Identification of an aspartic acid residue in the β subunit which is essential for catalysis and proton pumping by transhydrogenase from Escherichia coli

Abstract

Based on the alignment of 7 unknown amino acid sequences, including the recently determined sequences for the mouse and human enzymes, a highly conserved acidic domain was identified which in the Escherichia coli enzyme is located close to the C-terminal end of the predicted NADP(H)-binding site of the beta subunit. The effect of replacing the four conserved acidic residues, betaE361, betaE374, betaD383 and betaD392, in this domain on catalytic and proton-pumping activity was tested by site-directed mutagenesis. In addition, betaE371, which is not conserved but located in the same domain, was also mutated. Of these residues, betaAsp 392 proved to be the only residue which is essential for both activities. However, two betaAsp 392 mutants were still partly active in catalyzing the cyclic reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADPH, suggesting that the mutations did not cause a global change but rather a subtle local change influencing the dissociation of NADP(H). It is proposed that betaAsp 392 together with th previously identified betaHis91 form part of a proton wire in transhydrogenase.

Mapping of Residues in the NADP(H)-binding Site of Proton-translocating Nicotinamide Nucleotide Transhydrogenase fromEscherichia coli

“…rrI plus ecIIIH345C and ecIIIR350C (not shown). It has been hypothesized that ␤D392 could be directly involved in vectorial proton transport (44). The present results do not exclude this possibility.…”

“…3B) and NEM (not shown) and the fact that reaction with NEM yielded an enzyme derivative virtually unable to bind the NADPH substrate support the predicted location of ␤-Ala 390 at the end of ␤D in the NADP(H)-binding site. Necessarily, ␤-Asp 392 , which has been shown to be crucial for reverse transhydrogenation (44), must also be in or very near the NADP(H) site.…”

“…The ␤H345C mutant was previously shown to poorly assemble into membranes (42,43) and was here found to be difficult to purify (not shown). None of the various ␤-Asp 392 mutants analyzed so far catalyzed reverse transhydrogenation (44). Although not essential, ␤R350C seems to be an important residue for catalysis, but its role is unclear (43).…”

Review and Hypothesis. New insights into the reaction mechanism of transhydrogenase: Swivelling the dIII component may gate the proton channel

“…The protruding feature designated helix D/loop D is conformationally mobile [28], and along with the interacting loop E, is thought to have a central role in the transhydrogenase energy-coupling mechanism, perhaps in the crucial transition between the open and occluded states of the enzyme (see below). Mutations of EcbK424, EcbR425 and EcbY431 in helix E and of EcbD392 in helix D/loop D in the intact E. coli enzyme lead to inhibition of transhydrogenation activity [29][30][31][32]. Studies on these and other mutants in loop E and helix D/loop D of isolated E. coli dIII reveal further consequences of interactions of the protein with NADP + and NADPH [33,34].…”

“…Conservative single-site substitutions of only EcaH450 (in TM3), EcbH91 (TM9), EcbS139 (TM10), EcbD213 (in the cytoplasmic loop between TM12 and 13), EcbN222 (TM13), EcbG252 (TM14) and in a run of residues from EcbK261-bR265 (in the hinge at the C-terminus of TM14 which connects dII to dIII -see Fig. 1) of many examined in E. coli dII lead to strong inhibition of transhydrogenase activity [30,31,[41][42][43][44][45][46][47][48]. EcbH91 has often provoked interest in that it is the only conserved protonatable residue close to the centre of the membrane dielectric whose substitution significantly deactivates transhydrogenation.…”