Comparative analysis of xenobiotic metabolising N-acetyltransferases from ten non-human primates as in vitro models of human homologues

Xenobiotic metabolising N-acetyltransferases (NATs) perform biotransformation of drugs and carcinogens. Human NAT1 is associated with endogenous metabolic pathways of cells and is a candidate drug target for cancer. Human NAT2 is a well-characterised polymorphic xenobiotic metabolising enzyme, modulating susceptibility to drug-induced toxicity. Human NATs are difficult to express to high purification yields, complicating large-scale production for high-throughput screens or use in sophisticated enzymology assays and crystallography. We undertake comparative functional investigation of the NAT homologues of ten non-human primates, to characterise their properties and evaluate their suitability as models of human NATs. Considering the amount of generated recombinant protein, the enzymatic activity and thermal stability, the NAT homologues of non-human primates are demonstrated to be a much more effective resource for in vitro studies compared with human NATs. Certain NAT homologues are proposed as better models, such as the NAT1 of macaques Macaca mulatta and M. sylvanus, the NAT2 of Erythrocebus patas, and both NAT proteins of the gibbon Nomascus gabriellae which show highest homology to human NATs. This comparative investigation will facilitate in vitro screens towards discovery and optimisation of candidate pharmaceutical compounds for human NAT isoenzymes, while enabling better understanding of NAT function and evolution in primates.

: Geographic distribution and nutritional habits of compared primate species, classified as New World monkeys, Old World monkeys or apes (including human). The information was compiled from the Animal Diversity Web of the University of Michigan Museum of Zoology in the USA (http://animaldiversity.org/accounts/Primates/classification/#Primates).

Species
Taxonomic group Geographic range Diet    The Neighbour-Joining method was applied and the optimal tree with the sum of branch length =0.81734046 is shown. The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (2000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. There were a total of 290 positions in the final dataset with no gaps. Phylogenetic analysis was conducted in MEGA (Tamura et al (2007)  S4a: Gel filtration of four protein markers, shown as peaks coloured brown for bovine serum albumin (66.8 kDa, 3.5 ml elution volume), green for ovalbumin (44.3 kDa, 4.5 ml elution volume), pink for soybean trypsin inhibitor (21.5 kDa, 6 ml elution volume) and blue for bovine heart cytochrome c (12.4 kDa, 7 ml elution volume).

Allenopithecus nigroviridis
S4b-c: The (NOMGA)NAT proteins (~31 kDa) were eluted at volumes between 4.5 and 6 ml, as expected relative to the protein markers of Fig. S4a. The corresponding peaks are marked with a horizontal double arrow on gel filtration plots (top), which show successive runs of four aliquots of (NOMGA)NAT1 (Fig. S4b) and (NOMGA)NAT2 (Fig. S4c) preparations. The two proteins were then subjected to further purification by ion exchange chromatography (bottom).
In all ion exchange chromatography plots, blue is the eluted protein (OD at 280 nm), green is the salt concentration and brown is the conductivity.   Supplementary Fig. S6: Example of primate NAT proteins assayed by differential scanning fluorimetry (DSF).
Thermal stability of recombinant NAT proteins was assessed with or without acetyl-CoA, arylamine or combinations of the two types of substrates. The panels on the left show the generated thermal denaturation curves, with change in SyproOrange fluorescence provided in arbitrary units (AU). The panels on the right show the derivative of generated thermal profiles, with main peaks indicating protein Tm values. Two replicate experiments were performed, producing overlapping curves for which the average plot is shown. The proteins used were (NOMGA)NAT1 of Nomascus gabriellae (Fig. S6a, S6b) and (ERYPA)NAT2 of Erythrocebus patas (Fig. S6c, S6d). The NAT1-selective p-aminobenzoate (PABA) and the NAT2-selective sulphamethazine (SMZ) substrates were used with or without acetyl-CoA, as described in the graph legends (Tm values provided in o C).  Supplementary Fig. S7: Primate NAT1 protein (non-human) assayed for enzymatic activity with PABA and pABGlu.
Enzymatic release of coenzyme A (CoA) was monitored with Ellman's reagent over specific time points, in reactions containing 1μg of purified (NOMGA)NAT1 protein from the gibbon Nomascus gabriellae, 0.4 mM of acetyl-CoA and 0.5 mM of either p-aminobenzoate (PABA) or p-aminobenzoylglutamate (pABGlu). The calculated enzymatic specific activity measured with PABA (7940 nmol/min/mg) was about 2-fold higher than with pABGlu (4104 nmol/min/mg). Duplicate reactions were performed and each data point is the average±standard deviation. All preparations were generated (over a period of two months) in the U.K. lab using identical conditions, and gels were scanned with a conventional office scanner. Exceptions are the first two gels shown, which represent our most successful attempts to generate (HUMAN)NAT1 and (CHLTN/ERYPA)NAT1 recombinant proteins under the standard conditions applied. These preparations were produced later in the Greek lab and the gels were photographed on a standard white-light transilluminator apparatus.