Accommodation of GDP-linked sugars in the active site of GDP-perosamine synthase.
Perosamine (4-amino-4,6-dideoxy- d-mannose), or its N-acetylated form, is one of several dideoxy sugars found in the O-antigens of such infamous Gram-negative bacteria as Vibrio cholerae O1 and Escherichia coli O157:H7. It is added to the bacterial O-antigen via a nucleotide-linked version, namely GDP-perosamine. Three enzymes are required for the biosynthesis of GDP-perosamine starting from mannose 1-phosphate. The focus of this investigation is GDP-perosamine synthase from Caulobacter crescentus, which catalyzes the final step in GDP-perosamine synthesis, the conversion of GDP-4-keto-6-deoxymannose to GDP-perosamine. The enzyme is PLP-dependent and belongs to the aspartate aminotransferase superfamily. It contains the typically conserved active site lysine residue, which forms a Schiff base with the PLP cofactor. Two crystal structures were determined for this investigation: a site-directed mutant protein (K186A) complexed with GDP-perosamine and the wild-type enzyme complexed with an unnatural ligand, GDP-3-deoxyperosamine. These structures, determined to 1.6 and 1.7 A resolution, respectively, revealed the manner in which products, and presumably substrates, are accommodated within the active site pocket of GDP-perosamine synthase. Additional kinetic analyses using both the natural and unnatural substrates revealed that the K m for the unnatural substrate was unperturbed relative to that of the natural substrate, but the k cat was lowered by a factor of approximately 200. Taken together, these studies shed light on why GDP-perosamine synthase functions as an aminotransferase whereas another very similar PLP-dependent enzyme, GDP-4-keto-6-deoxy- d-mannose 3-dehydratase or ColD, catalyzes a dehydration reaction using the same substrate.
Cook
Paul D
PD
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Carney
Amanda E
AE
Holden
Hazel M
HM
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D016935:Caulobacter crescentus / Q000201:enzymology; D018360:Crystallography, X-Ray; D006156:Guanosine Diphosphate Sugars / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008358:Mannose / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D000637:Transaminases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D006156:Guanosine Diphosphate Sugars; C057033:4-amino-4,6-dideoxy-D-mannose; D000637:Transaminases; D002238:Carbohydrate Epimerases; C061272:perosamine synthetase; D008358:Mannose
Biochemistry. 2007 Dec 11;46(49):14215-24
17997582
Annu Rev Biochem. 2002;71:701-54
12045109
Curr Opin Chem Biol. 2002 Oct;6(5):590-7
12413542
FEMS Microbiol Rev. 2002 Mar;26(1):17-47
12007641
Acta Crystallogr D Biol Crystallogr. 1999 Feb;55(Pt 2):484-91
10089360
Annu Rev Microbiol. 1994;48:223-56
7826006
Methods Enzymol. 1997;276:307-26
27754618
Biochemistry. 2008 Mar 4;47(9):2833-40
18247575
Protein Sci. 2006 Sep;15(9):2093-106
16943443
Biochem Biophys Res Commun. 2007 Nov 23;363(3):525-30
17888872
Nature. 2007 Apr 26;446(7139):1008-16
17460661
Glycobiology. 2001 Aug;11(8):655-61
11479276
Annu Rev Biochem. 2004;73:383-415
15189147
Gene. 1995 Dec 1;166(1):33-42
8529891
J Biol Chem. 2008 Feb 15;283(7):4295-303
18045869
Curr Opin Struct Biol. 1998 Dec;8(6):759-69
9914259
Protein Sci. 2004 Nov;13(11):2992-3005
15498941
Annu Rev Biochem. 2002;71:635-700
12045108
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
15572765