Structures of aminoacylase 3 in complex with acetylated substrates

Abstract

Trichloroethylene (TCE) is one of the most widespread environmental contaminants, which is metabolized to N-acetyl-S-1,2-dichlorovinyl-L-cysteine (NA-DCVC) before being excreted in the urine. Alternatively, NA-DCVC can be deacetylated by aminoacylase 3 (AA3), an enzyme that is highly expressed in the kidney, liver, and brain. NA-DCVC deacetylation initiates the transformation into toxic products that ultimately causes acute renal failure. AA3 inhibition is therefore a target of interest to prevent TCE induced nephrotoxicity. Here we report the crystal structure of recombinant mouse AA3 (mAA3) in the presence of its acetate byproduct and two substrates: N α -acetyl-L-tyrosine and NA-DCVC. These structures, in conjunction with biochemical data, indicated that AA3 mediates substrate specificity through van der Waals interactions providing a dynamic interaction interface, which facilitates a diverse range of substrates.mercapturates | metalloprotein | X-ray structure A minoacylase 3 (AA3) is a member of the aminoacylase family of enzymes that deacylates a broad range of substrates including both N α -acetylated amino acids and S-cysteine conjugates of N-acetyl-L-cysteine (mercapturic acids) ( Fig. 1) (1). There are three types of aminoacylases: (i) aminoacylase 1 (AA1) deacetylates neutral aliphatic N-acyl-α-amino acids and mercapturic acids; (ii) aminoacylase 2 or aspartoacylase (AA2) has a strict specificity for N α -acetyl-L-aspartate (NAD); and (iii) aminoacylase 3 (AA3) preferentially deacetylates N α -acetylated aromatic amino acids and mercapturic acids that are usually not deacetylated by AA1 (1-6). Despite different substrate specificities, AA2 and AA3 have a high degree of sequence (42% identity) and structure homology but are both substantially different from AA1 (∼10% of sequence identity) (2, 6-10).AA3 is of particular interest for human health because it participates in mediating toxicity of the xenobiotic trichloroethylene (TCE). The United States produces in excess of 130,000 tons of TCE per year (11), making it the most widespread chemical contaminant in both soil and ground water. TCE is readily absorbed into the body where it can enter the glutathione conjugation detoxification pathway producing the mercapturic acid N-acetyl-S-1,2-dichlorovinyl-L-cysteine (NA-DCVC) for subsequent urinary excretion (12, 13). However, NA-DCVC can be deacetylated by AA3-which is highly expressed in the renal proximal tubule, liver, and brain-to generate S-1,2-dichlorovinyl-L-cysteine (DCVC) (2) and further transformed via β-lyases or flavin monooxygenases into lethal products capable of causing acute renal failure and toxicity to the liver and brain. (4, 13-22). Thus, inhibition of AA3 can decrease DCVC formation and ameliorate TCE toxicity, presenting a potential target for drug discovery.Generating specific inhibitors for AA3 would be greatly aided by high-resolution structural data, but structural studies of aminoacylases have been limited to AA1 (7) and AA2 (8, 9), which differ in both overall architecture and ac...

Carboxypeptidase O Is a Glycosylphosphatidylinositol-anchored Intestinal Peptidase with Acidic Amino Acid Specificity

“…Aminoacylase 2 (also called aspartoacylase) functions in the deacetylation of N-acetyl-L-aspartate through a CP-like mechanism (20). This is in contrast to its close relative, aminoacylase 3, which preferentially deacetylates N-acetylated aromatic amino acids (37). Mutations in aminoacylase 2 cause Canavan disease, a fatal and progressive neurodegenerative disease, due to greatly elevated levels of N-acetyl-L-aspartate (38).…”

Inhibition of aminoacylase 3 protects rat brain cortex neuronal cells from the toxicity of 4-hydroxy-2-nonenal mercapturate and 4-hydroxy-2-nonenal

“…Ebselen is a seleno-compound that easy reacts with reactive oxygen species and other cellular components, for example cysteine residues in proteins. Mouse AA3 has a cysteine residue (Cys175) in proximity to the active site (Hsieh et al, 2010), which modification by ebselen may decrease catalytic activity via restriction of substrate access to the active site. This possibility was excluded from consideration because purified human AA3 that has a glycine residue instead of cysteine (Pushkin et al, 2004; Tsirulnikov et al, 2009) was inhibited by ebselen to the same extent as mouse AA3 (data not shown).…”

“…Mouse and human AA3 were expressed in E. coli and purified as described (Tsirulnikov et al, 2009; Hsieh et al, 2010). Both purified enzymes were homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with MR-C1 and CH-4 antibodies (data not shown).…”

“…Mouse AA3 has a high sequence homology with rat and human AA3, and the identical amino acid residues form their active sites (Newman et al, 2007; Hsieh et al, 2010). Therefore the compounds generated in this study for mouse AA3 are expected to also inhibit rat AA3 and human AA3 at similar rates.…”

“…The atomic structures of AA1 (Lindner et al, 2003) and AA3 (Hsieh et al, 2010) have been studied by X-ray crystallography. An important structure/functional property of AA1 and AA3 is that they both are metalloenzymes inactivated by chelating agents (Anders, Dekant, 1994; Kördel, Schneider, 1977; Lindner et al, 2003, 2008; Perrier et al, 2005; Tsirulnikov et al, 2009; Wu, Tsou, 1993).…”