Extended Data Fig. 7: Human HPDL is an ortholog of A. orientalis HmaS.
From: The polar oxy-metabolome reveals the 4-hydroxymandelate CoQ10 synthesis pathway
a, b, Phylogenetic tree of HPD, HPDL, and HmaS cDNA (a) and protein (b) sequences across several model organisms. c, Protein sequence alignment of HPD, HPDL and HmaS. Catalytic histidines involved in coordinating the iron ion needed for activity are highlighted in red. Specific residues in Steptomyces avermitilis and Pseudomonas fluorescens HPD have been mutated in other studies, and the human equivalent mutations are as indicated; hydrophobic (blue), polar (green) amino acids and proline (yellow). The HPD P239T mutant decreases HGA production and generates oxopinone. The N241I/L mutation abolishes HGA production by HPD. The HPD S226A mutations blocks HGA production. However, the mutation in the equivalent site in HMS (S201A) does not affect the generation of 4-HMA. The F337V/L mutation in HPD decreases HGA synthesis and allows slight production of 4-HMA. d, Growth curve of MIAPACA2 cells with sgRNAs at 21% O2. (n = 3 technical replicates for each cell line, performed at least twice). e, Unlabelled and 13C8-labelled 4-HMA from PATU-8902 cells grown in 13C9-Tyr at 21% 16O2 for 24 h. (n = 3). Immunoblots of HPDL levels from PATU-8902 cells expressing control and HPDL sgRNAs. ERK2 serves as a loading control. f, Unlabelled and 13C-labelled Tyr, 4-HPPA, 4-HPLA from MIAPACA2 cells were grown in 13C9-Tyr at 21% 16O2 for 24 h (from Fig. 2a). (n = 5). g, Unlabelled and 13C-labelled Tyr, 4-HPPA, 4-HPLA from MIAPACA2 sgHPDL #3 cells were grown in 13C9-Tyr at 21% 16O2 for 24 h (from Fig. 2b). (n = 5). n represents the number of biologically independent replicates for each group and condition, unless indicated (d). Graphs are represented as mean ± s.d. (d) or s.e.m. (e–g) and were compared by two-tailed Student t-test (e), or two-way ANOVA (d, f, g), followed by Tukey post hoc test (*P < 0.05, ^P < 0.01, %P < 0.005, #P < 0.0001).
