(1/21) Construction and physiological studies on a stable bioengineered strain of shengjimycin.
Shengjimycin is a group of 4"-acylated spiramycins with 4"-isovalerylspiramycin as the major component, produced by recombinant S. spiramyceticus F21 harboring a 4"-O-acyltransferase gene from S. mycarofaciens 1748. A stable bioengineered strain of Streptomyces spiramyceticus WSJ-1 was constructed by integrating the 4"-O-acyltransferase gene (ist) by homologous recombination into the chromosome of the spiramycin-producing strain S. spiramyceticus F21. In this construction, a Streptomyces/E. coli shuttle plasmid pKC1139 (AmR) was used as the vector with the tsr gene used as selection marker for homologous recombination. The constructed strain, S. spiramyceticus WSJ-1,was genetically stable in production titer and proportion of components of shengjimycin as well as in maintaining the tsr selective marker when grown without selection. Southern hybridization confirmed the integrated status of the ist gene in the host genome. The production and the proportion of major component of 4"-isovalerylspiramycin of S. spiramyceticus WSJ-1 was also improved comparing with the strain harboring an autonomous plasmid -S. spiramyceticus F21/pIJ680(311) as shown by HPLC analysis. Physiological studies indicated that increase of the VDH ( valine dehydrogenase ) and LDH ( leucine dehydrogenase ) activities of WSJ-1 may be involved in this improvement. (+info)
(2/21) Leucine dehydrogenase from Corynebacterium pseudodiphtheriticum: purification and characterization.
Leucine dehydrogenase [EC 220.127.116.11] was purified to homogeneity from Corynebacterium pseudodiphtheriticum ICR 2210. The enzyme consisted of a single polypeptide with a molecular weight of about 34,000. Stepwise Edman degradation provided the N-terminal sequence of the first 24 amino acids, and carboxypeptidase Y digestion provided the C-terminal sequence of the last 2 amino acids. Although the enzyme catalyzed the reversible deamination of various branched-chain L-amino acids, L-valine was the best substrate for oxidative deamination at pH 10.9 and the saturated concentration. The enzyme, however, had higher reactivity for L-leucine, and the kcat/Km value for L-leucine was higher than that for L-valine. The enzyme required NAD+ as a natural coenzyme. The NAD+ analogs 3-acetylpyridine-NAD+ and deamino-NAD+ were much better coenzymes than NAD+. The enzyme activity was significantly reduced by sulfhydryl reagents and pyridoxal 5'-phosphate. D-Enantiomers of the substrate amino acids competitively inhibited the oxidation of L-valine. (+info)
(3/21) Leucine dehydrogenase from Bacillus stearothermophilus: identification of active-site lysine by modification with pyridoxal phosphate.
We have constructed an efficient expression plasmid for the leucine dehydrogenase gene previously cloned from Bacillus stearothermophilus. The recombinant enzyme was overproduced in Escherichia coli cells to a level of more than 30% of the total soluble protein upon induction with isopropyl beta-D-thiogalactopyranoside. The enzyme could be readily purified to homogeneity by heat treatment and a single step of ion-exchange chromatography. The purified enzyme was inactivated in a time-dependent manner upon incubation with pyridoxal 5'-phosphate (PLP) followed by reduction with sodium borohydride. The inactivation was completely prevented in the copresence of L-leucine and NAD+. Concomitantly with the inactivation, several molecules of PLP were incorporated into each subunit of the hexameric enzyme. Sequence analysis of the fluorescent peptides isolated from a proteolytic digest of the modified protein revealed that Lys80, Lys91, Lys206, and Lys265 were labeled. Among these residues, Lys80 was predominantly labeled and, in the presence of L-leucine and NAD+, was specifically protected from the labeling. Furthermore, a linear relationship of about 1:1 was observed between the extent of inactivation and the amount of PLP incorporated into Lys80. A slightly active mutant enzyme, in which Lys80 is replaced by Ala, was not inactivated at all by incubation with PLP, showing that the inactivation is correlated with the labeling of only Lys80. Lys80is conserved in the corresponding regions of all the amino acid dehydrogenase sequences reported to date. These results suggest that Lys80 is located at the active site and plays an important role in the catalytic function of leucine dehydrogenase. (+info)
(4/21) Identification and analysis of the genes coding for the putative pyruvate dehydrogenase enzyme complex in Acholeplasma laidlawii.
