Extracellular Ribonuclease fromBacillus licheniformis(Balifase), a New Member of the N1/T1 RNase Superfamily

Abstract

The N1/T1 RNase superfamily comprises enzymes with well-established antitumor effects, such as ribotoxins secreted by fungi, primarily by Aspergillus and Penicillium species, and bacterial RNase secreted by B. pumilus (binase) and B. amyloliquefaciens (barnase). RNase is regarded as an alternative to classical chemotherapeutic agents due to its selective cytotoxicity towards tumor cells. New RNase with a high degree of structural similarity with binase (73%) and barnase (74%) was isolated and purified from Bacillus licheniformis (balifase, calculated molecular weight 12421.9 Da, pI 8.91). The protein sample with enzymatic activity of 1.5 × 106 units/A280 was obtained. The physicochemical properties of balifase are similar to those of barnase. However, in terms of its gene organization and promoter activity, balifase is closer to binase. The unique feature of balifase gene organization consists in the fact that genes of RNase and its inhibitor are located in one operon. Similarly to biosynthesis of binase, balifase synthesis is induced under phosphate starvation; however, in contrast to binase, balifase does not form dimers under natural conditions. We propose that the highest stability of balifase among analyzed RNase types allows the protein to retain its structure without oligomerization.

The Native Monomer of Bacillus Pumilus Ribonuclease Does Not Exist Extracellularly

“…It consists of 109 amino acid residues with the molecular weight of 12.3 kDa. Binase was isolated from B. pumilus culture fluid and purified according to the procedure previously described for bacillary RNases [ 19 , 20 ]. Catalytic activity of binase was 1.4×10 7 U/mg when measured against high molecular weight yeast RNA at pH 8.5 and 37°C [ 4 ].…”

Supramolecular organization as a factor of ribonuclease cytotoxicity

“…We have improved a method for the isolation of bacillary RNases which enables preparation of a homogeneous protein in three stages. This method was used to isolate, chromatographically purify, and characterize guanyl-preferring RNases from B. pumilus 7P (binase), B. altitudinis B-388 (balnase), and B. licheniformis (balifase) [ 30 , 40 , 41 ]. Among the presented species, the most active RNase producer is B. pumilus secreting binase.…”

“…The B. licheniformis RNase balifase has a primary structure similar to that of binase (73%) and barnase (74%). Balifase synthesis is induced under phosphate starvation conditions, which brings the enzyme closer to binase and balnase, but the physico-chemical properties of balifase are closer to those of barnase [ 41 ].…”

“…Investigation of the structural organization of the RNases isolated by us – binase, balnase, and balifase – has revealed that all of them dimerize in vivo and are natural dimers [ 41 , 44 , 45 ]. Probably, the formation of RNase dimers is one of the key processes necessary for the enzymes to perform their functions and manifest their biological properties.…”

“…Some binase dimers are highly stable, apparently due to the exchange of N- or C-terminal regions, and do not dissociate under denaturing conditions; others are incapable of exchanging domains between monomers (swapping interactions), which leads to the dissociation of these dimers into monomers during electrophoresis under denaturing conditions [ 44 ]. Balnase and balifase constitute only the second type of dimers [ 22 , 41 , 45 ].…”