Examination of the Mechanism of Human Brain Aspartoacylase through the Binding of an Intermediate Analogue^,

Abstract

Canavan disease is a fatal neurological disorder caused by the malfunctioning of a single metabolic enzyme, aspartoacylase, that catalyzes the deacetylation of N-acetyl-L-aspartate to produce L-aspartate and acetate. The structure of human brain aspartoacylase has been determined in complex with a stable tetrahedral intermediate analogue, N-phosphonomethyl-L-aspartate. This potent inhibitor forms multiple interactions between each of its heteroatoms and the substrate binding groups arrayed within the active site. The binding of the catalytic intermediate analogue induces the conformational ordering of several substrate binding groups, thereby setting up the active site for catalysis. The highly ordered binding of this inhibitor has allowed assignments to be made for substrate binding groups and provides strong support for a carboxypeptidase-type mechanism for the hydrolysis of the amide bond of the substrate, N-acetyl-L-aspartate.Canavan disease (CD) 1 is a fatal autosomal recessive disorder that affects the central nervous system (1) and for which there is currently no effective treatment. The symptoms for CD can be noticed as early as 3-6 months of age and include rapid increase of the head circumference (megalocephaly), lack of head control, reduced visual responsiveness, abnormal muscle tone (i.e., floppiness or stiffness), and mental retardation (2). CD patients usually do not live past the first decade of their lives. Canavan disease is caused by a defect in the capability of the brain to metabolize N-acetyl-L-aspartate (NAA) (3), one of the most abundant amino acids in the brain (4). Aspartoacylase, whose function is to catalyze the deacetylation of NAA ( Figure 1), is located primarily in the white matter of the brain, more specifically in the cytosol of the oligodendrocytes (5). Analysis of the acy2 gene that encodes aspartoacylase taken from DNA isolated from CD patients has revealed more than 50 different mutations, including numerous deletions, missense mutations, and premature terminations (6). In most cases, the missense mutations result in nonconservative amino acid substitutions, leading to an altered enzyme that either is not expressed at all or is expressed but has little or no catalytic activity (6).Aspartoacylase was originally proposed to be member of the esterase family since it was shown to be inactivated by diisopropylfluorophosphate, a classic serine protease inactivator (7). On † This work was supported by a grant from the National Institutes of Health (NS45664), and C.X. was supported by Grant GM71790. ‡ The atomic coordinates for apo-aspartoacylase (2O53) and the tetrahedral intermediate analogue complex of human aspartoacylase (2O4H) the basis of these studies, conserved serine, histidine, and glutamate residues were identified and proposed to function as a catalytic triad. However, subsequent alignment studies showed few similarities between aspartoacylase and the esterase family, instead suggesting that aspartoacylase is a member of the zinc peptidase superfamily (8). Th...

Mouse aminoacylase 3: A metalloenzyme activated by cobalt and nickel

“…Nevertheless D114A as well as N70A and R71A were significantly activated by Co 2+ similar to wt-AA3. Arg71 has been proposed to be involved in binding of α-carboxyl group of Ac-Asp in the rat and human AA2 models [17,36] and was suggested to play a similar role in our first 3D model of AA3 (Fig. 1A).…”

“…Therefore it is likely that these conserved amino acids might be involved in metal coordination in mouse AA3. Several amino acid residues have been hypothesized to participate in N-acetyl-aspartate binding/catalysis of rat and human AA2 [17,35,36]. These amino acids are conserved not only in AA2 but also in AA3 (Arg63, Asp68, Asn70, Arg71, Asp114, Glu177 and Tyr287 in mouse AA3).…”

A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581T

“…(ii)The 342 aa protein (36.6 kDa, pI 4.6) encoded by doeB ( Helo_3664 , homologue to eutE ) is closely related to proteins of the succinyl-glutamate desuccinylase/aspartoacylase subfamily, which are part of the M14 family of metallocarboxypeptidases ( Makarova and Grishin, 1999 ). The desuccinylase is involved in arginine catabolism while the aspartoacylase cleaves N-acetyl-aspartate into aspartate and acetate ( Le Coq et al ., 2008 ). Deletion of doeB resulted in a mutant KB42 that could not utilize ectoine as carbon and nitrogen source.…”

Modification of aspartoacylase for potential use in enzyme replacement therapy for the treatment of Canavan disease

“…Human aspartoacylase is a functional dimer containing 23 lysines per monomer. Recent structures of this enzyme [24,25]reveal that all of these lysyl groups are solvent exposed to varying degrees (Fig. 1), and m odification of any of these lysines would be expected to alter the surface properties of aspartoacylase.…”