STRUCTURE AND FUNCTION OF MUTANT ARG44LYS OF 4-HYDROXYBENZOATE HYDROXYLASE - IMPLICATIONS FOR NADPH BINDING

被引:23
作者
EPPINK, MHM
SCHREUDER, HA
VANBERKEL, WJH
机构
[1] AGR UNIV WAGENINGEN,DEPT BIOCHEM,6703 HA WAGENINGEN,NETHERLANDS
[2] MARION MERRELL DOW RES INST,STRASBOURG,FRANCE
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1995年 / 231卷 / 01期
关键词
CRYSTAL STRUCTURE; FLAVOPROTEIN MONOOXYGENASE; NADPH BINDING; 4-HYDROXYBENZOATE HYDROXYLASE; SITE-SPECIFIC MUTAGENESIS;
D O I
10.1111/j.1432-1033.1995.0157f.x
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
Arg44, located at the si-face side of the flavin ring in 4-hydroxybenzoate hydroxylase, was changed to lysine by site-specific mutagenesis. Crystals of [R44K]4-hydroxybenzoate hydroxylase complexed with 4-hydroxybenzoate diffract to 0.22-nm resolution. The structure of [R44K]4-hydroxybenzoate hydroxylase is identical to the wild-type enzyme except for local changes in the vicinity of the mutation. The peptide unit between Ile43 and Lys44 is flipped by about 180 degrees in 50% of the molecules. The phi,psi angles in both the native and flipped conformation are outside the allowed regions and indicate a strained conformation. [R44K]4-Hydroxygenzoate hydroxylase has a decreased affinity for the flavin prosthetic group. This is ascribed to the lost interactions between the side chain of Arg44 and the diphosphoribose moiety of the FAD. The replacement of Arg44 by Lys does not change the position of the flavin ring which occupies the same interior position as in wild type. [R44K]4-Hydroxygenzoate hydroxylase fully couples flavin reduction to substrate hydroxylation. Stopped-flow kinetics showed that the effector role of 4-hydroxybenzoate is largely conserved in the mutant. Replacement of Arg44 by Lys however affects NADPH binding, resulting in a low yield of the charge-transfer species between reduced flavin and NADP(+). It is inferred from these data that Arg44 is indispensable for optimal catalysis.
引用
收藏
页码:157 / 165
页数:9
相关论文
共 46 条
  • [11] THE MOBILE FLAVIN OF 4-OH BENZOATE HYDROXYLASE
    GATTI, DL
    PALFEY, BA
    LAH, MS
    ENTSCH, B
    MASSEY, V
    BALLOU, DP
    LUDWIG, ML
    [J]. SCIENCE, 1994, 266 (5182) : 110 - 114
  • [12] HOSOKAWA K, 1966, J BIOL CHEM, V241, P2453
  • [13] HOWELL LG, 1972, J BIOL CHEM, V247, P4340
  • [14] HUSAIN M, 1979, J BIOL CHEM, V254, P6657
  • [15] JONES TA, 1985, METHOD ENZYMOL, V115, P157
  • [17] KUNKEL TA, 1987, METHOD ENZYMOL, V154, P367
  • [18] CRYSTAL-STRUCTURES OF MUTANT PSEUDOMONAS-AERUGINOSA P-HYDROXYBENZOATE HYDROXYLASES - THE TYR201PHE, TYR385PHE, AND ASN300ASP VARIANTS
    LAH, MS
    PALFEY, BA
    SCHREUDER, HA
    LUDWIG, ML
    [J]. BIOCHEMISTRY, 1994, 33 (06) : 1555 - 1564
  • [19] Lah MS, 1994, FLAVINS AND FLAVOPROTEINS 1993, P221
  • [20] ABSOLUTE STEREOCHEMISTRY OF FLAVINS IN ENZYME-CATALYZED REACTIONS
    MANSTEIN, DJ
    PAI, EF
    SCHOPFER, LM
    MASSEY, V
    [J]. BIOCHEMISTRY, 1986, 25 (22) : 6807 - 6816