was identified as a C-->G mutation at nucleotide 560 causing a Ser(187) to Cys(187) substitution. Replacing Ser(187) with cysteine, tyrosine or phenylalanine by site-directed mutagenesis not only reduced the enzymatic activity but also relieved its feedback inhibition by tyrosine, while Ser(187)Ala exhibited a comparable activity to that of wild-type enzyme and sensitized to allosteric regulation. The His(6)-tagged enzymes were expressed in Escherichia coli and purified to homogeneity by immobilized nickel-ion affinity chromatography. Kinetic analysis showed that tyrosine is a competitive inhibitor of phosphoenol pyruvate, one of the precursors for DAHP biosynthesis. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved

The role of a conserved histidine residue, His324, in Trigonopsis variabilis D-amino acid oxidase

To investigate the functional role of an invariant histidine residue in Trigonopsis variabilis D-amino acid oxidase (DAAO), a set of mutant enzymes with replacement of the histidine residue at position 324 was constructed and their enzymatic properties were examined, Wild-type and mutant enzymes have been purified to homogeneity using the His-bound column and the molecular masses were determined to be 39.2 kDa. Western blot analysis revealed that the in vivo synthesized mutant enzymes are immune-identical with that of the wild-type DAAO. The His324Asn and His324Gln mutants displayed comparable enzymatic activity to that of the wild-type enzyme, while the other mutant DAAOs showed markedly decreased or no detectable activity. The mutants, His324/Asn/Gln/Ala/Tyr/Glu, exhibited 38-181% increase in K-m and a 2-10-fold reduction in k(cat)/K-m. Based on the crystal structure of a homologous protein, pig kidney DAAO, it is suggested that His324 might play a structural role for proper catalytic function of T. variabilis DAAO. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved