Abstract
The alpha-aminoadipate reductase (alpha-AAR) of Penicillium chrysogenum, an enzyme that activates the alpha-aminoadipic acid by forming an alpha-aminoadipyl adenylate and reduces the activated intermediate to alpha-aminoadipic semialdehyde, was purified to homogeneity by immunoaffinity techniques, and the kinetics for alpha-aminoadipic acid, ATP, and NADPH were determined. Sequencing of the N-terminal end confirmed the 10 first amino acids deduced from the nucleotide sequence. Its domain structure has been investigated using limited proteolysis and active site labeling. Trypsin and elastase were used to cleave the multienzyme, and the location of fragments within the primary structure was established by N-terminal sequence analysis. Initial proteolysis generated two fragments: an N-terminal fragment housing the adenylation and the peptidyl carrier protein (PCP) domains (116 kDa) and a second fragment containing most of the reductive domain (28 kDa). Under harsher conditions the adenylation domain (about 64 kDa) and the PCP domain (30 kDa) become separated. Time-dependent acylation of alpha-AAR and of fragments containing the adenylation domain with tritiated alpha-aminoadipate occurred in vitro in the absence of NADPH. Addition of NADPH to the labeled alpha-AAR released most of the radioactive substrate. A fragment containing the adenylation domain was labeled even in absence of the PCP box. The labeling of this fragment (lacking PCP) was always weaker than that observed in the di-domain (adenylating and PCP) fragment suggesting that the PCP domain plays a role in the stability of the acyl intermediate. Low intensity direct acylation of the PCP box has also been observed. A domain structure of this multienzyme is proposed.
Highlights
The ␣-aminoadipate reductase (␣-AAR) of Penicillium chrysogenum, an enzyme that activates the ␣-aminoadipic acid by forming an ␣-aminoadipyl adenylate and reduces the activated intermediate to ␣-aminoadipic semialdehyde, was purified to homogeneity by immunoaffinity techniques, and the kinetics for ␣-aminoadipic acid, ATP, and NADPH were determined
Very similar results were obtained after treatment with formic or performic acid of a mixture of the labeled fragments obtained after limited tryptic digestion. These results clearly indicate that the ␣-AA molecule in the absence of NADPH remains bound as a thioester to the phosphopantetheine arm of the peptidyl carrier protein (PCP) domain rather than to a serine or threonine residue in the ␣-AA reductase
Some previous attempts for purification of ␣-AAR have been described for the S. cerevisiae enzyme [20] and for the P. chrysogenum enzyme [21, 32], but in both cases the purification protocols yielded enzyme preparations only partially pure
Summary
The ␣-aminoadipate reductase (␣-AAR) of Penicillium chrysogenum, an enzyme that activates the ␣-aminoadipic acid by forming an ␣-aminoadipyl adenylate and reduces the activated intermediate to ␣-aminoadipic semialdehyde, was purified to homogeneity by immunoaffinity techniques, and the kinetics for ␣-aminoadipic acid, ATP, and NADPH were determined. Time-dependent acylation of ␣-AAR and of fragments containing the adenylation domain with tritiated ␣-aminoadipate occurred in vitro in the absence of NADPH. A fragment containing the adenylation domain was labeled even in absence of the PCP box. Glutarate to form homocitrate [3], which is later subjected to isomerization [4],2 oxidative decarboxylation and amination to yield ␣-AA This intermediate is converted into ␣-AA-␦semialdehyde by the action of the ␣-aminoadipate reductase (␣-AAR, EC 1.2.1.31) encoded by the lys and lys genes. The ␣-AAR, called ␣-aminoadipate semialdehyde dehydrogenase, first activates the ␣-AA ␦-carboxyl group by an ATPdependent process through the formation of an ␣-AA-adenylate, a unique step among amino acid biosynthetic pathways, that is reduced by the reduction domain using NADPH to yield ␣-AA-␦-semialdehyde and AMP. This work provides structural and functional evidence of the domains of the ␣-AAR in comparison with the domains occurring in the ␣-AA activating domains of the five known ACV synthetases
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
