Open reading frame 3, which is adjacent to the mycocerosic acid synthase gene, is expressed as an acyl coenzyme A synthase in Mycobacterium bovis BCG

Abstract

The aim of this study was to test for expression of a 900-bp open reading frame (ORF), ORF3, located at the 5 end of the mycocerosic acid synthase gene in Mycobacterium bovis BCG and to determine the nature of the ORF3 protein. ORF3 was expressed as a 61-kDa C-terminal fusion protein with glutathione S-transferase in Escherichia coli. Polyclonal rabbit antiserum, prepared against this fusion protein, cross-reacted with a 65-kDa protein in M. bovis BCG crude extracts. Since this protein was larger than that predicted from the nucleotide sequence (32 kDa), ORF3 was resequenced, revealing an ORF of 1,749 bp that encodes a 64.8-kDa protein containing 583 amino acids. Reverse transcription-PCR revealed that ORF3 is expressed in M. bovis BCG. The ORF3 product has a high degree of similarity to the acyladenylate family of enzymes. Immunoaffinity absorption chromatography was used to isolate the 65-kDa cross-reacting protein from M. bovis BCG. This purified protein catalyzed coenzyme A (CoA) ester synthesis of n-C 10 to n-C 18 fatty acids but not mycocerosic acids. ORF3 antibodies severely inhibited acyl-CoA synthase activities of the purified protein and extracts of M. bovis BCG, Mycobacterium smegmatis, and E. coli. They also showed immunological cross-reactivity with proteins in these extracts. Both the ORF3 protein and the acyl-CoA synthase activity were located in the cell cytosol or were loosely associated with the cell membrane. These results indicate that ORF3 encodes an acyl-CoA synthase-like protein.Tuberculosis is the world's foremost cause of death from a single infectious agent. More than 30 million tuberculosisrelated deaths and 90 million new infections are expected to occur in the last decade of this century (11). The major difficulty in treating mycobacterial infections is that the cell walls of these organisms have a high lipid content (50 to 60%) and consequently are highly resistant to attack by both the host's own immune system and antimicrobial agents (7). With the advent of multidrug-resistant mycobacterial strains, identification of potential new drug targets has become a critical need. Components unique to pathogenic mycobacterial cell wall synthesis (such as the mycolic acids, mycocerosic acids, and phthiocerols) (6) may be such selective targets, and inhibitors of such processes may be used in conjunction with conventional antimicrobial therapies to treat mycobacterial diseases (9).In spite of accumulating evidence which highlights the importance of the nature and quantity of lipids in the cell wall, much basic information remains to be elucidated about the metabolism of such lipids. Mycocerosic acids are produced by a multifunctional synthase, mycocerosic acid synthase (MAS), which elongates n-C 20 acids by successive reactions involving the incorporation of four C 3 units from methylmalonyl-coenzyme A (CoA) (34). When the mas gene was cloned and sequenced, an adjacent open reading frame (ORF), ORF3, was identified that showed homology to the acyladenylate family of enzymes, suggesting a role...

An Acyl-CoA Synthase (acoas) Gene Adjacent to the Mycocerosic Acid Synthase (mas) Locus Is Necessary for Mycocerosyl Lipid Synthesis in Mycobacterium tuberculosisvar. bovis BCG

“…However, in vitro studies indicated that purified ACoAS protein catalyzed the activation of mycocerosic acids to their corresponding thioesters only at extremely low rates. Furthermore, ACoAS failed to stimulate MAS activity and did not cause measurable release of mycocerosic acids either in the free form or as phthiocerol derivatives when phenolphthiocerol or glycosylated phenolphthiocerol was provided as acceptor (11). Although such in vitro reconstitution experiments failed to reveal the function of ACoAS, additional factors may be necessary for this protein to function in the transfer of mycocerosic acids to the diol acceptors.…”

“…This laboratory has previously cloned and characterized the mycocerosic acid synthase gene (mas) (9) and has identified a gene cluster involved in phthiocerol and phenolphthiocerol synthesis (pps) in Mycobacterium bovis BCG (10). In addition, we recently reported that a small open reading frame, ORF3, is located at the 5Ј end of the mas gene; amino acid sequence homology and enzyme assays using purified ORF3 protein indicated that this open reading frame encoded an ACoAS 1 (11). Genes homologous to acoas have been identified adjacent to the mas gene in M. tuberculosis (GenBank TM accession number Z83858) and Mycobacterium leprae (GenBank TM accession number U00010), as well as 5Ј to a polyketide synthase gene and 3Ј to a mas-like gene in M. tuberculosis (GenBank TM accession numbers Z74697, U00024, and Z77826).…”

“…Given the high degree of homology among these genes (54 -99% identity; 69 -99% similarity), it seems likely that all these genes play a role in acyl transfer. The multiplicity and relative location of these genes adjacent to large polyketide synthase-like enzymes lead us to speculate that their gene products may be involved in the selective transfer of the acyl products of the neighboring synthase genes either directly or indirectly to the ultimate biological acceptors (11). Purified MAS fails to release mycocerosic acids, and mycocerosic acids are not found free in the cytosol but instead are esterified to the diols, which suggests the involvement of a separate transferase enzyme (12).…”

Biochemistry and molecular genetics of cell‐wall lipid biosynthesis in mycobacteria

“…The 65 kDa protein isolated from this extract by immunoaffinity purification with the antibodies showed acyl-CoA synthase activity. In view of the presence of the orf3 gene in close proximity to mas, a pks and a mas-like gene in several mycobacterial species, it is tempting to suggest that the products of the orf3-like genes function selectively with the products of the neighbouring synthase genes, presumably to transfer the synthase products to their biochemical acceptors (Fitzmaurice and Kolattukudy, 1997). Thus, for example, the ORF3 protein could either activate mycocerosic acids or function with a membrane-localized protein to transfer mycocerosyl groups from the synthase to the diols.…”

BCG Vaccines: Their mechanisms of attenuation and impact on safety and protective efficacy

“…21 52 The biosynthesis of PDIMs and PGLs has been a subject of intensive studies in recent years, 52 pioneered by Kolattukudy and co-workers. 53,54 The role of PDIMs and PGLs in virulence has now been well established. 52,55 Both of these molecules are thought to play a role in modulation of the host immune response.…”