Fig 1.
AMP deaminase reaction scheme.
Fig 2.
Relative rates of deamination.
Activity of native H. pomatia AMP deaminase on 100 μM 5’ AMP, 5’ ATP, and adenosine. The reaction volume was 0.4 ml. The change in absorbance at OD265 was followed in a spectrophotometer at 25° C.
Fig 3.
The amino acid sequence of the H. pomatia transcriptome contig 71391.
The sequence segments covered by LC-MS/MS derived peptides are shown in blue text.
Fig 4.
Phylogenetic tree of the adenyl-deaminase family of proteins.
The tree was generated with FastTree [16] from a MAFFT alignment. The numerical values at each branch represent substitutions per site. ADGF are the adenosine deaminase-related growth factors, AMPD are the AMP deaminases, ADE are the adenine deaminases, ADAL are the adenosine deaminase-like proteins, ADAR are the adenosine deaminases acting on RNA, and the ADA are the adenosine deaminases. The substrate assigned to each subfamily is indicated on the right. The accession numbers for each enzyme follow: ADGF human, AAF65941.1; ADGF Aplysia, AAD13112.1; ADGF Fish, AF384217.1; ADGF Lottia, XP_009053965.1; ADGF Chicken, AAX10953.1; ADGF Frog, AAX10952.1; ADAL Arabidopsis, NP_192397.2; ADAL human iso3, NP_001311295.1; ADE Aspergillus, AL56636.1; ADE Saccharomyces, NP_014258.1; ADA C elegans, NP_872091.1; ADA human iso1, NP_000013.2; AMPD human, NP_000471.1; AMPD Arabidopsis, NP_565886.1; AMPD2 human, NP_001244290.1; AMPD Drosophila, NP_727740.2.
Fig 5.
Alignment of the HPAMPD to other ADGF subfamily members.
Underlined residues are predicted signal sequences [19] and blue outlined boxes are catalytic residue positions defined by [18] in human ADA2. HPAMPD sequence is derived from cDNA.
Fig 6.
Structural Comparison of HPAMPD and human ADGF.
(A) A superimposition of the AlphaFold2 predicted HPAMPD monomer and a monomer of the human ADGF (PDB 3LGG). Human ADGF is colored pink and the HPAMPD is colored cream. Side chains of active site residues and coformycin (colored blue) are drawn as sticks. The zinc ion is represented as a sphere and dashed lines depict interactions with coordinating residues from human ADGF. (B) A close-up view of the active site residues, zinc and coformycin. The labeled residues correspond to the 3LGG structure.
Fig 7.
The amino acid sequence of the HPAMPD cDNA clone expressed in P. pastoris.
Table 1.
Relative rates of deamination.
Spectrophotometric assay of deamination of adenyl base monitored by loss of OD260 or OD265. The rates are linear initial rates and expressed as a percentage of the 5’ AMP rate. Reactions were at 25° C in 20 mM Tris-HCl pH 7.5, 50 mM NaCl and the indicated concentration of each substrate.
Fig 8.
Lineweaver-Burke plot for determination of Vmax and Km values for recombinant HPAMPD with AMP.
Table 2.
Relative initial rates of deamination of adenosine and AMP for each deaminase.
The deamination was measured by loss of OD265 in 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 50 mM NaCl buffer. The substrate concentrations were 100 μM for the HPAMPD and A. californica enzymes and 2 mM for the human enzyme. The reaction temperature for H. pomatia and A. californica ADGFs was 25° and 37° for human ADGF. All values were obtained with recombinant enzymes except for the value in parentheses which was obtained with the native preparation of HPAMPD.