Creation of salt-insensitive 3'(2'),5'-bisphosphate nucleotidase by modeling and mutagenesis approach.

3'(2'),5'-Bisphosphate nucleotidase, (EC 3.1.3.7) (BPntase) is a ubiquitous enzyme. Recently, these enzymes have drawn considerable attention as in vivo targets of salt toxicity as well as therapeutic targets of lithium that is used for the treatment of manic-depressive disorders. They belong to the Mg2+-dependent Li+-sensitive phosphomonoesterase super-family and are highly sensitive to lithium and sodium ions. However, the molecular mechanism of inhibition of this group of enzymes by monovalent cations has not been completely understood. Previously we have identified a BPntase (Dhal2p) from a highly halotolerant yeast Debaryomyces hansenii. Molecular characterization revealed a number of unique features in Dhal2p, indicating this is an extraordinary member of the family. In this study, we have carried out the structure-function analysis of Dhal2p through the combination of molecular modeling and in vitro mutagenesis approach. We have not only provided the explanation for the role played by the functionally important elements that are conserved among the members of this family but also identified important, novel structural elements in this enzyme. Our study for the first time unraveled the role of a flap as well as a loop region in the functioning of this enzyme. Most importantly, mutations in the loop region resulted in the creation of a BPntase that was insensitive to salt.

Role of N-terminal hydrophobic region in modulating the subcellular localization and enzyme activity of the bisphosphate nucleotidase from Debaryomyces hansenii.

3', 5'-Bisphosphate nucleotidase is a ubiquitous enzyme that converts 3'-phosphoadenosine-5'-phosphate to adenosine-5'-phosphate and inorganic phosphate. These enzymes are highly sensitive to sodium and lithium and, thus, perform a crucial rate-limiting metabolic step during salt stress in yeast. Recently, we have identified a bisphosphate nucleotidase gene (DHAL2) from the halotolerant yeast Debaryomyces hansenii. One of the unique features of Dhal2p is that it contains an N-terminal 54-amino-acid-residue hydrophobic extension. In this study, we have shown that Dhal2p exists as a cytosolic as well as a membrane-bound form and that salt stress markedly influences the accumulation of the latter form in the cell. We have demonstrated that the N-terminal hydrophobic region was necessary for the synthesis of the membrane-bound isoform. It appeared that an alternative translation initiation was the major mechanism for the synthesis of these two forms. Moreover, the two forms exhibit significant differences in their substrate specificity. Unlike the cytosolic form, the membrane-bound form showed very high activity against inositol-1,4-bisphosphate. Thus, the present study for the first time reports the existence of multiple forms of a bisphosphate nucleotidase in any organism.

Molecular cloning and biochemical characterization of a 3'(2'),5'-bisphosphate nucleotidase from Debaryomyces hansenii.

The enzyme 3'(2'),5'-bisphosphate nucleotidase catalyses a reaction that converts 3'-phosphoadenosine-5'-phosphate (PAP) to adenosine-5'-phosphate (AMP) and inorganic phosphate (Pi). The enzyme from Saccharomyces cerevisiae is highly sensitive to sodium and lithium and is thus considered to be the in vivo target of salt toxicity in yeast. In S. cerevisiae, the HAL2 gene encodes this enzyme. We have cloned a homologous gene, DHAL2, from the halotolerant yeast Debaryomyces hansenii. DNA sequencing of this clone revealed a 1260 bp open reading frame (ORF) that putatively encoded a protein of 420 amino acid residues. S. cerevisiae transformed with DHAL2 gene displayed higher halotolerance. Biochemical studies showed that recombinant Dhal2p could efficiently utilize PAP (K(m)17 microM) and PAPS (K(m)48 microM) as substrate. Moreover, we present evidence that, in comparison to other homologues from yeast, Dhal2p displays significantly higher resistance towards lithium and sodium ions.

Direct evidence for the oxidation of palmitic acid by host-grown Mycobacterium leprae.

Oxidation of palmitic acid by whole-cell suspensions of Mycobacterium leprae free from host tissues was investigated using manometric techniques. After a lag period of about 6-8 h, M. leprae suspensions catalysed an active oxidation of palmitic acid, and the oxidative process (oxygen uptake) was quite sensitive to rotenone, atabrine, amytal, antimycin A and cyanide. The spectrophotometric observations indicated that the M. leprae cytochrome system, under anaerobic conditions, was reduced in the presence of palmitic acid which was completely oxidized by oxygen. These data provide direct evidence that M. leprae cells are capable of oxidizing palmitic acid, and that oxidation is mediated by the electron transport system using oxygen as the terminal electron acceptor.

Respiration in Mycobacterium leprae.

Fairly pure leprosy bacilli were easily collected from nude mouse foot pad lepromas by the Ficoll density gradient centrifugation and alkali treatment methods. The yield of bacilli available for biochemical study was 42.6%. The density of Mycobacterium leprae was very heterogeneous. The percent of solid bacilli in the light bacilli fraction was 23%; that in the heavy bacilli fraction was 40%. The endogenous respiration activity in the heavy bacilli was greater than that in light bacilli. The average coefficient of respiration in M. leprae was 1 microliter O2/mg X hr. In the whole cells of M. leprae, a cytochrome b1 absorption peak and its Soret peak were detected at wavelengths of 560 nm and 426 nm, respectively. However, a cytochrome a2-like peak (which was observed in M. lepraemurium), and a cyt c and cyt a were not detected. Catalase activity was not found in whole cells, the cell-free extract, or particle fractions of M. leprae. Any catalase activity associated with M. leprae suspensions is a tissue contaminant. NAD-peroxidase activity was also not detected in the cell-free extract of the leprosy bacillus. These results would indicate that leprosy bacilli cannot degrade hydrogen peroxide.