Identification of a novel galactosyl transferase involved in biosynthesis of the mycobacterial cell wall.
The possibility of the Rv3782 protein of Mycobacterium tuberculosis being a putative galactosyl transferase (GalTr) implicated in galactan synthesis arose from its similarity to the known GalTr Rv3808c, its classification as a nucleotide sugar-requiring inverting glycosyltransferase (GT-2 family), and its location within the "possible arabinogalactan biosynthetic gene cluster" of M. tuberculosis. In order to study the function of the enzyme, active membrane and cell wall fractions from Mycobacterium smegmatis containing the overexpressed Rv3782 protein were incubated with endogenous decaprenyldiphosphoryl-N-acetylglucosaminyl-rhamnose (C(50)-P-P-GlcNAc-Rha) as the primary substrate for galactan synthesis and UDP-[(14)C]galactopyranose as the immediate precursor of UDP-[(14)C]galactofuranose, the ultimate source of all of the galactofuranose (Galf) units of galactan. Obvious increased and selective synthesis of C(50)-P-P-GlcNAc-Rha-Galf-Galf, the earliest product in the pathway leading to the fully polymerized galactan, was observed, suggesting that Rv3782 encodes a GalTr involved in the first stages of galactan synthesis. Time course experiments pointed to a possible bifunctional enzyme responsible for the initial synthesis of C(50)-P-P-GlcNAc-Rha-Galf, followed by immediate conversion to C(50)-P-P-GlcNAc-Rha-Galf-Galf. Thus, Rv3782 appears to be the initiator of galactan synthesis, while Rv3808c continues with the subsequent polymerization events.
Mikusová
Katarína
K
Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
Belánová
Martina
M
Korduláková
Jana
J
Honda
Kristine
K
McNeil
Michael R
MR
Mahapatra
Sebabrata
S
Crick
Dean C
DC
Brennan
Patrick J
PJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002240:Carbohydrate Sequence; D002473:Cell Wall / Q000378:metabolism*; D005685:Galactans / Q000096:biosynthesis*; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000201:enzymology / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D005685:Galactans; D005700:Galactosyltransferases