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. 2019 Nov 21;26(11):1623-1629.e3.
doi: 10.1016/j.chembiol.2019.09.009. Epub 2019 Oct 3.

Family-wide Annotation of Enzymatic Pathways by Parallel In Vivo Metabolomics

Joon T Kim  1 , Veronica L Li  1 , Stephanie M Terrell  1 , Curt R Fischer  2 , Jonathan Z Long  3
Affiliations

Family-wide Annotation of Enzymatic Pathways by Parallel In Vivo Metabolomics

Joon T Kim et al. Cell Chem Biol. .

Abstract

Enzymes catalyze fundamental biochemical reactions that control cellular and organismal homeostasis. Here we present an approach for de novo biochemical pathway discovery across entire mammalian enzyme families using parallel viral transduction in mice and untargeted liquid chromatography-mass spectrometry. Applying this method to the M20 peptidases uncovers both known pathways of amino acid metabolism as well as a previously unknown CNDP2-regulated pathway for threonyl dipeptide catabolism. Ablation of CNDP2 in mice elevates threonyl dipeptides across multiple tissues, establishing the physiologic relevance of our biochemical assignments. Taken together, these data underscore the utility of parallel in vivo metabolomics for the family-wide discovery of enzymatic pathways.

Keywords: acid; acy1; amino; cndp2; enzyme; in vivo; metabolomics; peptidase; pm20d1.

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Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Viral transduction of the M20 peptidases in vivo and depletion of carnosine and N-acetyl amino acids by CNDP1 and ACY1, respectively.
(A) Dendrogram showing murine M20 peptidase family. (B) Immunoblot using anti-flag (top) or anti-beta-tubulin (bottom) antibodies of livers from mice transduced with AAV-GFP or AAVs encoding the indicated flag-tagged enzymes. (C, D) Extracted ion chromatograms of the indicated metabolite from transduced livers, its structural assignment, and the inferred biochemical activity of the indicated enzyme (in red). All chromatograms were extracted at the exact mass ± 20 ppm. For C and D, n=3/group for the enzyme-transduced group and n=15 for all other viral transduction groups.
Figure 2.
Figure 2.. CNDP2 overexpression depletes Thr-Thr in vivo.
(A) Extracted ion chromatogram of an unknown metabolite at m/z = 219.099 from transduced livers in CNDP2-transduced (red) versus all other viral transduction groups (blue). (B-D) Structural assignment, co-elution, and tandem MS fragmentation of the endogenous m/z = 219.099 and an authentic Thr-Thr standard. For (A) and (C), chromatograms were extracted at the exact mass ± 20 ppm.
Figure 3.
Figure 3.. In vitro hydrolysis of threonyl dipeptides by CNDP2
(A) Immunoblot using anti-flag antibodies of the indicated purified recombinant mammalian enzyme from HEK293T cells. (B-D) In vitro Thr-Thr (B), Ser-Thr (C), and Thr-Ser (D) hydrolysis to the indicated dipeptides by CNDP2 or a catalytically dead mutant (CNDP2-E166A). For B-D, n=3–4/group. Data are shown as means ± SEM; * P < 0.05.
Figure 4.
Figure 4.. CNDP2 regulates the catabolism of threonyl dipeptides in vivo.
(A) Immunoblot using anti-CNDP2 (top) or anti-tubulin (bottom) antibodies of kidney tissue from CNDP2-WT or CNDP2-KO mice. (B-D) In vitro Thr-Thr (B), Ser-Thr (C), and Thr-Ser (D) hydrolysis of the indicated dipeptides in CNDP2-WT or KO kidney tissues. (E-G) Fold change in endogenous levels of the indicated metabolites from kidney (E), muscle (F), or blood (G) between CNDP2-WT or CNDP2-KO mice, with wild-type levels set to 1. For B-D, n=3/group; for E-G, n=4/group. Data are shown as means ± SEM; * P < 0.05, *** P < 0.001.
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