Random mutagenesis of the zinc-binding motif of betaine-homocysteine methyltransferase reveals that Gly 214 is essential
Random mutagenesis of the zinc-binding motif of betaine-homocysteine methyltransferase reveals that Gly 214 is essential
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DOI：
10.1006/abbi.2001.2751
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发表时间：
2002-03-01
影响因子：
3.9
通讯作者：
Garrow, TA
中科院分区：
文献类型：
--
作者：
Breksa, AP;Garrow, TA
甜菜碱-同型半胱氨酸S-甲基转移酶（BHMT; EC2.1.1.5）是一种锌金属酶，催化甲基从甜菜碱转移到同型半胱氨酸，分别产生二甲基甘氨酸和Met。这种酶是锌依赖性甲基转移酶家族的一员，该家族使用硫醇或硒醇作为甲基受体，并含有以下基序：G[IL-V]NCX（20，100）[ALV]X（2）[ILV]GGCCX（3）PX（2）I。我们最近报道，该基序中的三个半胱氨酸残基在BHMT中作为锌的配体起作用，因为将它们中的任何一个改变为丙氨酸都会破坏锌结合和酶活性（A. P. Breksa，III，and T. A. Garrow，1999，Biochemistry 38，13991-13998）。为了确定该基序中的其他氨基酸残基是否对酶功能至关重要，对人BHMT中由基序限定的两个区域GVNCH（218）和VRYIGGCCGFEPYHI（307）进行半随机和随机定点诱变。根据突变酶补充大肠杆菌菌株J5-3的Met营养缺陷型的能力，将突变酶分类为有活性或无活性。发现214位的Gly残基对于互补是绝对必需的。Gly 297，Gly 298和Gly 301占据的位置有利于取代小的氨基酸，如丙氨酸和丝氨酸。我们假设，这些甘氨酸残基提供了必要的灵活性，锌结合区，允许协调的金属。(C)2002 Elsevier Science（美国）。
Betaine-homocysteine S-methyltransferase (BHMT; EC2.1.1.5) is a zinc metalloenzyme that catalyzes the transfer of a methyl group from betaine to homocysteine to produce dimethylglycine and Met, respectively. This enzyme is a member of a family of zinc-dependent methyltransferases that use thiols or selenols as methyl acceptors and which contain the following motif: G[IL-V]NCX(20, 100)[ALV]X(2)[ILV]GGCCX(3)PX(2)I. We recently reported that the three cysteine residues within this motif function as ligands to zinc in BHMT because changing any of them to alanine abolished zinc-binding and enzyme activity (A. P. Breksa, III, and T. A. Garrow, 1999, Biochemistry 38, 13991-13998). To determine if other amino acid residues in this motif were critical for enzyme function, the two regions defined by the motif in human BHMT, GVNCH(218) and VRYIGGCCGFEPYHI(307), were subjected to semirandom and random site-directed mutagenesis. Mutant enzymes were classified as either active or inactive based on their ability to complement the Met auxotrophy of Escherichia coli strain J5-3. The Gly residue at position 214 was found to be absolutely essential for complementation. The positions occupied by Gly297, Gly298, and Gly301 favored substitutions of small amino acids like Ala and Ser. We hypothesize that these Gly residues provide the necessary flexibility to the Zn-binding region to permit coordination of the metal. (C) 2002 Elsevier Science (USA).