Characterization of 5'-nucleotidases secreted from Streptomyces

Abstract

To study the ability of Streptomyces to utilize environmental nucleotides, we screened for strains exhibiting extracellular 5'-inosine monophosphate (IMP)-dephosphorylating activity in our collection of soil isolates and obtained two producers: NE5-10 and Y2F8-2. The enzyme responsible for the activity was purified from the culture supernatant of each strain, and its mass spectral data were used to identify the coding sequence. The gene was successfully identified in the whole genome sequence of each strain; it was located in a conserved gene cluster of phosphate-related functions and encoded an approximately 600-amino acid long protein containing an N-terminal secretion signal. The mature part of the protein exhibited similarity to a known bacterial 5'-nucleotidase. The locus of the 5'-nucleotidase gene contained genes encoding proteins involved in phosphate utilization. The conserved gene arrangement of the locus in various Streptomyces genomes suggested the genetic region to be involved in phosphate-scavenging in this group of bacteria. Phylogenetic analysis demonstrated that the isolated Streptomyces enzymes represent an uncharacterized group of bacterial 5'-nucleotidases. Enzymatic characterization of the two Streptomyces enzymes demonstrated that both enzymes exhibited 5'-nucleotidase activity but differed in terms of optimal temperature and pH, dependence on divalent cations, and substrate specificity. The Km and Vmax values of the 5'-IMP-dephosphorylating activity were 0.239 mM and 9.47 U/mg, respectively, for NE5-10 and 0.221 mM and 38.17 U/mg, respectively, for Y2F8-2. Enzyme activity in the culture broth of the two Streptomyces producers occurred in a phosphate-limitation-dependent manner, supporting their involvement in the acquisition of phosphorus. KEY POINTS: • We purified and characterized nucleotidases from two Streptomyces. • Two nucleotidases were presumed to be involved in phosphate acquisition. • It showed diversity in phosphate acquisition among microorganisms.

Keywords: Adaptive response; Bacterial 5′-nucleotidase; Phosphorus; Secretion; Streptomyces.