Identification and characterization of two adenosine phosphorylase activities in Mycobacterium smegmatis

Abstract

Purine nucleoside phosphorylase (PNP) is an important enzyme in purine metabolism and cleaves purine nucleosides to their respective bases. Mycobacterial PNP is specific for 6-oxopurines and cannot account for the adenosine (Ado) cleavage activity that has been detected in M. tuberculosis and M. smegmatis cultures. In the current work, two Ado cleavage activities were identified from M. smegmatis cell extracts. The first activity was biochemically determined to be a phosphorylase that could reversibly catalyze adenosine + phosphate ↔ adenine + alpha-D-ribose-1-phosphate. Our purification scheme led to a 30-fold purification of this activity, with the removal of more than 99.9% of total protein. While Ado was the preferred substrate, inosine and guanosine were also cleaved, with 43% and 32% of the Ado activity, respectively. Our data suggest that M. smegmatis expresses two PNPs: a previously described trimeric PNP that can cleave inosine and guanosine only and a second, novel PNP (Ado-PNP) that can cleave Ado, inosine, and guanosine. Ado-PNP had an apparent K(m) (K(m) ( app)) of 98 ± 6 μM (with Ado) and a native molecular mass of 125 ± 7 kDa. The second Ado cleavage activity was identified as 5'-methylthioadenosine phosphorylase (MTAP) based on its biochemical properties and mass spectrometry analysis. Our study marks the first report of the existence of MTAP in any bacterium. Since human cells do not readily convert Ado to Ade, an understanding of the substrate preferences of these enzymes could lead to the identification of Ado analogs that could be selectively activated to toxic products in mycobacteria.

Fig. 1.

Enzymes involved in adenosine metabolism. (1) 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (not present in human cells) or 5′-methylthioadenosine phosphorylase; (2) adenine phosphoribosyl transferase; (3) adenosine kinase; (4) adenosine phosphorylases (not present in human cells) and most bacterial purine nucleoside phosphorylases; (5) adenosine deaminase; (6) purine nucleoside phosphorylase; (7) hypoxanthine/guanine phosphoribosyl transferase.

Fig. 2.

Elution of Ado, MTA, and Ino cleavage activities from M. smegmatis cell extract applied to the HiTrap Q anion-exchange column. M. smegmatis cell extract was applied to the HiTrap Q anion-exchange column and eluted with 150 to 750 mM NaCl gradient. Every other fraction was tested for Ade formation from Ado or MTA and Hx formation from Ino by the xanthine oxidase assay. The formation of Ade and Hx is expressed as the percent change in initial absorbance at 305 nm (for Ado and MTA) and 293 nm (for Ino) after a 2-hour incubation with Ado, MTA, and Ino.

Fig. 3.

Ado cleavage requires phosphate and is reversible. (A) Fraction 15 was incubated with Ado in the presence or absence of 50 mM phosphate, and Ade formation was monitored by HPLC. (B) The enzyme was incubated with Ado and 50 mM phosphate, and the formation of Ade was monitored by HPLC. The enzyme was also incubated with Ade and ribose-1-phosphate, and the formation of Ado was monitored by HPLC. Each data point represents the mean ± standard deviation for 3 determinations. Some error bars are too small to be seen or are hidden by the symbol.