Biosynthesis of the Prosthetic Group of Citrate Lyase

Schneider, Karin; Dimroth, Peter; Bott, Michael

doi:10.1021/bi000401r

Cited by 45 publications

(53 citation statements)

References 42 publications

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“…Three additional gene products are involved in the maturation of the CL complex. CitG is involved in the modification of the CoA cofactor, CitX in the attachment of the prosthetic group to the ␥ subunit, and CitC in the activation of the group by catalyzing the initial acetylation (124).…”

Section: Physiological Functionmentioning

confidence: 99%

“…Since the latter is not present, it is likely that carbon dioxide is released freely by the ␣ subunit in F. nucleatum. Moreover, in the OAD complex the ␣ subunit is peripherally bound to the membrane through a specific interaction mediated by the ␥ subunit (oadG), an integral membrane protein (124,127). It follows that in F. nucleatum, OadA is a cytoplasmic enzyme that does not conserve the free energy released upon decarboxylation of oxaloacetate, in much the same way as the malic enzyme in C. perfringens ( Fig.…”

Section: Physiological Functionmentioning

confidence: 99%

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The 2-Hydroxycarboxylate Transporter Family: Physiology, Structure, and Mechanism

Sobczak

Lolkema

2005

Microbiol Mol Biol Rev

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SUMMARY The 2-hydroxycarboxylate transporter family is a family of secondary transporters found exclusively in the bacterial kingdom. They function in the metabolism of the di- and tricarboxylates malate and citrate, mostly in fermentative pathways involving decarboxylation of malate or oxaloacetate. These pathways are found in the class Bacillales of the low-CG gram-positive bacteria and in the gamma subdivision of the Proteobacteria. The pathways have evolved into a remarkable diversity in terms of the combinations of enzymes and transporters that built the pathways and of energy conservation mechanisms. The transporter family includes H+ and Na+ symporters and precursor/product exchangers. The proteins consist of a bundle of 11 transmembrane helices formed from two homologous domains containing five transmembrane segments each, plus one additional segment at the N terminus. The two domains have opposite orientations in the membrane and contain a pore-loop or reentrant loop structure between the fourth and fifth transmembrane segments. The two pore-loops enter the membrane from opposite sides and are believed to be part of the translocation site. The binding site is located asymmetrically in the membrane, close to the interface of membrane and cytoplasm. The binding site in the translocation pore is believed to be alternatively exposed to the internal and external media. The proposed structure of the 2HCT transporters is different from any known structure of a membrane protein and represents a new structural class of secondary transporters.

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Section: Physiological Functionmentioning

confidence: 99%

Section: Physiological Functionmentioning

confidence: 99%

The 2-Hydroxycarboxylate Transporter Family: Physiology, Structure, and Mechanism

Sobczak

Lolkema

2005

Microbiol Mol Biol Rev

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“…Note that this ratio is near unity for other fermentations with TCA pathway intermediates, which provides a clue to why citrate is ineffective. Addition of citrate may induce citrate lyase (18,30,39), an enzyme that cleaves citrate into an equimolar mixture of oxaloacetate and acetate. Oxaloacetate is readily metabolized to fumarate.…”

Section: Vol 68 2002 Flux Through Citrate Synthase Limits Growth 1075mentioning

confidence: 99%

Flux through Citrate Synthase Limits the Growth of EthanologenicEscherichia coliKO11 during Xylose Fermentation

Underwood

Buszko

Shanmugam

et al. 2002

Appl Environ Microbiol

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Previous studies have shown that high levels of complex nutrients (Luria broth or 5% corn steep liquor) were necessary for rapid ethanol production by the ethanologenic strain Escherichia coli KO11. Although this strain is prototrophic, cell density and ethanol production remained low in mineral salts media (10% xylose) unless complex nutrients were added. The basis for this nutrient requirement was identified as a regulatory problem created by metabolic engineering of an ethanol pathway. Cells must partition pyruvate between competing needs for biosynthesis and regeneration of NAD ؉ . Expression of low-K m Zymomonas mobilis pdc (pyruvate decarboxylase) in KO11 reduced the flow of pyruvate carbon into native fermentation pathways as desired, but it also restricted the flow of carbon skeletons into the 2-ketoglutarate arm of the tricarboxylic acid pathway (biosynthesis). In mineral salts medium containing 1% corn steep liquor and 10% xylose, the detrimental effect of metabolic engineering was substantially reduced by addition of pyruvate. A similar benefit was also observed when acetaldehyde, 2-ketoglutarate, or glutamate was added. In E. coli, citrate synthase links the cellular abundance of NADH to the supply of 2-ketoglutarate for glutamate biosynthesis. This enzyme is allosterically regulated and inhibited by high NADH concentrations. In addition, citrate synthase catalyzes the first committed step in 2-ketoglutarate synthesis. Oxidation of NADH by added acetaldehyde (or pyruvate) would be expected to increase the activity of E. coli citrate synthase and direct more carbon into 2-ketoglutarate, and this may explain the stimulation of growth. This hypothesis was tested, in part, by cloning the Bacillus subtilis citZ gene encoding an NADH-insensitive citrate synthase. Expression of recombinant citZ in KO11 was accompanied by increases in cell growth and ethanol production, which substantially reduced the need for complex nutrients.The engineering of metabolic pathways for production of industrial chemicals from renewable carbohydrates offers an alternative to petroleum-based processes and chemical synthesis (3,12,22). Genetically altering metabolic pathways, however, often causes unanticipated changes which may limit utility. Undesirable changes, such as reduced growth, decreased glycolytic flux, and low volumetric productivity, are generally attributed to a lack of ATP (19, 50), creation of futile cycles (10,11,37), changes in intracellular metabolite pools, or a metabolic imbalance (2,7,9,13,28,51,53). Often, these detrimental effects are masked by abundant complex nutrients in laboratory media and are apparent only in more minimal media (7,10,11,31).Salmonella enterica serovar Typhimurium was engineered for succinate production by increasing expression of pyc encoding pyruvate carboxylase (50). Although succinate production increased, the growth rate declined by 18% and the glycolytic flux decreased by 40%. Similar results were reported for an analogous construction in Escherichia coli (19). Donnelly and cow...

