Nucleotide sequence of theHansenula anomalagene encoding flavocytochrome b2(L-lactate:cytochromecoxidoreductase)

Risler, Yanick; Tegoni, Mariella; Gervais, Michel

doi:10.1093/nar/17.20.8381

Cited by 21 publications

(9 citation statements)

References 4 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Significant homology was found between LctD and several flavin mononucleotide (FMN)-dependent enzymes (Fig. 5), including the glycolate oxidase of spinach (41), the (S)-mandelate dehydrogenase of P. putida (40), the L-lactate 2-monooxygenase of Mycobacterium smegmatis (14), and flavocytochromes b2 (L-lactate dehydrogenases) of Saccharomyces cerevisiae (21) and Hansenula anomala (33). These proteins probably all contain an eight-stranded ce43 barrel with conserved sites for FMN-substrate binding and catalysis (24,25,42).…”

Section: Resultsmentioning

confidence: 99%

Three overlapping lct genes involved in L-lactate utilization by Escherichia coli

et al. 1993

View full text Add to dashboard Cite

In Escherichia coli, the let locus at min 80 on the chromosome map is associated with ability to grow on L-lactate and to synthesize a substrate-inducible flavin-linked dehydrogenase. Similar to that of the glpD-encoded aerobic glycerol-3-phosphate dehydrogenase, the level of induced enzyme activity is elevated by aerobiosis. Both of these controls are mediated by the two-component signal transduction system ArcB/ArcA, although sensitivity to the control is much more striking for L-lactate dehydrogenase. This study disclosed that the kct locus contained three overlapping genes in the clockwise order of lktD (encoding a flavin mononucleotide-dependent dehydrogenase), IctR (encoding a putative regulator), and kctP (encoding a permease) on the chromosomal map. These genes, however, are transcribed in the counterclockwise direction. No homology in amino acid sequence was found between aerobic glycerol-3-phosphate dehydrogenase and L-lactate dehydrogenase. A F(kctD-lac) mutant was inducible by L-lactate but not D-lactate. Although the mutant lost the ability to grow on L-lactate, growth on D-lactate, known to depend on a different enzyme, remained normal.Growth of Escherichia coli on L-lactate results in the induction of a flavin-linked L-lactate dehydrogenase (9,13,29), and this trait is associated with the Ict locus at min 80.8 on the chromosome map (3,19,31 Bacterial strains, phages, and plasmids. All strains used were E. coli K-12 derivatives. The genotypes and sources of the bacterial strains, phages, and plasmids are given in Table 1.Growth conditions. Overnight cultures were grown in LB medium (28). Minimal agar media (38) were supplemented with thiamine. When used, the following compounds were added to the media at the following concentrations unless otherwise specified: D-or L-lactate, 20 mM; D-xylose, 10 mM; CAA, 1%; thiamine, 2 ug/ml; kanamycin (kan), 40 ,ug/ml; and ampicillin, 100 ,ug/ml. Mannitol (1%)-MacConkey agar plates were used for screening the inheritance of the mtl allele.Cultures for enzyme and permease assays were grown in 300-ml flasks containing 10 ml of MOPS (0.1 M) mineral medium at pH 7.6 with appropriate supplements (15). Ample aeration was ensured by vigorous agitation, and cells were harvested when growth reached the mid-exponential phase.Enzyme and permease assays. For L-and D-lactate dehydrogenase assays, cells were harvested by centrifugation at 5,000 x g for 15 min and washed once in cold 10 mM potassium phosphate buffer (pH 7.0). The pellet was weighed and suspended in 4 volumes of the same buffer. The suspended cells were lysed for 1 min/ml in a model 60 W ultrasonic disintegrator (MSE) at 1.5 A while being chilled in a dry ice-ethanol bath. Lysates were cleared by centrifugation for 30 min at 10,000 x g. Enzyme assays were performed in a manner similar to that for glycerol-3-phosphate dehydrogenase by measuring the reduction of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl-tetrazolium bromide mediated by phenazine methosulfate (20)

show abstract

Section: Resultsmentioning

confidence: 99%

Three overlapping lct genes involved in L-lactate utilization by Escherichia coli

et al. 1993

View full text Add to dashboard Cite

show abstract

“…The recently determined amino acid sequence of rat kidney long-chain hydroxy acid oxidase (HAO) (L& and Lederer, 1991) established homology between this protein and other FMN-dependent a-hydroxy-acid-oxidizing enzymes, including glycolate oxidase from spinach (Volokita and Somerville, 1987;Cederlund et al, 1988), lactate oxidase from Mycobacterium smegmatis (Giegel et al, 1990), mandelate dehydrogenase from Pseudomonas putida (Tsou et al, 1990) and flavocytochrome b, or L-lactate dehydrogenase from yeasts Guiard, 1985;Black et al, 1989;Risler et al, 1989). The reaction catalyzed by all these enzymes occurs in two stages.…”

mentioning

confidence: 99%

Molecular cloning and nucleotide sequence of cDNA encoding rat kidney long‐chain l‐2‐hydroxy acid oxidase

