Domain structure characterization of the multifunctional alpha-aminoadipate reductase from Penicillium chrysogenum by limited proteolysis. Activation of alpha-aminoadipate does not require the peptidyl carrier protein box or the reduction domain.
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.
Hijarrubia
Maria José
MJ
Insituto de Biotecnologia, Parque Cientifico de León, Avda. del Real no. 1, Spain.
Aparicio
Jesús F
JF
Martín
Juan F
JF
D015074:2-Aminoadipic Acid / Q000378:metabolism*; D000215:Acylation; D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D017931:DNA Primers; D006868:Hydrolysis; D007700:Kinetics; D050647:L-Aminoadipate-Semialdehyde Dehydrogenase; D010408:Penicillium chrysogenum / Q000201:enzymology*; D011487:Protein Conformation; D013379:Substrate Specificity
D002352:Carrier Proteins; D017931:DNA Primers; D015074:2-Aminoadipic Acid; D000445:Aldehyde Oxidoreductases; D050647:L-Aminoadipate-Semialdehyde Dehydrogenase