YhfJ: a Lipoyl transferase Involved in the Exogenous Protein Lipoylation Pathway in Bacillus subtilis

MARTIN, N.; DE MENDOZA D; MANSILLA MC

Rosario, Argentina

Congreso; V Congreso Argentino de Microbiología General; 2008

SAMIGE

YhfJ: a Lipoyl transferase Involved in the Exogenous ProteinLipoylation Pathway in Bacillus subtilisNatalia Martin1, Diego de Mendoza1, María C. Mansilla11 IBR, CONICET (nmartin@ibr.gov.ar)Lipoic acid (LA) is a sulfur-containing cofactor found in mostprokaryotic and eukaryotic organisms. In Escherichia coli andother organisms lipoic acid is essential for the function ofseveral key enzymes involved in oxidative and single carbonmetabolism including pyruvate dehydrogenase (PDH), 2-oxoglutarate dehydrogenase (2OGDH), branched-chain 2-oxoacid dehydrogenase (BCKADH), acetoin dehydrogenase(ADH) and the glycine cleavage system (GCS). The currentmodel for protein lipoylation in E. coli involves two pathways.The exogenous pathway, in which LA enters the cell bydiffusion and is attached to the apoproteins by lipoyl proteinligase A (LplA), provides a scavenging pathway for utilization ofexogenous LA. The endogenous pathway is the major route ofLA synthesis. It consists in two steps: the first one is thetransfer of octanoate from octanoyl-ACP to the 2-oxoacid lipoyldomains and to H protein catalyzed by octanoyl-[acyl carrierprotein]-protein transferase (LipB), and the second, is theconversion of octanoylated domains into lipoylated derivativesby lipoyl synthase (LipA), which catalyzes the insertion of sulfuratoms into the six- and eight-carbon positions of thecorresponding fatty acids. In bacteria, enzymes involved inlipoylation have gained increasing attention because of theirimplication in pathogenicity.We have previously constructed a Bacillus subtilis ¢ywfLmutant, NM51, which was dependent of the addition of LA forgrowth in minimal media. Its growth was also restored by theaddition of acetate and branched-chain fatty acid precursors,indicating that this strain was deficient in PDH and BCKADHactivities. However, 2OGDH activity is not affected in thismutant. NM51 sporulates poorly in Schaeffer´s sporulationmedium, and this phenotype was partially reverted by theaddition of LA. These results showed that YwfL is involved inthe endogenous protein lipoylation pathway in B. subtilis, soywfL was called lipL. The aim of this work was to identify theenzyme/s of B. subtilis involved in its exogenous lipoylationpathway. Analysis of the genome sequence revealed that thismicroorganism possess two ORFs which encode products withhomology to LplAs (yhfJ and yqhM). We constructed an yhfJnull mutant, NM60, which did not present any growth defectsin minimal media nor was deficient in sporulation, as expectedfor a lipoyl ligase. However, a lipL yhfJ double mutant, NM67,was unable to grow in minimal media even if LA was added.Addition of acetate and branched-chain fatty acid precursorspartially restored its growth, but the growth rate was muchslower than the observed for the wild type. Activities ofdehydrogenase complexes of NM51 and NM67 strains grownwith or without LA were measured. These results indicate thatYhfJ has lipoyl transferase activity which is responsible of thelipoylation of the dehydrogenase complex.