Overproduction and nucleotide sequence of the respiratory D-lactate dehydrogenase of Escherichia coli

Abstract

Recombinant DNA plasmids containing the gene for the membrane-bound D-lactate dehydrogenase (D-LDH) of Escherichia coli linked to the promoter PL from lambda were constructed. After induction, the levels of D-LDH were elevated 3Q0-fold over that of the wild type and amounted to 35 % of the total cellular protein. The nucleotide sequence of the D-LDH gene was determined and shown to agree with the amino acid composition and the amino-terminal sequence of the purified enzyme. Removal of the amino-terminal formyl-Met from D-LDH was not inhibited in cells which contained these high levels of D-LDH.

Membrane-bound lactate dehydrogenases and mandelate dehydrogenases of Acinetobacter calcoaceticus. Purification and properties

“…Similar results were obtained with authentic FAD, corresponding to stoichiometric conversion of FAD into FMN by the phosphodiesterase in the venom; no change in fluorescence was observed when venom was added to a solution of FMN. Preliminary quantitative analysis of the flavin content suggests that there is between 0.5 and 1 mol of FAD/mol of enzyme, depending on whether protein concentration was measured by the method of Bradford (1976) or Lowry et al (1951) respectively. [Rule et al (1985) noted that estimates of the amounts of D-lactate dehydrogenase from E. coli varied by a factor of 2-fold depending on the method of protein estimation.] A tryptic digest of the trichloroacetic acid precipitate contained no detectable flavin, suggesting that the FAD in these enzymes is all non-covalently bound.…”

Neisseria gonorrhoeae possesses two nicotinamide adenine dinucleotide-independent lactate dehydrogenases

“…Analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that six or more major bands plus several minor bands of protein were present in both iLDH-I and iLDH-II peak fractions (data not shown). Although protein bands similar in size to M r 64520 calculated from the gene sequence of the E. coli D( --)-iLDH [15] (the best characterized iLDH to date) were present, we were unable to deduce proteins corresponding to either iLDH-I or iLDH-II. Comparison with the crude iLDH extracts also failed to provide insight, probably because the concentrations of iLDH enzymes in cells are generally low [14].…”

A 19F‐NMR study of the membrane‐binding region of D‐lactate dehydrogenase of escherichia coli

“…D-Lactate dehydrogenase can be labeled with 0-, m-, or p-fluorophenylalanine (F-Phe), or rn-fluorotyrosine (F-Tyr); the resulting enzyme is active (Table 1 ; Rule et al, 1987a). There are 26 native Phe and 23 native Tyr residues in the wild-type enzyme (Rule et al, 1985). The I9F-NMR spectra of the 0-, m-, and p-F-Phe-labeled D-LDH are complex ( Fig.…”

“…As with many membrane-associated proteins, it has proven difficult to produce satisfactory crystals of D-LDH for X-ray crystallography, and the size of D-LDH pre-cludes the determination of its structure by two-or threedimensional NMR spectroscopy. We have been using a combination of site-specific mutagenesis and I9F-NMR spectroscopy to investigate the structure, dynamics, and interactions of D-LDH labeled with fluorinated amino acids (Rule et al, 1985(Rule et al, , 1987aHo et al, 1989;Peersen et al, 1990;Truong et al, 1991a,b). The wild-type native enzyme contains five tryptophan residues (Rule et al, 1985;Ho et al, 1988), and the 19F-NMR spectrum of 5-fluorotryptophan (SF-Trp)-labeled wild-type D-LDH shows five peaks (Rule et al, 1987a,b).…”

“…We have been using a combination of site-specific mutagenesis and I9F-NMR spectroscopy to investigate the structure, dynamics, and interactions of D-LDH labeled with fluorinated amino acids (Rule et al, 1985(Rule et al, , 1987aHo et al, 1989;Peersen et al, 1990;Truong et al, 1991a,b). The wild-type native enzyme contains five tryptophan residues (Rule et al, 1985;Ho et al, 1988), and the 19F-NMR spectrum of 5-fluorotryptophan (SF-Trp)-labeled wild-type D-LDH shows five peaks (Rule et al, 1987a,b). Tryptophan has been substituted for phenylalanine, tyrosine, isoleucine, and leucine at various positions throughout the enzyme, and the fluorinated native and substituted tryptophan residues of the active mutants have been used as probes of the local environment.…”