Optimization of the Expression of Human Aldehyde Oxidase for Investigations of Single-Nucleotide Polymorphisms

Abstract

Aldehyde oxidase (AOX1) is an enzyme with broad substrate specificity, catalyzing the oxidation of a wide range of endogenous and exogenous aldehydes as well as N-heterocyclic aromatic compounds. In humans, the enzyme's role in phase I drug metabolism has been established and its importance is now emerging. However, the true physiologic function of AOX1 in mammals is still unknown. Further, numerous single-nucleotide polymorphisms (SNPs) have been identified in human AOX1. SNPs are a major source of interindividual variability in the human population, and SNP-based amino acid exchanges in AOX1 reportedly modulate the catalytic function of the enzyme in either a positive or negative fashion. For the reliable analysis of the effect of amino acid exchanges in human proteins, the existence of reproducible expression systems for the production of active protein in ample amounts for kinetic, spectroscopic, and crystallographic studies is required. In our study we report an optimized expression system for hAOX1 in Escherichia coli using a codon-optimized construct. The codon-optimization resulted in an up to 15-fold increase of protein production and a simplified purification procedure. The optimized expression system was used to study three SNPs that result in amino acid changes C44W, G1269R, and S1271L. In addition, the crystal structure of the S1271L SNP was solved. We demonstrate that the recombinant enzyme can be used for future studies to exploit the role of AOX in drug metabolism, and for the identification and synthesis of new drugs targeting AOX when combined with crystallographic and modeling studies.

Statistical analysis of disease‐causing and neutral mutations in human membrane proteins

“…In buried regions, Cys has a high preference to change to nonpolar residues. This is in line with the observation that the C44W mutation destabilizes and affects the function of aldehyde oxidase, an enzyme which plays an important role in drug metabolism . A comparison between inside and exposed regions showed that changes in nonpolar residues are dominant in the inside region, whereas charged (Asp, Glu) and nonpolar (Pro, Gly) residues are frequently exchanged in the exposed region (Figure ).…”

“…This is in line with the observation that the C44W mutation destabilizes and affects the function of aldehyde oxidase, an enzyme which plays an important role in drug metabolism. 73 A comparison between inside and exposed regions showed that changes in nonpolar residues are dominant in the inside region, whereas charged (Asp, Glu) and nonpolar (Pro, Gly) residues are frequently exchanged in the exposed region ( Figure 6). On the other hand, changes in Cys and positively charged residues (Lys, Arg) are preferred in the outside and exposed regions, respectively (Figure 7).…”

Human aldehyde oxidase (hAOX1): structure determination of the Moco‐free form of the natural variant G1269R and biophysical studies of single nucleotide polymorphisms

“…The results suggest that the amino acid exchanges in the variants G1269R and S1271L have affected the stability of the protein. In fact, the G1269R variant corresponds to a Moco-free form of the enzyme where the whole cofactor is absent, as reported here, and, as expected, this variant has no measurable activity [16]. In contrast, the structure of the S1271L variant previously reported (PDB ID: 5EPG) [16] did not show any significant structural differences when compared to the WT enzyme.…”

“…In fact, the G1269R variant corresponds to a Moco-free form of the enzyme where the whole cofactor is absent, as reported here, and, as expected, this variant has no measurable activity [16]. In contrast, the structure of the S1271L variant previously reported (PDB ID: 5EPG) [16] did not show any significant structural differences when compared to the WT enzyme. Nevertheless, the replacement of a polar amino acid (Ser) by a hydrophobic residue (Leu) in the core of the protein that is surrounded by a hydrophobic neighbourhood affects the protein dynamics.…”

Systematic exploration of predicted destabilizing nonsynonymous single nucleotide polymorphisms (nsSNPs) of human aldehyde oxidase: A Bio‐informatics study

“…The nsSNPs near the Moco active site and surrounding region are the most relevant ones for enzyme activity and substrate specificity. Some of these nsSNPs have been expressed and characterized, namely R802C, R921H, G1269R, and S1271L (Table and Figure C) . Interestingly, in the first report concerning the presence of nsSNPs in hAOX1, it was shown that the R802C nsSNP was predominantly purified in its monomeric form in solution, resulting in a higher proportion of the inactive form of the enzyme .…”