Sequence divergence and diversity suggests ongoing functional diversification of vertebrate NAD metabolism

Abstract

NAD is not only an important cofactor in redox reactions but has also received attention in recent years because of its physiological importance in metabolic regulation, DNA repair and signaling. In contrast to the redox reactions, these regulatory processes involve degradation of NAD and therefore necessitate a constant replenishment of its cellular pool. NAD biosynthetic enzymes are common to almost all species in all clades, but the number of NAD degrading enzymes varies substantially across taxa. In particular, vertebrates, including humans, have a manifold of NAD degrading enzymes which require a high turnover of NAD. As there is currently a lack of a systematic study of how natural selection has shaped enzymes involved in NAD metabolism we conducted a comprehensive evolutionary analysis based on intraspecific variation and interspecific divergence. We compare NAD biosynthetic and degrading enzymes in four eukaryotic model species and subsequently focus on human NAD metabolic enzymes and their orthologs in other vertebrates. We find that the majority of enzymes involved in NAD metabolism are subject to varying levels of purifying selection. While NAD biosynthetic enzymes appear to experience a rather high level of evolutionary constraint, there is evidence for positive selection among enzymes mediating NAD-dependent signaling. This is particularly evident for members of the PARP family, a diverse protein family involved in DNA damage repair and programmed cell death. Based on haplotype information and substitution rate analysis we pinpoint sites that are potential targets of positive selection. We also link our findings to a three dimensional structure, which suggests that positive selection occurs in domains responsible for DNA binding and polymerization rather than the NAD catalytic domain. Taken together, our results indicate that vertebrate NAD metabolism is still undergoing functional diversification.

Keywords: NAD metabolism; PARP; Pathway evolution; Positive selection.

Fig. 1

Schematic representation of the NAD pathway model. Enzymes are subdivided into biosynthetic (green) and degrading enzymes (red). NAD key metabolites are circled, remaining substrates and products are indicated by reaction arrows. For further details and abbreviations see . (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Evolutionary rates for 50 NAD metabolic enzymes measured as d_n/d_s based on human NAD enzymes and their orthologs in other vertebrate species. Genes that show significant evidence for positive selection based on site test (see Section 2) are boxed in red. Parameter estimates for the site test results are shown in Table 2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Alignment of PARP14 homologs covering macro domain 1 and macro domain 2 (human residues 800–1200). Red boxes indicate sites that have been identified to be evolving under positive selection. Green blocks indicate sites for substrate binding and cyan blocks indicate macro domain 1 and 2 interacting sites . Site 903 has nonpolar interactions with sites 1105 and 1143. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Sliding window analysis (3 Kb overlapping windows for FST and 10 Kb for H, step size 0.5 Kb) of human PARP4 variation. The upper panel shows FST, a measurement of population differentiation along with confidence intervals (dashed black line, obtained from extreme values of 100 subsamples) and average FST of human chromosome 13 . The middle panel shows Fay and Wu's H statistic, a test statistic for positive selection in presence of frequent recombination. The lower panel indicates the PARP4 exon/intron distribution along with the proposed protein domains (InterPro Accession Q9UKK3 [80]) and MVP interacting region , . (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)