Cyclopropane fatty acid synthase of Escherichia coli: Deduced amino acid sequence, purification, and studies of the enzyme active site

Wang, Ai Yu; Grogan, Dennis W.; Cronan, John E.

doi:10.1021/bi00160a011

Cited by 89 publications

(76 citation statements)

References 45 publications

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“…The decrease in the conversion of C,,., to C,,:o,y, following induction of alkB, even in the stationary phase, is probably due to a lower CFA synthetase activity. This could result from a reduced access of the soluble CFA synthetase to its substrate in the lipid bilayer, possibly by 'shielding' of lipid by AlkB molecules, or it could be caused by inactivation of CFA synthetase, which has been shown to be an extremely labile enzyme (Wang et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

Overproduction of a Foreign Membrane Protein in Escherichia Coli Stimulates and Depends on Phospholipid Synthesis

Nieboer¹,

Vis²

1996

European Journal of Biochemistry

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When the Pseudomonas oleovorans alk system, consisting of the alkBFGHJKL and alkST genes, is expressed in Escherichia coli W3110, significant changes in phospholipid metabolism of the host are observed. A major role seems to be played by the cytoplasmic membrane protein alkane hydroxylase (AlkB), which is synthesized as up to 10-15% of the total protein in this strain [Nieboer, M., Kingma, J. & Witholt, B. (1993) The alkane oxidation system of Pseudomonas oleovorans: induction of the alk genes in Escherichia coli W311 O[pGEc47] affects membrane biogenesis and results in overexpression of alkane hydroxylase in a distinct cytoplasmic membrane subfraction, Mol. Microbiol. 8, 1039-10511. In the present paper, we have studied the link between synthesis of the membrane protein and the synthesis of phospholipids and fatty acids by examining the kinetics of these processes.Using [35S]methionine labeling, it was shown that induction of AlkB was maximal within 30-60 min after addition of inducer, when up to 15 % of all newly synthesized protein is AlkB. Phospholipid synthesis was followed by measuring the incorporation of 14C-labeled acetate and '3P-labeled phosphoric acid into phospholipids. Despite a negative effect of the inducer on the growth rate of W31?0[pGEc47], net phospholipid synthesis was significantly enhanced as a result of the expression of alkB. Synthesis of all three major phospholipids were stimulated to comparable extents by the induction of alkB. Induction did not increase "P incorporation into lipids in the control recombinant alk' strain which lacked alkB.Simultaneous with AlkB synthesis, the conversion of unsaturated 9-hexadecenoic acid (C,6 ,) into 9,lO-methylene hexadecanoic acid (C17 ocyc) was reduced in the alk' recombinant. Overall, these data show that the production of a foreign membrane protein in E. coli can engender a response of the phospholipid-synthesizing system of the host. In the absence of such a response, induction of the alk system would be much more toxic to the cells. Apparently, the increased phospholipid synthesis plays an important role in enabling the AlkB overproducing strain to grow.

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Section: Discussionmentioning

confidence: 99%

Overproduction of a Foreign Membrane Protein in Escherichia Coli Stimulates and Depends on Phospholipid Synthesis

Nieboer¹,

Vis²

1996

European Journal of Biochemistry

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“…The CFA synthase of E. coli has been well characterized (Huang et al, 2002;Wang et al, 1992) and the crystal structures of M. tuberculosis CFA synthases have been solved (Huang et al, 2002). In amino acid sequence alignments, Cfa1 shared 45 % amino acid sequence identity with Cfa2, and both these proteins shared 35-40 % amino acid identities with the Cfa proteins from E. coli, P. putida and M. tuberculosis proteins (Huang et al, 2002;MunozRojas et al, 2006;Wang et al, 1992).…”

Section: Nd* Nddmentioning

confidence: 99%

“…In amino acid sequence alignments, Cfa1 shared 45 % amino acid sequence identity with Cfa2, and both these proteins shared 35-40 % amino acid identities with the Cfa proteins from E. coli, P. putida and M. tuberculosis proteins (Huang et al, 2002;MunozRojas et al, 2006;Wang et al, 1992). There were several regions that are conserved across all the Cfa proteins.…”

Section: Nd* Nddmentioning

confidence: 99%

“…Cyclopropanation is a postsynthetic modification of phospholipids which reaches a maximum during the stationary growth phase (Grogan & Cronan, 1997). While CFA synthase in Escherichai coli has been well studied (Courtois et al, 2004;Courtois & Ploux, 2005;Wang et al, 1992), there appear to have been no studies as yet in S. meliloti. Two putative CFA synthases (SMc00358 and SMc02645) have been annotated as cfa1 and cfa2 genes in S. meliloti but no genetic or metabolite analyses have been conducted to support these functional assignments.…”

