A Dynamic Loop at the Active Center of the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Modulates Substrate Utilization and Chemical Communication with the E2 Component

Sachin Kale,Palaniappa Arjunan,William Furey,Frank Jordan

doi:10.1074/jbc.m704326200

Abstract

Our crystallographic studies have shown that two active center loops (an inner loop formed by residues 401-413 and outer loop formed by residues 541-557) of the E1 component of the Escherichia coli pyruvate dehydrogenase complex become organized only on binding a substrate analog that is capable of forming a stable thiamin diphosphate-bound covalent intermediate. We showed that residue His-407 on the inner loop has a key role in the mechanism, especially in the reductive acetylation of the E. coli dihydrolipoamide transacetylase component, whereas crystallographic results showed a role of this residue in a disorder-order transformation of these two loops, and the ordered conformation gives rise to numerous new contacts between the inner loop and the active center. We present mapping of the conserved residues on the inner loop. Kinetic, spectroscopic, and crystallographic studies on some inner loop variants led us to conclude that charged residues flanking His-407 are important for stabilization/ordering of the inner loop thereby facilitating completion of the active site. The results further suggest that a disorder to order transition of the dynamic inner loop is essential for substrate entry to the active site, for sequestering active site chemistry from undesirable side reactions, as well as for communication between the E1 and E2 components of the E. coli pyruvate dehydrogenase multienzyme complex.

Highlights

Our crystallographic studies have shown that two active center loops of the E1 component of the Escherichia coli pyruvate dehydrogenase complex become organized only on binding a substrate analog that is capable of forming a stable thiamin diphosphate-bound covalent intermediate
We showed that residue His-407 on the inner loop has a key role in the mechanism, especially in the reductive acetylation of the E. coli dihydrolipoamide transacetylase component, whereas crystallographic results showed a role of this residue in a disorder-order transformation of these two loops, and the ordered conformation gives rise to numerous new contacts between the inner loop and the active center
The results further suggest that a disorder to order transition of the dynamic inner loop is essential for substrate entry to the active site, for sequestering active site chemistry from undesirable side reactions, as well as for communication between the E1 and E2 components of the E. coli pyruvate dehydrogenase multienzyme complex

Summary

EXPERIMENTAL PROCEDURES

Materials—Thiamin diphosphate, NADϩ, coenzyme A, dithiothreitol, and isopropyl 1-thio-␤-D-galactopyranoside were from U. It was exciting to 1-lipoyl E2ec vector transformed in E. coli BL21(DE3) cells was discover that in the crystal structure of E1ec in complex with used for overexpression of 1-lipoyl domain E2ec (1-lip E2ec) phosphonolactyl-ThDP (PLThDP), a stable analog of LThDP in [13] This single lipoyl E2ec construct is virtually indistinguishsupplemental Scheme I, both the inner and the outer loop (res- able in its biochemical behavior from the three-lipoyl wild type idues 541–557) become organized [11]. Fluorescence Measurements—The binding affinity of ThDP to E1ec and variants was measured by quenching of the intrinsic protein fluorescence by the coenzyme as described previously [15]. The initial model included 1602 amino acids without ThDP Both variant structures were refined using the program CNS [20]. Refinement statistics Resolution range R-factor (last shell) Rfree (last shell) No of residues No of waters

RESULTS

Substitution DCPIP Overall kcat

DISCUSSION