α-Lactalbumin (LA) stimulates milk β-1,4-galactosyltransferase I (β4Gal-T1) to transfer glucose from UDP-glucose to N-acetylglucosamine -: Crystal structure of β4Gal-T1-LA complex with UDP-Glc

被引:95
作者
Ramakrishnan, B
Shah, PS
Qasba, PK
机构
[1] NCI, Struct Glycobiol Sect, LECB, CCR,NIH, Ft Detrick, MD 21702 USA
[2] NCI, Intramural Res Support Program, SAIC, Lab Expt & Computat Biol,Ctr Canc Res,NIH, Ft Detrick, MD 21702 USA
关键词
D O I
10.1074/jbc.M102458200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
beta -1,4-Galactosyltransferase 1 (Gal-T1) transfers galactose (Gal) from UDP-Gal to N-acetylglucosamine (Glc-NAc), which constitutes its normal galactosyltransferase (Gal-T) activity. In the presence of alpha -lactalbumin (IA), it transfers Gal to Glc, which is its lactose synthase (LS) activity. It also transfers glucose (Glc) from UDP-Glc to GlcNAc, constituting the glucosyltransferase (Glc-T) activity, albeit at an efficiency of only 0.3-0.4% of Gal-T activity. In the present study, we show that LA increases this activity almost 30-fold. It also enhances the Glc-T activity toward various N-acyl substituted glucosamine acceptors. Steady state kinetic studies of Glc-T reaction show that the K-m for the donor and acceptor substrates are high in the absence of LA. In the presence of LA, the K-m for the acceptor substrate is reduced 30-fold, whereas for UDP-Glc it is reduced only 5-fold. In order to understand this property, we have determined the crystal structures of the Gal-TI-IA complex with UDP-Glc.Mn2+ and with N-butanoyl-glucosamine (N-butanoyl-GlcN), a preferred sugar acceptor in the Glc-T activity. The crystal structures reveal that although the binding of UDP-Glc is quite similar to UDP-Gal, there are few significant differences observed in the hydrogen bonding interactions between UDP-Glc and Gal-T1. Based on the present kinetic and crystal structural studies, a possible explanation for the role of LA in the Glc-T activity has been proposed.
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页码:37665 / 37671
页数:7
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共 35 条
  • [1] Cloning and expression of a proteoglycan UDP-galactose:β-xylose β1,4-galactosyltransferase I -: A seventh member of the human β4-galactosyltransferase gene family
    Almeida, R
    Levery, SB
    Mandel, U
    Kresse, H
    Schwientek, T
    Bennett, EP
    Clausen, H
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (37) : 26165 - 26171
  • [2] A family of human β4-galactosyltransferases -: Cloning and expression of two novel UDP-galactose:: β-N-acetylglucosamine β1,4-galactosyltransferases, β4Gal-T2 and β4Gal-T3
    Almeida, R
    Amado, M
    David, L
    Levery, SB
    Holmes, EH
    Merkx, G
    van Kessel, AG
    Rygaard, E
    Hassan, H
    Bennett, E
    Clausen, H
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1997, 272 (51) : 31979 - 31991
  • [3] ANDREE PJ, 1978, BIOCHIM BIOPHYS ACTA, V544, P489
  • [4] Ausubel FM, 1995, SHORT PROTOCOLS MOL
  • [5] BERLINER LJ, 1984, MOL CELL BIOCHEM, V62, P37, DOI 10.1007/BF00230075
  • [6] EXPRESSION OF DELETION CONSTRUCTS OF BOVINE BETA-1,4-GALACTOSYLTRANSFERASE IN ESCHERICHIA-COLI - IMPORTANCE OF CYS134 FOR ITS ACTIVITY
    BOEGGEMAN, EE
    BALAJI, PV
    SETHI, N
    MASIBAY, AS
    QASBA, PK
    [J]. PROTEIN ENGINEERING, 1993, 6 (07): : 779 - 785
  • [7] Crystallography & NMR system:: A new software suite for macromolecular structure determination
    Brunger, AT
    Adams, PD
    Clore, GM
    DeLano, WL
    Gros, P
    Grosse-Kunstleve, RW
    Jiang, JS
    Kuszewski, J
    Nilges, M
    Pannu, NS
    Read, RJ
    Rice, LM
    Simonson, T
    Warren, GL
    [J]. ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 1998, 54 : 905 - 921
  • [8] ULTRAVIOLET PHOTOINACTIVATION OF GALACTOSYLTRANSFERASE - PROTECTION BY SUBSTRATES
    CLYMER, DJ
    GEREN, CR
    EBNER, KE
    [J]. BIOCHEMISTRY, 1976, 15 (05) : 1093 - 1097
  • [9] ALPHA-LACTALBUMIN INDUCES BOVINE-MILK BETA-1,4-GALACTOSYLTRANSFERASE TO UTILIZE UDP-GALNAC
    DO, KY
    DO, SI
    CUMMINGS, RD
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (31) : 18447 - 18451
  • [10] Crystal structures of the bovine β4galactosyltransferase catalytic domain and its complex with uridine diphosphogalactose
    Gastinel, LN
    Cambillau, C
    Bourne, Y
    [J]. EMBO JOURNAL, 1999, 18 (13) : 3546 - 3557