Biochemical and structural characterization of WlbA from Bordetella pertussis and Chromobacterium violaceum: enzymes required for the biosynthesis of 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid.
The unusual sugar 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, or ManNAc3NAcA, has been observed in the lipopolysaccharides of both pathogenic and nonpathogenic Gram-negative bacteria. It is added to the lipopolysaccharides of these organisms by glycosyltransferases that use as substrates UDP-ManNAc3NAcA. Five enzymes are ultimately required for the biosynthesis of UDP-ManNAc3NAcA starting from UDP-N-acetylglucosamine. The second enzyme in the pathway, encoded by the wlba gene and referred to as WlbA, catalyzes the NAD-dependent oxidation of the C-3' hydroxyl group of the UDP-linked sugar. Here we describe a combined structural and functional investigation of the WlbA enzymes from Bordetella pertussis and Chromobacterium violaceum. For this investigation, ternary structures were determined in the presence of NAD(H) and substrate to 2.13 and 1.5 Å resolution, respectively. Both of the enzymes display octameric quaternary structures with their active sites positioned far apart. The octamers can be envisioned as tetramers of dimers. Kinetic studies demonstrate that the reaction mechanisms for these enzymes are sequential and that they do not require α-ketoglutarate for activity. These results are in sharp contrast to those recently reported for the WlbA enzymes from Pseudomonas aeruginosa and Thermus thermophilus, which function via ping-pong mechanisms that involve α-ketoglutarate. Taken together, the results reported here demonstrate that there are two distinct families of WlbA enzymes, which differ with respect to amino acid sequences, quaternary structures, active site architectures, and kinetic mechanisms.
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, United States.
James B
JB
Thoden
Hazel M
HM
Holden
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001886:Bordetella pertussis / Q000201:enzymology*; D002236:Carbohydrate Conformation; D002861:Chromobacterium / Q000201:enzymology*; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D008958:Models, Molecular; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014574:Uronic Acids / Q000378:metabolism*
D001426:Bacterial Proteins; D014574:Uronic Acids; C035278:2,3-diacetamido-2,3-dideoxymannuronic acid
J Bacteriol. 2008 Sep;190(18):6060-9
18621892
Structure. 1996 Dec 15;4(12):1413-28
8994968
Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55
15299926
Biochemistry. 2010 Sep 14;49(36):7939-48
20690587
J Biol Chem. 2005 Mar 11;280(10):9662-70
15590630
J Bacteriol. 1946 Jul;52(1):145-6
16561146
Biokhimiia. 1994 Dec;59(12):1784-851
7533007
Biochemistry. 2009 Jun 16;48(23):5446-55
19348502
Infect Immun. 2000 Dec;68(12):6720-8
11083787
Acta Crystallogr D Biol Crystallogr. 2006 Aug;62(Pt 8):859-66
16855301
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
15572765
Acta Crystallogr D Biol Crystallogr. 1999 Apr;55(Pt 4):849-61
10089316
J Immunol. 2004 Aug 1;173(3):1959-65
15265930
J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674
19461840
J Biol Chem. 2009 May 1;284(18):11854-62
19282284