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Analysis of gluconeogenic and anaplerotic enzymes in Campylobacter jejuni: an essential role for phosphoenolpyruvate carboxykinase

Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK¹

Author for correspondence: David J. Kelly. Tel: +44 114 224414. Fax: +44 114 2728697. e-mail: d.kelly{at}sheffield.ac.uk

Campylobacter jejuni is unable to utilize glucose as a carbon source due to the absence of the key glycolytic enzyme 6-phosphofructokinase. The genome sequence of C. jejuni NCTC 11168 indicates that homologues of all the appropriate enzymes for gluconeogenesis from phosphoenolpyruvate (PEP) are present, in addition to the anaplerotic enzymes pyruvate carboxylase (PYC), phosphoenolpyruvate carboxykinase (PCK) and malic enzyme (MEZ). Surprisingly, a pyruvate kinase (PYK) homologue is also present. To ascertain the role of these enzymes, insertion mutants in pycA, pycB, pyk and mez were generated. However, this could not be acheived for pckA, indicating that PCK is an essential enzyme in C. jejuni. The lack of PEP synthase and pyruvate orthophosphate dikinase activities confirmed a unique role for PCK in PEP synthesis. The pycA mutant was unable to grow in defined medium with pyruvate or lactate as the major carbon source, thus indicating an important role for PYC in anaplerosis. Sequence and biochemical data indicate that the PYC of C. jejuni is a member of the ₄ß₄, acetyl-CoA-independent class of PYCs, with a 65·8 kDa subunit containing the biotin moiety. Whereas growth of the mez mutant was comparable to that of the wild-type, the pyk mutant displayed a decreased growth rate in complex medium. Nevertheless, the mez and pyk mutants were able to grow with pyruvate, lactate or malate as carbon sources in defined medium. PYK was present in cell extracts at a much higher specific activity [>800 nmol min^-1 (mg protein)^-1] than PYC or PCK [<65 nmol min^-1 (mg protein)^-1], was activated by fructose 1,6-bisphosphate and displayed other regulatory properties strongly indicative of a catabolic role. It is concluded that PYK may function in the catabolism of unidentified substrates which are metabolized through PEP. In view of the high K_m of MEZ for malate (9 mM) and the lack of a growth phenotype of the mez mutant, MEZ seems to have only a minor anaplerotic role in C. jejuni.

Keywords: pyruvate carboxylase, pyruvate kinase, malic enzyme

Abbreviations: CAC, citric acid cycle; F1,6-BP, fructose 1,6-bisphosphate; G6-P, glucose 6-phosphate; MEZ, malic enzyme; OAA, oxaloacetate; PCK, phosphoenolpyruvate carboxykinase; PEP, phosphoenolpyruvate; PYC, pyruvate carboxylase; PYK, pyruvate kinase

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