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Crystal structures of ethanolamine ammonia-lyase complexed with coenzyme B12 analogs and substrates.

ABSTRACT: N-terminal truncation of the Escherichia coli ethanolamine ammonia-lyase beta-subunit does not affect the catalytic properties of the enzyme (Akita, K., Hieda, N., Baba, N., Kawaguchi, S., Sakamoto, H., Nakanishi, Y., Yamanishi, M., Mori, K., and Toraya, T. (2010) J. Biochem. 147, 83-93). The binary complex of the truncated enzyme with cyanocobalamin and the ternary complex with cyanocobalamin or adeninylpentylcobalamin and substrates were crystallized, and their x-ray structures were analyzed. The enzyme exists as a trimer of the (alphabeta)(2) dimer. The active site is in the (beta/alpha)(8) barrel of the alpha-subunit; the beta-subunit covers the lower part of the cobalamin that is bound in the interface of the alpha- and beta-subunits. The structure complexed with adeninylpentylcobalamin revealed the presence of an adenine ring-binding pocket in the enzyme that accommodates the adenine moiety through a hydrogen bond network. The substrate is bound by six hydrogen bonds with active-site residues. Argalpha(160) contributes to substrate binding most likely by hydrogen bonding with the O1 atom. The modeling study implies that marked angular strains and tensile forces induced by tight enzyme-coenzyme interactions are responsible for breaking the coenzyme Co-C bond. The coenzyme adenosyl radical in the productive conformation was modeled by superimposing its adenine ring on the adenine ring-binding site followed by ribosyl rotation around the N-glycosidic bond. A major structural change upon substrate binding was not observed with this particular enzyme. Glualpha(287), one of the substrate-binding residues, has a direct contact with the ribose group of the modeled adenosylcobalamin, which may contribute to the substrate-induced additional labilization of the Co-C bond.

SUBMITTER: Shibata N

PROVIDER: S-EPMC2924083 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Crystal structures of ethanolamine ammonia-lyase complexed with coenzyme B12 analogs and substrates.

Shibata Naoki N Tamagaki Hiroko H Hieda Naoki N Akita Keita K Komori Hirofumi H Shomura Yasuhito Y Terawaki Shin-Ichi S Mori Koichi K Yasuoka Noritake N Higuchi Yoshiki Y Toraya Tetsuo T

The Journal of biological chemistry 20100601 34

N-terminal truncation of the Escherichia coli ethanolamine ammonia-lyase beta-subunit does not affect the catalytic properties of the enzyme (Akita, K., Hieda, N., Baba, N., Kawaguchi, S., Sakamoto, H., Nakanishi, Y., Yamanishi, M., Mori, K., and Toraya, T. (2010) J. Biochem. 147, 83-93). The binary complex of the truncated enzyme with cyanocobalamin and the ternary complex with cyanocobalamin or adeninylpentylcobalamin and substrates were crystallized, and their x-ray structures were analyzed. ...[more]

PMID: 20519496

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Project description:The adenosylcobalamin- (coenzyme B12) dependent ethanolamine ammonia-lyase (EAL) plays a key role in aminoethanol metabolism, associated with microbiome homeostasis and Salmonella- and Escherichia coli-induced disease conditions in the human gut. To gain molecular insight into these processes toward development of potential therapeutic targets, reactions of the cryotrapped (S)-2-aminopropanol substrate radical EAL from Salmonella typhimurium are addressed over a temperature (T) range of 220-250 K by using T-step reaction initiation and time-resolved, full-spectrum electron paramagnetic resonance spectroscopy. The observed substrate radical reaction kinetics are characterized by two pairs of biexponential processes: native decay to diamagnetic products and growth of a non-native radical species and Co(II) in cobalamin. The multicomponent low-T kinetics are simulated by using a minimal model, in which the substrate-radical macrostate, S?, is partitioned by a free-energy barrier into two sequential microstates: 1) S1?, a relatively high-entropy/high-enthalpy microstate with a protein configuration that captures the nascent substrate radical in the terminal step of radical-pair separation; and 2) S2?, a relatively low-enthalpy/low-entropy microstate with a protein configuration that enables the rearrangement reaction. The non-native, destructive reaction of S1? at T ? 250 K is caused by a prolonged lifetime in the substrate-radical capture state. Monotonic S? decay over 278-300 K indicates that the free-energy barrier to S1? and S2? interconversion is latent at physiological T-values. Overall, the low-temperature studies reveal two protein-configuration microstates and connecting protein-configurational transitions that specialize the S? macrostate for the dual functional roles of radical capture and rearrangement enabling. The identification of new, to our knowledge, intermediate states and specific protein-fluctuation contributions to the reaction coordinate represent an advance toward development of novel therapeutic targets in EAL.

Project description:The decay kinetics of the aminoethanol-generated Co(II)-substrate radical pair catalytic intermediate in ethanolamine ammonia-lyase from Salmonella typhimurium have been measured on timescales of <10(5) s in frozen aqueous solution from 190 to 217 K. X-band continuous-wave electron paramagnetic resonance (EPR) spectroscopy of the disordered samples has been used to continuously monitor the full radical pair EPR spectrum during progress of the decay after temperature step reaction initiation. The decay to a diamagnetic state is complete and no paramagnetic intermediate states are detected. The decay exhibits three kinetic regimes in the measured temperature range, as follows. i), Low temperature range, 190 < or = T < or = 207 K: the decay is biexponential with constant fast (0.57 +/- 0.04) and slow (0.43 +/- 0.04) phase amplitudes. ii), Transition temperature range, 207 < T < 214 K: the amplitude of the slow phase decreases to zero with a compensatory rise in the fast phase amplitude, with increasing temperature. iii), High temperature range, T > or = 214 K: the decay is monoexponential. The observed first-order rate constants for the monoexponential (k(obs,m)) and the fast phase of the biexponential decay (k(obs,f)) adhere to the same linear relation on an lnk versus T(-1) (Arrhenius) plot. Thus, k(obs,m) and k(obs,f) correspond to the same apparent Arrhenius prefactor and activation energy (logA(app,f) (s(-1)) = 13.0, E(a,app,f) = 15.0 kcal/mol), and therefore, a common decay mechanism. We propose that k(obs,m) and k(obs,f) represent the native, forward reaction of the substrate through the radical rearrangement step. The slow phase rate constant (k(obs,s)) for 190 < or = T < or = 207 K obeys a different linear Arrhenius relation (logA(app,s) (s(-1)) = 13.9, E(a,app,s) = 16.6 kcal/mol). In the transition temperature range, k(obs,s) displays a super-Arrhenius increase with increasing temperature. The change in E(a,app,s) with temperature and the narrow range over which it occurs suggest an origin in a liquid/glass or dynamical transition. A discontinuity in the activation barrier for the chemical reaction is not expected in the transition temperature range. Therefore, the transition arises from a change in the properties of the protein. We propose that a protein dynamical contribution to the reaction, which is present above the transition temperature, is lost below the transition temperature, owing to an increase in the activation energy barrier for protein motions that are coupled to the reaction. For both the fast and slow phases of the low temperature decay, the dynamical transition in protein motions that are obligatorily coupled to the reaction of the Co(II)-substrate radical pair lies below 190 K.

Dataset Information

Crystal structures of ethanolamine ammonia-lyase complexed with coenzyme B12 analogs and substrates.

Publications

Crystal structures of ethanolamine ammonia-lyase complexed with coenzyme B12 analogs and substrates.

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