Biomedical subjects

H Kallwass

Publications and source records attributed to H Kallwass.

3 recordsLinked to original sources

PubMed1992

THR246 mutations decrease substrate inhibition in lactate dehydrogenase.

Threonine 246 in Bacillus stearothermophilus L-lactate dehydrogenase has been changed to valine, serine, and alanine by site-directed mutagenesis. Kinetic analyses show a decrease in substrate inhibition for pyruvate reduction with the T246S mutant and virtual elimination of substrate inhibition for the T246A and T246V mutants. The results indicate that the absence of substrate inhibition in the 246A/V-catalyzed reactions is due to the elimination of a key hydrogen bond between the hydroxyl group of threonine and pyruvate in the wild-type complex that is an important contributor in the formation of the abortive enzyme-NAD(+)-pyruvate complex responsible for substrate inhibition.

Escherichia coli↗

PubMed1992

Crystallization and preliminary X-ray diffraction studies of two mutants of lactate dehydrogenase from Bacillus stearothermophilus.

Bacillus stearothermophilus lactate dehydrogenase, one of the most thermostable bacterial enzymes known, has had its three-dimensional structure solved, the gene coding for it has been cloned, and the protein can be readily overexpressed. Two mutants of the enzyme have been prepared. In one, Arg171 was changed to Trp (R171W) and Gln102 was changed to Arg (Q102R). In the other, the mutation Q102R was maintained, but Arg171 was changed to Tyr (R171Y). In addition, an inadvertent C97G mutant was present. Both mutants have been crystallized by the hanging drop vapor diffusion method at room temperature. Bipyrimidal crystals have been obtained against (NH4)2SO4 in 50 mM piperazine HCl buffer. The crystals belong to space group P6(2)22 (P6(4)22) (whereas the native enzyme, the structure of which has been solved by Piontek et al., Proteins 7:74-92, 1990) crystallized in the space group P6(1)) with a = 102.3 A, c = 168.6 A for the R171W, Q102R, C97G triple mutant, and a = 98.2 A; c = 162.1 A for the R171Y, Q102R, C97G mutant. These crystal forms appear to contain one-quarter of a tetramer (M(r) 135,000) in the asymmetric unit and have VM values of 3.8 and 3.3 A3/dalton, respectively). The R171W mutant diffracts to 2.5 A and the R171 Y mutant to approximately 3.5 A.

Bacterial Proteins↗

PubMed1989

Cloning, sequencing and expression in Escherichia coli of the D-2-hydroxyisocaproate dehydrogenase gene of Lactobacillus casei.

D-2-Hydroxyisocaproic acid dehydrogenase (D-HicDH) from Lactobacillus casei was purified and partially sequenced. A 65-mer oligodeoxyribonucleotide probe corresponding to the N-terminal 23 amino acids was synthesized and a physical map was made of the genomic region which hybridized most strongly. A strongly hybridising restriction fragment was highly purified and eventually cloned at low frequency in pBR322. The original clones spontaneously produced D-HicDH at about 0.05% of total protein and showed viability problems in that 10- to 12-h growth-lag periods occurred after diluting stationary cultures into fresh medium. Subcloning into pGEM3 plasmids for sequencing with concomitant ExoIII deletion led to clones which no longer exhibited the growth inhibition characteristics but now made D-HicDH as 3 to 5% of total protein. Subcloning downstream from a double PL PR promoter in plasmid pJLA601 gave a highly inducible clone that builds large inclusion bodies of largely denatured D-HicDH. The gene transcript was mapped for L. casei and Escherichia coli hosts. The promoter, terminator and Shine-Dalgarno sequence are functional in both organisms. The gene encodes a protein subunit of 38 kDa, whereby 67% of the sequence could be checked by correlation with partial peptide sequences from the original enzyme. So far no Lactobacillus gene has been found to utilize the Arg codons AGG and AGA.

Alcohol Oxidoreductases↗