Michel Gervais

Nitrite reductase from Pseudomonas aeruginosa: Sequence of the gene and the protein

The gene coding for nitrite reductase of Pseudomonas aeruginosa has been cloned and its sequence determined. The coding region is 1707 bp long and contains information for a polypeptide chain of 568 amino acids. The sequence of the mature protein has been confirmed independently by extensive amino acid sequencing. The amino‐terminus of the mature protein is located at Lys‐26; the preceding 25 residue long extension shows the features typical of signal peptides. Therefore the enzyme is probably secreted into the periplasmic space. The mature protein is made of 543 amino acid residues and has a molecular mass of 60204 Da. The c‐heme‐binding domain, which contains the only two Cys of the molecule, is located at the amino‐terminal region. Analysis of the protein sequence in terms of hydrophobicity profile gives results consistent with the fact that the enzyme is fully water soluble and not membrane bound; the most hydrophilic region appears to correspond to the c‐heme domain. Secondary structure predictions are in general agreement with previous analysis of circular dichroic data.

Dissection of flavocytochrome b2 — a bifunctional enzyme — into a cytochrome core and a flavoprotein molecule

Summary After a cleavage of the two trypsin-sensitive zones characterized along the folded polypeptide chain of the Hansenula anomala flavocytochrome b2 (yielding three fragments N, e and β), the tetramer molecule (MW 4 × 60 000) divides into two kinds of stable molecules showing no affinity for each other. One is the cytochrome b2 core (MW ca 14 000) which is very similar to its bakers yeast homologue and which corresponds to fragment N. The other molecule is a T-flavoprotein which up to recently escaped detection and which is characterized in the present paper. It is a tetramer of MW ca 160 000, each protomer being made of one e (MW 18 300) and one β (MW 21 600) fragment and of the flavin binding sites.

Oxygen-Dependent Transcriptional Regulator Hap1p Limits Glucose Uptake by Repressing the Expression of the Major Glucose Transporter Gene RAG1 in Kluyveromyces lactis

ABSTRACT The HAP1 (CYP1) gene product of Saccharomyces cerevisiae is known to regulate the transcription of many genes in response to oxygen availability. This response varies according to yeast species, probably reflecting the specific nature of their oxidative metabolism. It is suspected that a difference in the interaction of Hap1p with its target genes may explain some of the species-related variation in oxygen responses. As opposed to the fermentative S. cerevisiae, Kluyveromyces lactis is an aerobic yeast species which shows different oxygen responses. We examined the role of the HAP1-equivalent gene (KlHAP1) in K. lactis. KlHap1p showed a number of sequence features and some gene targets (such as KlCYC1) in common with its S. cerevisiae counterpart, and KlHAP1 was capable of complementing the hap1 mutation. However, the KlHAP1 disruptant showed temperature-sensitive growth on glucose, especially at low glucose concentrations. At normal temperature, 28°C, the mutant grew well, the colony size being even greater than that of the wild type. The most striking observation was that KlHap1p repressed the expression of the major glucose transporter gene RAG1 and reduced the glucose uptake rate. This suggested an involvement of KlHap1p in the regulation of glycolytic flux through the glucose transport system. The ΔKlhap1 mutant showed an increased ability to produce ethanol during aerobic growth, indicating a possible transformation of its physiological property to Crabtree positivity or partial Crabtree positivity. Dual roles of KlHap1p in activating respiration and repressing fermentation may be seen as a basis of the Crabtree-negative physiology of K. lactis.

How the loss of several residues, at the level of one interglobule junction, modulates the lactate dehydrogenase activity of yeast flavocytochrome b2: A study of the nicked enzymes resulting from clostripain and trypsin action

The native chain of flavocytochrome b2 is folded into three globules linked together by two protease-sensitive bridges a and cd. We show in this paper that zone a of H-flavocytochrome b2 is the first to be cleaved under clostripain action. The alpha c and beta c fragments thus formed are homologous to alpha T and betaT trypsic fragments. The remaining activities of the resulting (alpha c beta c) and alpha T betaT) forms are only 25 per cent and 4 per cent of the native flavocytochrome b2 one. The study of the catalytic properties of (alpha c betaT) and (alpha T beta c) species resulting from the crossed reassociation of the isolated fragments show that the beta type fragment plays a critical role in the catalytic process. A dramatic activity decrease may be correlated with the loss of 6 amino acid residues at the N-terminal of beta c. Our best hypothesis is that these amino acids are involved in the active site, which may be located in the contact zone between alpha and beta. These results are in agreement with previous results obtained in this laboratory which showed the necessity of both alpha T and betaT fragments for the correct conformation of the flavin binding site.