Protein–Protein Interactions and Substrate Channeling in Orthologous and Chimeric Aldolase–Dehydrogenase Complexes

Abstract

Bacterial aldolase-dehydrogenase 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 °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-dehydrogenase complex are highly conserved among homologues, but coevolution of partner enzymes is required to fine-tune this interaction to allow for efficient substrate channeling.

The urea carboxylase and allophanate hydrolase activities of urea amidolyase are functionally independent

“…The k cat and K M of an enzyme may be altered by the formation of a protein complex. 20,21 For example, if PsUC and PsAH coordinate their activities during catalysis through either intermediate channeling or direct physical interactions, the presence of one enzyme could alter substrate access or catalytic turnover of the other, and thus alter its kinetic parameters. To assess this possibility, the overall kinetic parameters were determined for UC, AH, Figure 1.…”

“…(A) Representative chromatograms from size exclusion chromatography. In each case, a 200 lL injection of 1 mg mL 21 PsAH (dotted lines), 1 mg mL 21 PsUC (dashed lines) or preincubated 1 mg mL 21 PsAH/PsUC (solid lines) was applied to a superpose 6 HR 10/ 30 column. The elution peak at 15 mL corresponds to the predicted molecular weight for both the wild-type PsAH dimer (MW 5 130 kDa), and the PsUC monomer (MW 5 130 kDa).…”

“…The proteins were eluted using buffer B with a gradient from 100 to 750 mM NaCl. The proteins were concentrated using a 30 kDa-molecular weight cutoff centrifugal filter to final concentrations ranging from 6 to 16.5 mg mL 21 , and drop frozen in liquid nitrogen. The purity of the protein samples were confirmed by SDS-PAGE.…”

Sequestration of a highly reactive intermediate in an evolving pathway for degradation of pentachlorophenol

“…Trapping of a reaction intermediate that has been released to the solvent by a very fast reaction is one of the most rigorous tests for channeling. This approach has been used to measure the efficiency of channeling between the active sites of BphI and BphJ (36), dihydroxyacetone kinase and aldolase in a fused bifunctional enzyme (37), and orthologous and chimeric aldolasedehydrogenase complexes (38). In the latter case, channeling occurs in orthologous complexes, but not in chimeric complexes, even though stable complexes are formed in both cases.…”

Importance of Hydrophobic Cavities in Allosteric Regulation of Formylglycinamide Synthetase: Insight from Xenon Trapping and Statistical Coupling Analysis

“…An efficient strategy employed by nature to sequester unstable intermediates along various biosynthetic pathways is by evolving separate enzymes with coupled reactions that are only active in consort as multiprotein complexes [1], [2]. On the other hand, in some instances analogous systems have emerged that retain these activities together via synthesis of the enzymes as a single polypeptide chain [3], [4].…”