Section: Introductionmentioning

confidence: 99%

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Cyclopropane fatty acyl synthase in Sinorhizobium meliloti

et al. 2009

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Cyclopropane fatty acyl synthases (CFA synthases) are enzymes that catalyse the addition of a methylene group across cis double bonds of monounsaturated fatty acyl chains in lipids. We have investigated the function of two putative genes, cfa1 and cfa2, proposed to code for CFA synthases in Sinorhizobium meliloti. Total fatty acid composition and fatty acid distributions within lipid classes for wild-type and cfa1 and cfa2 mutant strains grown under P i starvation and in acidic culture conditions were obtained by GC/MS and by infusion ESI/MS/MS, respectively. For wildtype cells and the cfa1 mutant, total cyclopropane fatty acids (CFAs) increased by 10 % and 15 % under P i starvation and acidic conditions, respectively; whereas in the cfa2 mutant, CFAs were less than 0.1 % of wild-type under both growth conditions. Reporter gene fusion experiments revealed that cfa1 and cfa2 were expressed at similar levels in free-living cells. Thus under the conditions we examined, cfa2 was required for the cyclopropanation of lipids in S. meliloti whereas the role of cfa1 remains to be determined. Analysis of intact lipids revealed that cyclopropanation occurred on cis-11-octadecenoic acid located in either the sn-1 or the sn-2 position in phospholipids and that cyclopropanation in the sn-2 position occurred to a greater extent in phosphatidylcholines and sulfoquinovosyldiacylglycerols under acidic conditions than under P i starvation. The cfa2 gene was also required for cyclopropanation of non-phosphoruscontaining lipids. Principal components analysis revealed no differences in the cyclopropanation of four lipid classes. We concluded that cyclopropanation occurred independently of the polar head group. Neither cfa1 nor cfa2 was required for symbiotic nitrogen fixation.

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“…There are at least three mycolic acid cyclopropane synthases (CmaA1, PcaA and CmaA2) that are responsible for the site-specific modifications of mycolic acids. The first one to be identified was CmaA1, based on its homology to the E. coli enzyme CFA synthase (Wang et al, 1992). At least seven homologous genes have been identified in the genome sequence of H37Rv .…”

Section: Cell Wallmentioning

confidence: 99%

Comparative proteome analysis of Mycobacterium tuberculosis grown under aerobic and anaerobic conditions

Starck

Källenius

Marklund³

et al. 2004

Microbiology

135

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Data are presented from two-dimensional (2-D) PAGE analysis of Mycobacterium tuberculosis strain Harlingen grown during aerobic and anaerobic culture, according to a modified Wayne dormancy model. M. tuberculosis cultures were grown to the transition point between exponential growth and stationary phase in the presence of oxygen (7 days) and then part of the cultures was shifted to anaerobic conditions for 16 days. Growth declined similarly during aerobic and anaerobic conditions, whereas the ATP consumption rapidly decreased in the anaerobic cultures. 2-D PAGE revealed 50 protein spots that were either unique to, or more abundant during, anaerobic conditions and 16 of these were identified by MALDI-TOF. These proteins were the a-crystalline homologue (HspX), elongation factor Tu (Tuf), GroEL2, succinyl-CoA : 3-oxoacid-CoA transferase (ScoB), mycolic acid synthase (CmaA2), thioredoxin (TrxB2), b-ketoacyl-ACP synthase (KasB), L-alanine dehydrogenase (Ald), Rv2005c, Rv2629, Rv0560c, Rv2185c and Rv3866. Some protein spots were found to be proteolytic fragments, e.g. HspX and GroEL2. These data suggest that M. tuberculosis induces expression of about 1 % of its genes in response to dormancy. INTRODUCTIONHuman tuberculosis (TB) is caused by an intracellular pathogen, Mycobacterium tuberculosis, which causes both latent and acute illness. Approximately eight million active cases are reported annually with 2 million deaths. In addition, about one-third of the global population is estimated to suffer from latent tuberculosis (Dye et al., 1999), which can be reactivated even after several decades. The fact that the bacilli can shift into a dormant state is of therapeutic importance, since this dormant state induces resistance to the two most important TB-drugs, rifampicin and isoniazide (Wayne & Sramek, 1994). One of the most challenging problems in TB research is to reveal the mechanisms behind latency.Latency is believed to involve a non-replicating persistence of mycobacteria or a very slow growth. One important condition believed to contribute to latency is reduced access to oxygen. M. tuberculosis is generally regarded as a strictly aerobic bacillus, although it can survive for a long time in a micro-aerophilic environment as long as the shift is not too abrupt (Wayne & Hayes, 1996). An in vitro model to study this persistent state is the so-called Wayne dormancy model, in which the bacteria downregulate their metabolism due to reduced access to oxygen (Wayne & Hayes, 1996). Another dormancy model utilizes nutrient starvation to induce a state where M. tuberculosis arrests growth and decreases its respiration rate (Betts et al., 2002). Recently it was demonstrated that inhibition of respiration by nitric oxide might induce a dormant state in M. tuberculosis . One should recognize, however, that the evidence linking in vitro dormant states of M. tuberculosis to human latent infection still remains circumstantial.The vaccine strain Mycobacterium bovis BCG has been reported to occasionally persist in a latent state in ...

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Cyclopropane fatty acid synthase of Escherichia coli: Deduced amino acid sequence, purification, and studies of the enzyme active site

Cited by 89 publications

References 45 publications

Overproduction of a Foreign Membrane Protein in Escherichia Coli Stimulates and Depends on Phospholipid Synthesis

Overproduction of a Foreign Membrane Protein in Escherichia Coli Stimulates and Depends on Phospholipid Synthesis

Cyclopropane fatty acyl synthase in Sinorhizobium meliloti

Comparative proteome analysis of Mycobacterium tuberculosis grown under aerobic and anaerobic conditions

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