A monospecific antibody recognizing two membrane proteins in Acholeplasma laidlawii identified a plasmid clone from a genomic library. The nucleotide sequence of the 4.6-kbp insert contained four sequential genes coding for proteins of 39 kDa (E1 alpha, N terminus not cloned), 36 kDa (E1 beta), 57 kDa (E2), and 36 kDa (E3; C terminus not cloned). The N termini of the cloned E2, E1 beta, and native A. laidlawii E2 proteins were verified by amino acid sequencing. Computer-aided searches showed that the translated DNA sequences were homologous to the four subenzymes of the pyruvate dehydrogenase complexes from gram-positive bacteria and humans. The plasmid-encoded 57-kDa (E2) protein was recognized by antibodies against the E2 subenzymes of the pyruvate and oxoglutarate dehydrogenase complexes from Bacillus subtilis. A substantial fraction of the E2 protein as well as part of the pyruvate dehydrogenase enzymatic activity was associated with the cytoplasmic membrane in A. laidlawii. In vivo complementation with three different Escherichia coli pyruvate dehydrogenase-defective mutants showed that the four plasmid-encoded proteins were able to restore pyruvate dehydrogenase enzyme activity in E. coli. Since A. laidlawii lacks oxoglutarate dehydrogenase and most likely branched-chain dehydrogenase enzyme complex activities, these results strongly suggest that the sequenced genes code for the pyruvate dehydrogenase complex. (+info)
(5/21) A combinatorial approach to detect coevolved amino acid networks in protein families of variable divergence.
(6/21) An enzymatic cyclopentyl[b]indole formation involved in scytonemin biosynthesis.
(7/21) Enzymatic in situ determination of stereospecificity of NAD-dependent dehydrogenases.
Amino acid racemases inherently catalyze the exchange of alpha-hydrogen of amino acids with deuterium during racemization in 2H2O. When the reactions catalyzed by alanine racemase (EC 18.104.22.168) and L-alanine dehydrogenase (EC 22.214.171.124), which is pro-R specific for the C-4 hydrogen transfer of NADH, are coupled in 2H2O, [4R-2H]NADH is exclusively produced. Similarly, [4S-2H]NADH is made in 2H2O with amino-acid racemase with low substrate specificity (EC 126.96.36.199) and L-leucine dehydrogenase (EC 188.8.131.52), which is pro-S specific. We have established a simple procedure for the in situ analysis of stereospecificity of C-4 hydrogen transfer of NADH by an NAD-dependent dehydrogenase by combination with either of the above two couples of enzymes in the same reaction mixture. When the C-4 hydrogen of NAD+ is fully retained after sufficient incubation, the stereospecificity of hydrogen transfer by a dehydrogenase is the same as that of alanine dehydrogenase or leucine dehydrogenase. However, when the C-4 hydrogen of NAD+ is exchanged with deuterium, the enzyme to be examined shows the different stereospecificity from alanine dehydrogenase or leucine dehydrogenase. Thus, we can readily determine the stereospecificity by 1H NMR measurement without isolation of the coenzymes and products. (+info)
(8/21) A specific kinetic assay for tripeptide aminopeptidase in serum.
This is a method for measuring tripeptide aminopeptidase (EC 184.108.40.206) activity in serum. L- Leucylglycylglycine is used as substrate, and the reaction is followed by monitoring the absorbance increase at 340 nm when NAD+ is reduced to NADH in the presence of an excess of leucine dehydrogenase. This principle allows kinetic determination of the enzyme without interference by carboxypeptidases. Amastatin is added to the reaction mixture to prevent nonspecific hydrolysis of the substrate catalyzed by other aminopeptidases. As final reaction concentrations we recommend (per liter): 100 mmol of Tris buffer (pH 8.2), 4.0 mmol of L- leucylglycylglycine , 10 kU of leucine dehydrogenase, 3.8 mmol of NAD+, and 85 mumol of amastatin . The assay is suited to modern enzyme analyzers and has high precision. (+info)