Structural characterization of sialic acid synthase by electrospray mass spectrometry--a tetrameric enzyme composed of dimeric dimers.
Sialic acid synthase (NeuB) encoded by the neuB gene catalyzes the condensation of N-acetylmannosamine and phospho(enol)pyruvate to form N-acetylneuraminic acid. The enzyme is essential for the biosynthesis of polysialic acid, a capsular sugar polymer functioning as a virulent factor and antiphagocytic barrier. This report demonstrates the first characterization on the quaternary structure of NeuB from Escherichia coli (EcNeuB) and Streptococcus agalactiae (SaNeuB) by nanoflow electrospray ionization mass spectrometry (ESI-MS). Under non-denaturing conditions, Tris buffer was observed to induce a higher ratio of tetramer/dimer of NeuB in the ESI mass spectra, providing supportive evidence for the existence of a "structurally-specific" tetramer. The instrument parameters were found to significantly affect the ratio of detected tetramer/dimer in ESI mass spectra. The harshest conditions, including high desolvation voltages and pressure in the collision cell, led to enhanced detection of the 160 kDa tetramer. The prevalence of dimeric form is likely the cause in loss of tetramer stability in gas-phase arising from insufficient collisional cooling, which implies an asymmetric assembly, possibly composed of dimeric dimers. Most interestingly, the hypothesis was further supported by chemical cross-linking of SaNeuB, in which the reaction of shorter linker yielded mainly the dimer whereas that of longer linker produced both dimer and tetramer. Furthermore, the ESI-MS analysis can reflect dramatic change of pH-dependent quaternary structure in association with enzyme activity, suggesting the tetrameric form may be the primary species responsible for the enzyme catalysis.
Huang
Hsin-Hung
HH
Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan. yjchen@chm.sinica.edu.tw
Liao
Hsin-Kai
HK
Chen
Yu-Ju
YJ
Hwang
Tzann-Shun
TS
Lin
Yi-Han
YH
Lin
Chun-Hung
CH
D019281:Dimerization; D008958:Models, Molecular; D007652:Oxo-Acid-Lyases / Q000032:analysis* / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013329:Structure-Activity Relationship
C018730:N-acetylneuraminate synthase; D007652:Oxo-Acid-Lyases
Biochemistry. 2002 Nov 19;41(46):13767-73
12427039
J Bacteriol. 2000 Aug;182(16):4466-77
10913080
Anal Biochem. 2001 Apr 1;291(1):48-61
11262155
J Mass Spectrom. 2003 Mar;38(3):315-20
12644993
J Biol Chem. 2004 Apr 2;279(14):13311-6
14726533
Rapid Commun Mass Spectrom. 2001;15(8):596-601
11312509
Proc Natl Acad Sci U S A. 1970 Jul;66(3):651-6
4913206
Mass Spectrom Rev. 1997 Jan-Feb;16(1):1-23
9414489
J Am Chem Soc. 2004 May 19;126(19):5950-1
15137744
Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):13-20
8552589
J Biol Chem. 2004 Oct 8;279(41):42750-7
15294894
Protein Sci. 2001 Jul;10(7):1293-304
11420431
Protein Expr Purif. 2003 Feb;27(2):346-56
12597896
Proc Natl Acad Sci U S A. 1987 Oct;84(20):7208-12
2823254
Structure. 1994 May 15;2(5):361-9
8081752
Proc Natl Acad Sci U S A. 2003 Sep 16;100(19):10611-6
12947045
J Am Soc Mass Spectrom. 2004 Oct;15(10):1435-43
15465356
J Biol Chem. 2000 Mar 31;275(13):9476-84
10734095
Structure. 1999 Jul 15;7(7):865-75
10425687
J Am Chem Soc. 2003 Mar 5;125(9):2817-26
12603172
J Am Soc Mass Spectrom. 1999 Jul;10(7):635-47
10384727
Anal Chem. 2004 Jan 1;76(1):211-7
14697053
Mass Spectrom Rev. 1997 Jul-Aug;16(4):165-79
9449832
Proc Natl Acad Sci U S A. 2000 Dec 19;97(26):14151-5
11087821
J Mol Biol. 1998 Aug 21;281(3):553-64
9698569
Curr Opin Struct Biol. 1999 Feb;9(1):135-41
10047587
J Am Soc Mass Spectrom. 2003 May;14(5):419-29
12745211
Biochemistry. 1989 Jun 13;28(12):5306-10
2669973
Curr Opin Chem Biol. 2004 Oct;8(5):519-26
15450495
Biochim Biophys Acta. 1988 Nov 23;957(2):313-7
3191147
J Bacteriol. 1995 Jan;177(2):312-9
7814319
Anal Chem. 2001 Dec 15;73(24):6040-6
11791577
Biochem Biophys Res Commun. 2002 Jul 5;295(1):167-73
12083785
J Mol Biol. 2000 Aug 11;301(2):389-99
10926516
Proteins. 1998;Suppl 2:3-11
9849905
J Biol Chem. 1959 Aug;234(8):1971-5
13672998