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Identification of apolipoprotein n-acyltransferase (LNT) in mycobacteria


Tschumi, A; Nai, C; Auchli, Y; Hunziker, P; Gehrig, P; Keller, P; Grau, T; Sander, P (2009). Identification of apolipoprotein n-acyltransferase (LNT) in mycobacteria. Journal of Biological Chemistry, 284(40):27146-27156.

Abstract

Lipoproteins of Gram-negative and Gram-positive bacteria carry a thioether bound diacylglyceryl but differ by a fatty acid amide-bound to the alpha-amino group of the universally conserved cysteine. In Escherichia coli the N-terminal acylation is catalyzed by the N-acyltransferase Lnt. Using E. coli Lnt as a query in a BLASTp search, we identified putative lnt genes also in Gram-positive mycobacteria. The Mycobacterium tuberculosis lipoprotein LppX, heterologously expressed in Mycobacterium smegmatis, was N-acylated at the N-terminal cysteine, whereas LppX expressed in a M. smegmatis lnt::aph knock-out mutant was accessible for N-terminal sequencing. Western blot analyses of a truncated and tagged form of LppX indicated a smaller size of about 0.3 kDa in the lnt::aph mutant compared to the parental strain. MALDI-TOF/TOF analyses of a trypsin digest of LppX proved the presence of the diacylglyceryl modification in both strains, parental strain and lnt::aph mutant. N-acylation was found exclusively in the M. smegmatis parental strain. Complementation of the lnt::aph mutant with M. tuberculosis ppm1 restored N-acylation. The substrate for N-acylation is a C16 fatty acid while the two fatty acids of the diacylglyceryl residue were identified as C16 and C19:0 fatty acid, the latter most likely tuberculostearic acid. We demonstrate that mycobacterial lipoproteins are triacylated. For the first time to our knowledge, we identify Lnt activity in Gram-positive bacteria and assigned the responsible genes. In M. smegmatis and M. tuberculosis the open reading frames are annotated as MSMEG_3860 and M. tuberculosis ppm1, respectively.

Lipoproteins of Gram-negative and Gram-positive bacteria carry a thioether bound diacylglyceryl but differ by a fatty acid amide-bound to the alpha-amino group of the universally conserved cysteine. In Escherichia coli the N-terminal acylation is catalyzed by the N-acyltransferase Lnt. Using E. coli Lnt as a query in a BLASTp search, we identified putative lnt genes also in Gram-positive mycobacteria. The Mycobacterium tuberculosis lipoprotein LppX, heterologously expressed in Mycobacterium smegmatis, was N-acylated at the N-terminal cysteine, whereas LppX expressed in a M. smegmatis lnt::aph knock-out mutant was accessible for N-terminal sequencing. Western blot analyses of a truncated and tagged form of LppX indicated a smaller size of about 0.3 kDa in the lnt::aph mutant compared to the parental strain. MALDI-TOF/TOF analyses of a trypsin digest of LppX proved the presence of the diacylglyceryl modification in both strains, parental strain and lnt::aph mutant. N-acylation was found exclusively in the M. smegmatis parental strain. Complementation of the lnt::aph mutant with M. tuberculosis ppm1 restored N-acylation. The substrate for N-acylation is a C16 fatty acid while the two fatty acids of the diacylglyceryl residue were identified as C16 and C19:0 fatty acid, the latter most likely tuberculostearic acid. We demonstrate that mycobacterial lipoproteins are triacylated. For the first time to our knowledge, we identify Lnt activity in Gram-positive bacteria and assigned the responsible genes. In M. smegmatis and M. tuberculosis the open reading frames are annotated as MSMEG_3860 and M. tuberculosis ppm1, respectively.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Medical Microbiology
04 Faculty of Medicine > Functional Genomics Center Zurich
08 University Research Priority Programs > Systems Biology / Functional Genomics
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:6 October 2009
Deposited On:05 Oct 2009 13:06
Last Modified:05 Oct 2016 07:37
Publisher:American Society for Biochemistry and Molecular Biology
ISSN:0021-9258
Additional Information:This research was originally published in Tschumi, A; Nai, C; Auchli, Y; Hunziker, P; Gehrig, P; Keller, P; Grau, T; Sander, P (2009). Identification of apolipoprotein n-acyltransferase (LNT) in mycobacteria. Journal of Biological Chemistry, 284(40):27146-27156. © the American Society for Biochemistry and Molecular Biology
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1074/jbc.M109.022715
PubMed ID:19661058
Permanent URL: https://doi.org/10.5167/uzh-21082

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