Branched-chain amino acid catabolism in bacteria.

Pseudomonas aeruginosa is a gram-negative, rod-shaped bacterium that belongs to the gamma-proteobacteria. This clinically challenging, opportunistic pathogen occupies a wide range of niches from an almost ubiquitous environmental presence to causing infections in a wide range of animals and plants. P. aeruginosa is the single most important pathogen of the cystic fibrosis (CF) lung. It causes serious chronic infections following its colonisation of the dehydrated mucus of the CF lung, leading to it being the most important cause of morbidity and mortality in CF sufferers. The recent finding that steep O2 gradients exist across the mucus of the CF-lung indicates that P. aeruginosa will have to show metabolic adaptability to modify its energy metabolism as it moves from a high O2 to low O2 and on to anaerobic environments within the CF lung. Therefore, the starting point of this review is that an understanding of the diverse modes of energy metabolism available to P. aeruginosa and their regulation is important to understanding both its fundamental physiology and the factors significant in its pathogenicity. The main aim of this review is to appraise the current state of knowledge of the energy generating pathways of P. aeruginosa. We first look at the organisation of the aerobic respiratory chains of P. aeruginosa, focusing on the multiple primary dehydrogenases and terminal oxidases that make up the highly branched pathways. Next, we will discuss the denitrification pathways used during anaerobic respiration as well as considering the ability of P. aeruginosa to carry out aerobic denitrification. Attention is then directed to the limited fermentative capacity of P. aeruginosa with discussion of the arginine deiminase pathway and the role of pyruvate fermentation. In the final part of the review, we consider other aspects of the biology of P. aeruginosa that are linked to energy metabolism or affected by oxygen availability. These include cyanide synthesis, which is oxygen-regulated and can affect the operation of aerobic respiratory pathways, and alginate production leading to a mucoid phenotype, which is regulated by oxygen and energy availability, as well as having a role in the protection of P. aeruginosa against reactive oxygen species. Finally, we consider a possible link between cyanide synthesis and the mucoid switch that operates in P. aeruginosa during chronic CF lung infection.

Oxygen, Cyanide and Energy Generation in the Cystic Fibrosis Pathogen Pseudomonas aeruginosa

Using both rational and random mutagenesis, we have created the first known broad substrate range, nicotinamide cofactor dependent, and highly stereoselective d-amino acid dehydrogenase. This new enzyme is capable of producing d-amino acids via the reductive amination of the corresponding 2-keto acid with ammonia. This biocatalyst was the result of three rounds of mutagenesis and screening performed on the enzyme meso-diaminopimelate d-dehydrogenase. The first round targeted the active site of the wild-type enzyme and produced mutants that were no longer strictly dependent on the native substrate. The second and third rounds produced mutants that had an increased substrate range including straight- and branched-aliphatic amino acids and aromatic amino acids. The very high selectivity towards the d-enantiomer (95 to > 99% e.e) was shown to be preserved even after the addition of the five mutations found in the three rounds of mutagenesis and screening. This new enzyme could complement and improve upon current methods for d-amino acid synthesis.

Creation of a broad-range and highly stereoselective D-amino acid dehydrogenase for the one-step synthesis of D-amino acids.

In the environment bacteria share their habitat with a great diversity of organisms, from microbes to humans, animals and plants. In these complex communities, the production of extracellular effec ...

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New insights into the mechanisms and biological roles of D-amino acids in complex eco-systems

Purification and properties of D-amino acid dehydrogenase, an inducible membrane-bound iron-sulfur flavoenzyme from Escherichia coli B.

A particulate d-amino acid dehydrogenase has been partially purified from cell free extracts of Pseudomonas aeruginosa grown on dl-valine as the source of carbon and energy A standard assay was developed which utilized 2,6-dichlorophenol-indophenol as the electron acceptor The pH optimum for enzyme activity ranged from 60 to 80, depending on the amino acid assayed The enzyme was most active with monoamino-monocarboxylic amino acids and histidine The Michaelis constant for d-phenylalanine was found to be 13 × 10-3m d-phenylalanine Constants could not be calculated for the other amino acids oxidized because anomalous plots of V as a function of V/S were obtained Spectra of enzyme preparations reduced with d-valine or sodium hydrosulfite exhibited adsorption bands typical of the α, β, and γ bands of cytochromes as well as bleaching in the flavin region of the spectrum When dl-valine was added to a medium with glycerol as the energy source, d-amino acid dehydrogenase was detected after the addition of valine and was produced at a rate directly proportional to the synthesis of total protein The enzyme was formed when d-valine, l-valine, or dl-alanine was the source of carbon and energy, but not when glucose, glycerol, or succinate was the energy source

Vincent P. Marshall

Papers

Oxidation of d-Amino Acids by a Particulate Enzyme from Pseudomonas aeruginosa