George D. Hegeman

Oxidation of carbon monoxide by bacteria.

Publisher Summary This chapter discusses the oxidation of carbon monoxide (CO) by bacteria. The CO generated in soil and in the lower layers of the atmosphere is oxidized to CO 2 locally by biological agents, principally microbes. CO is added to the atmosphere in significant amounts through the incomplete combustion of fossil fuels and by atmospheric reactions. Bacteria that oxidize CO may be subdivided according to their ability to use CO as an energy source for growth (utilitarian oxidation) or based on whether the oxidation process is a gratuitous one (nonutilitarian oxidation) resulting from the acceptance of CO as a pseudosubstrate for an enzyme system evolved to catalyze another process. CO is inhibitory for all aerobic respiratory organisms. Even in aerobic carboxydobacteria, the high concentrations of CO reduce the growth rate and cellular yield, thereby indicating that CO tolerance is a necessary part of the ability to use CO at higher concentrations. Phototrophic bacteria may remove significant amounts of CO formed during the degradation of photosynthetic pigments in decaying vegetative materials in anaerobic sediments. The chapter also discusses the applications of the bacterial CO oxidation.

Selective disadvantage of non-functional protein synthesis inEscherichia coli

SummaryComparison of growth rates of isogenic strains that synthesize varying levels ofβ-galactosidase during continuous culture on non-inducing medium indicates that synthesis of low levels of non-functional protein has a small but possibly significant effect upon growth rate.

A birect kinetic assay for mandelate racemase using circular dichroic measurements

Abstract A new, direct kinetic assay for mandelate racemase has been developed. The assay depends upon changes with time in circular dichroic ellipticities of mandelate solutions at 262 nm. The technique should allow more precise kinetic measurements of the mandelate racemase reaction than have heretofore been possible. Also, the approach to equilibrium can be measured from either ( R )- or ( S )-mandelate with equal facility. The technique, should, moreover, be extendible to other enzyme-catalyzed reactions involving changes with time in circular dichroic ellipticities.

The effect of a non-metabolizable analog on mandelate catabolism in Pseudomonas putida.

Dl-2,3,4,5,6-pentafluoromandelic acid (PFM) specifically inhibits the growth of Pseudomonas putida (ATCC 12633) on medium containing mandelate as sole carbon and energy source by competitive inhibition of mandelate dehydrogenase. PFM is not metabolized and is neither an inducer of the mandelate catabolic enzymes nor an antagonist of induction. Mutants resistant to the inhibitory effects of PFM (PFMr) were isolated; most prove to be superinducible, i.e. synthesize coordinately the mandelatespecific catabolic enzymes at elevated levels following induction. In at least one case the PFMr mutation maps very near the structural genes that encode the enzymes functional in the first two steps of mandelate catabolism. It is reasoned that the PFMr mutation is of the promotor type. Resistance to substrate analogs such as PFM offers a general method for isolation of regulatory mutants in catabolic metabolism.

Benzyl alcohol metabolism by Pseudomonas putida: a paradox resolved

Evidence is presented for the existence in Pseudomonas putida of two NAD-linked dehydrogenases that function sequentially to oxidize benzyl alcohol. Induction of muconate lactonizing enzyme, a 3-oxoadipate pathway enzyme, indicated that P. putida oxidized benzyl alcohol to benzoate. Polyacrylamide gel electrophoresis with activity staining and enzymatic assays for an NAD-dependent dehydrogenase both showed that cells contained a single, constitutive alcohol dehydrogenase capable of oxidizing benzyl alcohol. This enzyme was shown to have the same specificity in extracts of glucose-grown as in benzy alcoholgrown cells. An NAD-aldehyde dehydrogenase oxidized benzaldehyde but was most active with normal alkyl aldehydes. This aldehyde dehydrogenase was shown to be induced, by enzymatic assays and by activity staining of polyacrylamide gel electropherograms, not only in cells grown on benzyl alcohol, but also in cells grown on ethanol. These experiments suggested that the aldehyde dehydrogenase was induced by the alcohol being oxidized rather than the substrate aldehyde.In sum, the evidence from enzyme assays and polyacrylamide gel electrophoresis of extracts indicates that Pseudomonas putida catabolizes benzyl alcohol slowly when it is the sole carbon and energy source, by the action of a constitutive, nonspecific, alcohol dehydrogenase and an alcohol-induced, nonspecific aldehyde dehydrogenase to yield benzoate, which is further metabolized via the 3-oxoadipate (beta-ketoadipate) pathway.

A method for measuring natural abundance intramolecular stable carbon isotopic distributions in malic acid

Abstract A method for the quantitative conversion of the individual carbon atoms of l -malic acid to CO 2 for high-precision carbon isotope ratio determination is presented. Malic acid is decarboxylated sequentially at C-4 and C-1 using malic enzyme and pyruvate decarboxylase. The acetaldehyde remaining following these reactions is oxidized to acetic acid, which is analyzed by pyrolysis and/or combustion. These methods permit measurement of the natural abundance of 13 C in the individual carbon atoms of malic acid.

[62] Mandelate racemase

Publisher Summary This chapter explains Mandelate racemase (EC 5.1.2.2) of Pseudomonas putida. It catalyzes the reversible interconversion of the D and L enantiomers of mandelate. The enzyme has been isolated in a high state of purity and has been shown to be a tetramer composed of identieal subunits, each with a molecular weight of 69,500. The enzyme acts without flavin or pyridine nucleotide cofactor, but has an absolute divalent metal ion requirement for activity. Mechanistic studies have indicated that the hydrogen alpha to the carboxylate is removed as a proton in the course of the enzyme-catalyzed racemization, generating a carbanion intermediate; the intermediate can be represented by the resonance structures. It followed that a basic group on the enzyme itself was participating in the generation of the carbanion intermediate by abstracting the a-hydrogen from mandelate. In an attempt to identify such a basic group on the enzyme, structural analogs of mandelate were sought that could also serve as alkylating agents. The chapter further explains the design of the affinity labeling reagent, synthesis of radioactivity labeled affinity label, determination of the stoichiometry of binding of the affinity label to the racemase, kinetic studies of the irreversible inhibition, effect of divalent metal ion on the affinity labeling process and attempts to identify the residue of the racemase alkylated by the affinity label.