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“…It was recently demonstrated (29,30) that synthesis and attachment of the prosthetic group involve two reactions catalyzed by CitG and CitX (Fig. 2).…”

mentioning

confidence: 97%

Identification of a Gene Cluster in Klebsiella pneumoniae Which Includes citX , a Gene Required for Biosynthesis of the Citrate Lyase Prosthetic Group

et al. 2002

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The biosynthesis of the 2-(5؆-phosphoribosyl)-3-dephospho-coenzyme A (CoA) prosthetic group of citrate lyase (EC 4.1.3.6), a key enzyme of citrate fermentation, proceeds via the initial formation of the precursor 2-(5؆-triphosphoribosyl)-3-dephospho-CoA and subsequent transfer to apo-citrate lyase with removal of pyrophosphate. In Escherichia coli, the two steps are catalyzed by CitG and CitX, respectively, and the corresponding genes are part of the citrate lyase gene cluster, citCDEFXG. In the homologous citCDEFG operon of Klebsiella pneumoniae, citX is missing. A search for K. pneumoniae citX led to the identification of a second genome region involved in citrate fermentation which comprised the citWX genes and the divergent citYZ genes. The citX gene was confirmed to encode holo-citrate lyase synthase, whereas citW was shown to encode a citrate carrier, the third one identified in this species. The citYZ genes were found to encode a two-component system consisting of the sensor kinase CitY and the response regulator CitZ. Remarkably, both proteins showed >40% sequence identity to the citrate-sensing CitA-CitB two-component system, which is essential for the induction of the citrate fermentation genes in K. pneumoniae. A citZ insertion mutant was able to grow anaerobically with citrate, indicating that CitZ is not essential for expression of citrate fermentation genes. CitX synthesis was induced to a basal level under anaerobic conditions, independent of citrate, CitB, and CitZ, and to maximal levels during anaerobic growth with citrate as the sole carbon source. Similar to the other citrate fermentation enzymes, CitX synthesis was apparently subject to catabolite repression.Many species of enterobacteria, such as Klebsiella pneumoniae and Escherichia coli, are able to utilize citrate under anoxic, fermentative conditions. Whereas K. pneumoniae can grow with citrate as the sole carbon and energy source (for a review, see reference 6), E. coli is dependent on the presence of an oxidizable cosubstrate (18), due to the lack of oxaloacetate decarboxylase. The initial step in all known citrate fermentation pathways is the Mg 2ϩ -dependent cleavage of citrate to acetate and oxaloacetate, a reaction catalyzed by citrate lyase (2, 10, 33).In K. pneumoniae, the structural genes for citrate lyase are part of the citCDEFG operon, which is located divergent to citS (Fig. 1). The proteins deduced from citC, citD, citE, and citF are citrate lyase ligase and the ␥, ␤, and ␣ subunits of citrate lyase, respectively (7). The citC operon is induced under anoxic conditions in the presence of citrate and Na ϩ ions. Its expression is strictly dependent on the citrate-sensing CitACitB two-component regulatory system (8,16,20) and is subject to catabolite repression, presumably by the cyclic AMP receptor protein (19).In E. coli, a citCDEFXG gene cluster between 13.9 and 14.2 min (Fig. 1) that exhibited high similarity to the K. pneumoniae citrate lyase cluster (5) but differed by the presence of an additional gene, designated citX (...

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Biosynthesis of the Prosthetic Group of Citrate Lyase

Cited by 45 publications

References 42 publications

The 2-Hydroxycarboxylate Transporter Family: Physiology, Structure, and Mechanism

The 2-Hydroxycarboxylate Transporter Family: Physiology, Structure, and Mechanism

Flux through Citrate Synthase Limits the Growth of EthanologenicEscherichia coliKO11 during Xylose Fermentation

Identification of a Gene Cluster in Klebsiella pneumoniae Which Includes citX , a Gene Required for Biosynthesis of the Citrate Lyase Prosthetic Group

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