Verdoheme formation in Proteus mirabilis catalase

Background

Heme oxidative degradation has been extensively investigated in peroxidases but not in catalases. The verdoheme formation, a product of heme oxidation which inactivates the enzyme, was studied in Proteus mirabilis catalase.

Methods

The verdoheme was generated by adding peracetic acid and analyzed by mass spectrometry and spectrophotometry.

Results

Kinetics follow-up of different catalase reactional intermediates shows that i) the formation of compound I always precedes that of verdoheme, ii) compound III is never observed, iii) the rate of compound II decomposition is not compatible with that of verdoheme formation, and iv) dithiothreitol prevents the verdoheme formation but not that of compound II, whereas NADPH prevents both of them. The formation of verdoheme is strongly inhibited by EDTA but not increased by Fe³⁺ or Cu²⁺ salts. The generation of verdoheme is facilitated by the presence of protein radicals as observed in the F194Y mutated catalase. The inability of the inactive variant (H54F) to form verdoheme, indicates that the heme oxidation is fully associated to the enzyme catalysis.

Conclusion

These data, taken together, strongly suggest that the verdoheme formation pathway originates from compound I rather than from compound II.

General significance

The autocatalytic verdoheme formation is likely to occur in vivo.     

Unfolding of in planta activity of anti-rep ribozyme in presence of a RNA silencing suppressor

Antisense RNA ribozymes have intrinsic endonucleolytic activity to effect cleavage of the target RNA. However, this activity in vivo is often controlled by the dominance of antisense or other double-stranded RNA mechanism. In this work, we demonstrate the in planta activity of a hammerhead ribozyme designed to target rep-mRNA of a phytopathogen Mungbean Yellow Mosaic India virus (MYMIV) as an antiviral agent. We also found RNA-silencing is induced on introduction of catalytically active as well as inactive ribozymes. Using RNA-silencing suppressors (RSS), we demonstrate that the endonucleolytic activity of ribozymes is a true phenomenon, even while a mutated version may demonstrate a similar down-regulation of the target RNA. This helps to ease the confusion over the action mechanism of ribozymes in vivo.     

Complementation and characterization of the nested Rz and Rz1 reading frames in the genome of bacteriophage lambda

Transposon insertions in the Rz gene of bacteriophage λ block lysis if the medium contains divalent cations at concentrations greater than 5 mM, but otherwise cause no change in phenotype. The Rz protein is thought to have an endopeptidase activity, previously reported in λ lysates, which might be involved in cleavage of oligopeptide crosslinks between glycosidic strands in the peptidoglycan and the Lpp lipoproteins of the outer bacterial membrane. Recently, a small lipoprotein has been reported as the product of a short reading frame, designated Rz1, in the +1 register within Rz. This protein has been detected in membranes of induced λ lysogens. To determine whether Rz1 has a function in the λ vegetative cycle, amber nonsense alleles of Rz and Rz1 have been constructed by site-directed mutagenesis and used for complementation and suppression analysis. Both Rzam and Rz1am alleles have phenotypes identical to those of the original Rz insertion alleles, and complement and are fully suppressed in a supE host, indicating that the two genes are independent, trans-acting genes encoding proteins required for lysis in the presence of cations. Moreover, supF suppresses Rzam but not the Rz1am mutation, and the defective Rz1am product in the supF host shows a partially dominant character and significantly retards lysis even in the absence of additional cations in the medium. Rz and Rz1 represent a unique example of two genes located in different reading frames in the same nucleotide sequence, which encode different proteins that are both required in the same physiological pathway. Received: 21 May 1999 / Accepted: 24 August 1999