Roles of Ala-149 in the catalytic activity of diadenosine tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv

Figures & data

Fig. 1. Multiple sequence alignment of the active site region of MtAPA and HIT superfamily proteins (S. cerevisiae Ap₄A phosphorylases 1 and 2; APA1_Yeast and APA2_Yeast, Schizosaccharomyces pombe HIT family Ap_nA hydrolase; APH1_Schpo, H. sapiens HIT family Ap_nA hydrolase; Fhit_Human, Arabidopsis thaliana galactose-1-phosphate uridylyltransferases; GalT_Arath, and Oryctolagus cuniculus AMP-lysine hydrolase; Hint1_Rabbit).

Notes: Sequence alignment was performed using the ClustalW 2.1 software.¹²⁾ The HIT motif is underlined. The numbers along the top refer to the positions of amino acids in the sequence of MtAPA. The Ala-149 in MtAPA and the corresponding positions in other HIT superfamily proteins are shown in the box. In addition to Ala-149, the amino acids corresponding to Asn-139, Gly-146, Ser-147, His-153, and His-155 in MtAPA are shown in black, since these amino acids were previously presented as important residues for enzymatic activity of MtAPA,²⁾ and other amino acids are gray. The secondary structure of the active site based on the crystal structure of MtAPA is shown. The SWISS-PROT or TrEMBL accession numbers are as follows: MtAPA (P9WMK9), APA1_Yeast (P16550), APA2_Yeast (P22108), APH1_SCHPO (P49776), Fhit_Human (P49789), Galt_Arath (Q9FK51), and Hint1_Rabbit (P80912).

Table 1. Nucleotide substrate utilization by wild-type MtAPA and ∆149A-MtAPA.

Nucleotides^a	Relative activity (%)
	Wild-type MtAPA^b	∆149A-MtAPA^c
5′,5′′′-P^1,P^3-triphosphate (Ap3A)	19^d	10
Ap4A	100^d	34
Ap5A	106^d	100
5′,5′′′-P^1,P^6-hexaphosphate (Ap6A)	25^d	<1^e
P^1-(5′-adenosyl)P^4-(5′-guanosyl) tetraphosphate (Ap4G)	100^d	23
P^1-(5′-adenosyl)P^5-(5′-guanosyl) pentaphosphate (Ap5G)	76^d	7
diguanosine 5′,5′′′-P^1,P^4-tetraphosphate (Gp4G)	107^d	18
diguanosine 5′,5′′′-P^1,P^5-pentaphosphate (Gp5G)	106^d	33
P^1-(5′-adenosyl)P^4-(5′-uridyl) tetraphosphate (Ap4U)	53	12
P^1-(5′-adenosyl)P^5-(5′-uridyl) pentaphosphate (Ap5U)	66	7
P^1-(5′-adenosyl)P^4-(5′-(2′-deoxy-thymidyl)) tetraphosphate (Ap4dT)	6	<1
P^1-(5′-adenosyl)P^5-(5′-(2′-deoxy-thymidyl)) pentaphosphate (Ap5dT)	41	24

^a Substrate concentration 0.1 mM; the structures of the nucleotides are described in Supplemental Fig. 2.

^b The relative activity in the presence of 0.1 mM Ap₄A was taken as 100%.

^c The relative activity in the presence of 0.1 mM Ap₅A was taken as 100%.

^d Values are from¹⁾.

^e Relative activity was below 1%.

Fig. 2. Ribbon model of the active site of MtAPA (A) and the comparison of the loop conformation within it (B).

Notes: Ribbon diagrams were generated by PyMOL (W.L. DeLano, http://www.pymol.org). The atomic coordinates of crystal structures were downloaded from PDB (www.rcsb.org). It is likely that the phosphate ion-binding site of MtAPA (PDB ID; 3ANO) is the bona fide active site. In this site, Asn-139, Ser-147, His-153, and His-155 coordinate the phosphate ion, whereas Gly-146 is in close proximity.²⁾ (A) The loop in the active site is shown in green, with the start and end of the loop indicated by arrows. Asn-139, Gly-146, Ser-147, Ala-149, His-153, His-155, and the phosphate ion are represented by sticks. Carbon, nitrogen, oxygen, and phosphorus atoms are shown in green, blue, red, and orange, respectively. (B) The ribbon models of the active site of MtAPA, Fhit_Human (PDB ID; 6FIT), and GalT_Arath (PDB ID; 1Z84) are shown in green, blue, and red, respectively. The position of Ala-149 in MtAPA is shown in black. The phosphate ion in the active site of MtAPA is represented by sticks. The positions of the start and end of the loop in the active site are indicated by arrows.

Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23:2947–2948.10.1093/bioinformatics/btm404

 PubMed Web of Science ®Google Scholar

Mori S, Shibayama K, Wachino J, Arakawa Y. Structural insights into the novel diadenosine 5′,5‴-p1,p4-tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv. J. Mol. Biol. 2011;410:93–104.10.1016/j.jmb.2011.04.059

 PubMed Web of Science ®Google Scholar

Mori S, Shibayama K, Wachino J, Arakawa Y. Purification and molecular characterization of a novel diadenosine 5′,5′′′-P1,P4-tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv. Protein Express. Purif. 2010;69:99–105.10.1016/j.pep.2009.09.010

 PubMed Web of Science ®Google Scholar