Purification, general properties and two other catalytic activities of α-ketoglutarate:glyoxylate carboligase of Mycobacterium phlei

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Abstract

  • 1.

    1. An α-ketoglutarate; glyoxylate carboligase was purified 200- to 250-fold from the sonic extracts of Mycobacterium phlei by (NH4)2 SO4 fractionation, pH precipitation, starch block electrophoresis and column chromatography.

  • 2.

    2. From studies on decarboxylation from 14C-labelled substrates, the enzyme seemed to catalyze the condensation of α-ketoglutarate and glyoxylate to form α-hydroxy-β-ketoadipic acid in the presence of thiamine pyrophosphate. α-Hydroxy-β-ketoadipic acid was spontaneously decarboxylated to δ-hydroxylevulinic acid in the presence of acid.

  • 3.

    3. The enzyme had an optimum pH of 6.3 in potassium phosphate buffer at 37°. Km values for α-ketoglutarate and glyoxylate were 2.0 mM and 3.2 mM, respectively. The isoelectric point, determined by isoelectric focusing, was 5.6.

  • 4.

    4. The enzyme activity was markedly activated by Mn2+ and was activated to a small extent by Mg2+

  • 5.

    5. π-Chloromercuribenzene sulfonic acid, monoiodoacetic acid. EDTA and Zn2+ inhibited the enzyme activity.

  • 6.

    6. The purified enzyme catalyzed α-ketoglutarate decarboxylase and α-ketoglutarate:acetaldehyde carboligase activities as well as α-ketoglutarate:glyoxylate carboligase activity.

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    To our knowledge, no carboligase or decarboxylase activities had been previously reported for the E1o component of the E. coli KDH complex. Nevertheless, our findings are consistent with the biochemical detection of these different ThDP-dependent activities in cell extracts from a number of bacterial species, including E. coli and mycobacteria (Yamasaki and Moriyama, 1970, 1971), and can also account for early reports describing multiple activities associated with bacterial and mammalian KDH complexes (Bunik and Fernie, 2009; O'Fallon and Brosemer, 1977; Schlossberg et al., 1970). The FHA domain-containing protein GarA has been recently identified as a phospho-dependent regulator of metabolic enzymes (including KGD) involved in glutamate synthesis under control of the Ser/Thr protein kinase PknG in both corynebacteria and mycobacteria (Niebisch et al., 2006; Bott, 2007; O'Hare et al., 2008; Nott et al., 2009).

  • Activity-Based Metabolomic Profiling of Enzymatic Function: Identification of Rv1248c as a Mycobacterial 2-Hydroxy-3-oxoadipate Synthase

    2010, Chemistry and Biology
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    HOAS activity (EC 2.2.1.5) has been described as a side reaction of the E1 component of KDH complex that occurs in the absence of a functional KDH complex (O'Fallon and Brosemer, 1977; Schlossberg et al., 1970). Before DNA sequencing and cloning were widespread, HOA and HLA were detected in animals, plants, fungi, and bacteria (O'Fallon and Brosemer, 1977; Saito et al., 1971; Schlossberg et al., 1970; Yamasaki and Moriyama, 1970a, 1971), where it was speculated that they might be involved in GLX detoxification or synthesis of secondary metabolites. Moreover, early studies of protein extracts of Mycobacterium phlei identified the synthesis of HOA and its conversion to HLA, which was dependent on α-KG, GLX, TDP, and Mg2+, though the gene or protein encoding this activity was never assigned (Yamasaki and Moriyama, 1970b, 1971).

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