Identification of the open reading frame for the Pseudomonas putida D-hydantoinase gene and expression of the gene in Escherichia coli.

A DNA fragment containing the gene for D-hydantoinase was cloned from Pseudomonas putida CCRC 12857 into Escherichia coli. The cloned gene contained an open reading frame (ORF) of 1485 nucleotides encoding a protein of 53.4 kDa in which the carboxyl terminal end is longer than that previously deduced from strain DSM 84. This ORF was verified by amino acid sequencing of amino and carboxyl termini, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino acid sequence comparison. Deletion analysis revealed that 32 amino acids from the carboxyl terminal end were essential for D-hydantoinase activity. Tagging of six consecutive histidyl residues to the amino terminus or to the carboxyl terminus of the enzyme did not significantly affect D-hydantoinase activity. Under the control of T5lac promoter and lactose induction, the D-hydantoinase activity of transformed E. coli reached 200 U l-1 which is about 20-fold higher than that of gene donor strain.

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.

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 immuno-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 Km and a 2-10-fold reduction in kcat/Km. 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.

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 more 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.

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. Cys145Ser mutant retained about 16% of the enzyme activity, while DAHP synthase activity was abolished in Cys67Ser mutant. Kinetic analysis of Cys145Ser mutant with PEP as a substrate revealed a marked increase in Km with significant change in kcat, resulting in a 13.6-fold decrease in kcat/KmPEP. Cys334 was found to be nonessential for catalytic activity, although it is highly conserved in DAHP synthases. From these studies, Cys67 appears important for synthase activity, while Cys145 plays a crucial role in the catalytic efficiency through affecting the mode of substrate binding.

Efficient utilization of starch by a recombinant strain of Saccharomyces cerevisiae producing glucoamylase and isoamylase.

Two plasmids, designated pRTI and pTI, were constructed to allow the integration of a bacterial isoamylase gene (iso) into Saccharomyces cerevisiae G23-8 chromosome. The integrative plasmid pRTI comprises the iso gene from Pseudomonas amyloderamosa, a portion of S. cerevisiae ribosomal DNA (rDNA), S. cerevisiae trp1 gene deficient in promoter and the bacterial vector pSP72. The structure of plasmid pTI is similar to that of pRTI, except that it lacks an rDNA segment. The Aspergillus awamori glucoamylase and P. amyloderamosa isoamylase genes were expressed in the recombinant strain of S. cerevisiae under the control of the yeast alcohol dehydrogenase gene (adh1) promoter. Southern-blot analysis showed that these plasmids were integrated into the yeast chromosome in tandem repeat and dispersion copies. The recombinant strains could assimilate starch more efficiently than the recipient strain with a conversion rate of greater than 95%.