Belmouden

Lederer

et al. 1993

European Journal of Biochemistry

View full text Add to dashboard Cite

Long-chain L-a-hydroxy acid oxidase from rat kidney is a member of the family of FMNdependent a-hydroxy-acid-oxidizing enzymes. With the knowledge of the recently determined amino acid sequence, the cDNA encoding the enzyme has now been cloned using the polymerase chain reaction. The 1648-bp cDNA contains an open reading frame coding for the 352 residues of the previously determined sequence, preceded by a methionine codon. In addition, several clones were found to present a nine-base insertion, predicting the existence of an isoform with a tripeptide VRK inserted between residues 188 and 189 of the mature protein. The presence of about 10% of this isoform in the oxidase purified from rat kidney was indeed identified by amino acid sequencing. A recombinant active enzyme was obtained as a protein fused to glutathione S-transferase using the bacterial expression plasmid pGEX-3X. Physico-chemical characterization indicated, for the fused enzyme, properties similar to those of the rat kidney protein. When the chimaera was submitted to factor Xa, proteolysis at the engineered cleavage point was poor. Separation of hydroxy acid oxidase from glutathione S-transferase could not be achieved with trypsin either. With both proteases, the initial cleavage point appeared to be in a peptide loop internal to the hydroxy acid oxidase sequence, close to or in the tripeptide insertion locus and not at the engineered factor-Xa-cleavage point. Comparative tryptic proteolysis of the rat kidney enzyme yielded a form cleaved in the same loop.

show abstract

“…I .3.15) (Lindqvist & Branden, 1985;Lindqvist, 1989). Amino acid sequence data (Lederer et aI., 1985;Cederlund et al, 1988;Black et al, 1989;Risler et al, 1989) show that these two proteins belong to a family of FMN-dependent a-hydroxy acid-oxidizing enzymes, which also includes long chain L-a-hydroxy acid oxidase (isozyme B) from higher organisms (EC 1.1.3.15) LO & Lederer, 1991), lactate oxidase from Mycobacterium smegmatis (L-lactate 2-monooxygenase E C 1.13.12.4) (Giegel et al, 1990), and mandelate dehydrogenase from Pseudornonasputida (Tsou et al, 1990). Reoxidation of the cofactor occurs for the oxidases at the expense of oxygen with formation of H202, whereas for flavocytochrome b2 it occurs by intramolecular single electron transfer to heme b~.…”

mentioning

confidence: 99%

Extreme pK_a displacements at the active sites of FMN‐dependent α‐hydroxy acid‐oxidizing enzymes

Lederer

1992

Protein Science

View full text Add to dashboard Cite

Flavocytochrome b2 (or L-lactate dehydrogenase) from baker's yeast is thought to operate by the initial formation of a carbanion, as do the evolutionarily related alpha-hydroxy acid-oxidizing FMN-dependent oxidases. Previous work has shown that, in the active site of the unligated reduced flavocytochrome b2, the group that has captured the substrate alpha-proton has a high pKapp, calculated to lie around 15 through the use of Eigen's equation. A detailed inspection of the now known three-dimensional structure of the enzyme leads to the conclusion that the high pKa belongs to His 373, an active site group that plays the role of general base in the forward reaction and of general acid in the reverse direction. Moreover, consideration of the kinetics of proton transfer during the catalytic cycle suggests that the pKa of the reduced FMN N5 position should be lowered by several pH units compared to its pKa of 20 or more when free. The features of the three-dimensional structure possibly responsible for these pK shifts are analyzed; they are proposed to consist of a network of hydrogen bonds with the solvent and of a mutual electrostatic stabilization of anionic reduced flavin and the imidazolium ion. Finally, it is suggested that similar pK shifts affect the active sites of the alpha-hydroxy acid-oxidizing flavooxidases, which are homologous to flavocytochrome b2. The functional significance of these pK shifts in terms of catalysis and semiquinone stabilization is discussed.

show abstract

Nucleotide sequence of theHansenula anomalagene encoding flavocytochrome b₂(L-lactate:cytochromecoxidoreductase)

Cited by 21 publications

References 4 publications

Three overlapping lct genes involved in L-lactate utilization by Escherichia coli

Three overlapping lct genes involved in L-lactate utilization by Escherichia coli

Molecular cloning and nucleotide sequence of cDNA encoding rat kidney long‐chain l‐2‐hydroxy acid oxidase

Extreme pK_a displacements at the active sites of FMN‐dependent α‐hydroxy acid‐oxidizing enzymes

Contact Info

Product

Resources

About

Nucleotide sequence of theHansenula anomalagene encoding flavocytochrome b2(L-lactate:cytochromecoxidoreductase)

Cited by 21 publications

References 4 publications

Three overlapping lct genes involved in L-lactate utilization by Escherichia coli

Three overlapping lct genes involved in L-lactate utilization by Escherichia coli

Molecular cloning and nucleotide sequence of cDNA encoding rat kidney long‐chain l‐2‐hydroxy acid oxidase

Extreme pKa displacements at the active sites of FMN‐dependent α‐hydroxy acid‐oxidizing enzymes

Contact Info

Product

Resources

About

Nucleotide sequence of theHansenula anomalagene encoding flavocytochrome b₂(L-lactate:cytochromecoxidoreductase)

Extreme pK_a displacements at the active sites of FMN‐dependent α‐hydroxy acid‐oxidizing enzymes