Protein鈥揚rotein Interactions and Substrate Channeling in Orthologous and Chimeric Aldolase鈥揇ehydrogenase Complexes

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• 作者：Perrin Baker ; Colleen Hillis ; Jason Carere ; Stephen Y. K. Seah
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：March 6, 2012
• 年：2012
• 卷：51
• 期：9
• 页码：1942-1952
• 全文大小：447K
• 年卷期：v.51,no.9(March 6, 2012)
• ISSN：1520-4995

文摘

Bacterial aldolase鈥揹ehydrogenase complexes catalyze the last steps in the meta cleavage pathway of aromatic hydrocarbon degradation. The aldolase (TTHB246) and dehydrogenase (TTHB247) from Thermus thermophilus were separately expressed and purified from recombinant Escherichia coli. The aldolase forms a dimer, while the dehydrogenase is a monomer; these enzymes can form a stable tetrameric complex in vitro, consisting of two aldolase and two dehydrogenase subunits. Upon complex formation, the K_m value of 4-hydroxy-2-oxopentanoate, the substrate of TTHB246, is decreased 4-fold while the K_m of acetaldehyde, the substrate of TTHB247, is increased 3-fold. The k_cat values of each enzyme were reduced by 2-fold when they were in a complex. The half-life of TTHB247 at 50 掳C increased by 4-fold when it was in a complex with TTHB246. The acetaldehyde product from TTHB246 could be efficiently channelled directly to TTHB247, but the channeling efficiency for the larger propionaldehyde was 40% lower. A single A324G substitution in TTHB246 increased the channeling efficiency of propionaldehyde to a value comparable to that of acetaldehyde. Stable and catalytically competent chimeric complexes could be formed between the T. thermophilus enzymes and the orthologous aldolase (BphI) and dehydrogenase (BphJ) from the biphenyl degradation pathway of Burkholderia xenovorans LB400. However, channeling efficiencies for acetaldehyde in these chimeric complexes were 10%. Structural and sequence analysis suggests that interacting residues in the interface of the aldolase鈥揹ehydrogenase complex are highly conserved among homologues, but coevolution of partner enzymes is required to fine-tune this interaction to allow for efficient substrate channeling.