Dataset Information

Disruption of the proton relay network in the class 2 dihydroorotate dehydrogenase from Escherichia coli.

ABSTRACT: Dihydroorotate dehydrogenases (DHODs) are FMN-containing enzymes that catalyze the conversion of dihydroorotate (DHO) to orotate in the de novo synthesis of pyrimidines. During the reaction, a proton is transferred from C5 of DHO to an active site base and the hydrogen at C6 of DHO is transferred to N5 of the isoalloxazine ring of the flavin as a hydride. In class 2 DHODs, a hydrogen bond network observed in crystal structures has been proposed to deprotonate the C5 atom of DHO. The active site base (Ser175 in the Escherichia coli enzyme) hydrogen bonds to a crystallographic water molecule that sits on a phenylalanine (Phe115 in the E. coli enzyme) and hydrogen bonds to a threonine (Thr178 in the E. coli enzyme), residues that are conserved in class 2 enzymes. The importance of these residues in the oxidation of DHO was investigated using site-directed mutagenesis. Mutating Ser175 to alanine had severe effects on the rate of flavin reduction, slowing it by more than 3 orders of magnitude. Changing the size and/or hydrophobicity of the residues of the hydrogen bond network, Thr178 and Phe115, slowed flavin reduction as much as 2 orders of magnitude, indicating that the active site base and the hydrogen bond network work together for efficient deprotonation of DHO.

SUBMITTER: Kow RL

PROVIDER: S-EPMC2780000 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Disruption of the proton relay network in the class 2 dihydroorotate dehydrogenase from Escherichia coli.

Kow Rebecca L RL Whicher Jonathan R JR McDonald Claudia A CA Palfey Bruce A BA Fagan Rebecca L RL

Biochemistry 20091001 41

Dihydroorotate dehydrogenases (DHODs) are FMN-containing enzymes that catalyze the conversion of dihydroorotate (DHO) to orotate in the de novo synthesis of pyrimidines. During the reaction, a proton is transferred from C5 of DHO to an active site base and the hydrogen at C6 of DHO is transferred to N5 of the isoalloxazine ring of the flavin as a hydride. In class 2 DHODs, a hydrogen bond network observed in crystal structures has been proposed to deprotonate the C5 atom of DHO. The active site ...[more]

PMID: 19694481

Similar Datasets

Project description:The bacterial lifestyle is plastic, requiring transcriptional, translational, and metabolic tailoring for survival. These dynamic cellular processes are energy intensive; therefore, flexible energetics is requisite for adaptive plasticity. An intricate network of complementary and supplementary pathways exists in bacterial energy metabolism. There are two main entry points for electrons in the aerobic electron transport system, NADH dehydrogenase (NDH) and succinate dehydrogenase (SDH), receiving electrons from NADH and succinate, respectively. Aerobic bacterial phyla have a non-proton-pumping NADH dehydrogenase, which is often the primary dehydrogenase under aerobiosis. Here, we report adaptive changes supporting growth restoration in an Escherichia coli strain lacking the primary dehydrogenase. Growth optimization is achieved by reducing the activity of succinate dehydrogenase, and thus we demonstrate a physiological discord between proton-pumping NADH dehydrogenase and succinate dehydrogenase in supporting growth. Beyond the fundamental understanding of the bioenergetic network, identifying this compensatory feature provides impetus to rational antimicrobial combinations for targeting the non-proton-pumping dehydrogenase. IMPORTANCE Energy generation pathways are a potential avenue for the development of novel antibiotics. However, bacteria possess remarkable resilience due to the compensatory pathways, which presents a challenge in this direction. NADH, the primary reducing equivalent, can transfer electrons to two distinct types of NADH dehydrogenases. Type I NADH dehydrogenase is an enzyme complex comprising multiple subunits and can generate proton motive force (PMF). Type II NADH dehydrogenase does not pump protons but plays a crucial role in maintaining the turnover of NAD+. To study the adaptive rewiring of energy metabolism, we evolved an Escherichia coli mutant lacking type II NADH dehydrogenase. We discovered that by modifying the flux through the tricarboxylic acid (TCA) cycle, E. coli could mitigate the growth impairment observed in the absence of type II NADH dehydrogenase. This research provides valuable insights into the intricate mechanisms employed by bacteria to compensate for disruptions in energy metabolism.

Dataset Information

Disruption of the proton relay network in the class 2 dihydroorotate dehydrogenase from Escherichia coli.

Publications

Disruption of the proton relay network in the class 2 dihydroorotate dehydrogenase from Escherichia coli.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets