The active site of Escherichia coli UDP-N-acetylglucosamine acyltransferase -: Chemical modification and site-directed mutagenesis
The active site of Escherichia coli UDP-N-acetylglucosamine acyltransferase -: Chemical modification and site-directed mutagenesis
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DOI：
10.1074/jbc.274.38.27047
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发表时间：
1999-09-17
影响因子：
4.8
通讯作者：
Raetz, CRH
中科院分区：
文献类型：
--
作者：
Wyckoff, TJO;Raetz, CRH
UDP-N-乙酰葡糖胺（UDP-GlcNAc）酰基转移酶（LpxA）在脂质A生物合成的第一步中催化R-3-羟酰基链从R-3-羟酰基-酰基载体蛋白可逆转移至UDP-GlcNAc的葡糖胺3-OH。脂质A是大多数革兰氏阴性菌生长和毒力所必需的，使其生物合成酶成为开发新抗菌剂的有趣靶点。LpxA是左手β-螺旋蛋白大家族的成员，其中许多是酰基或乙酰基转移酶。我们现在证明，组氨酸，赖氨酸和组氨酸特异性试剂有效地抑制大肠杆菌LpxA，而丝氨酸和半胱氨酸特异性试剂不。利用这些信息结合多序列比对，我们构建了保守的组氨酸，赖氨酸和精氨酸残基的定点丙氨酸取代突变。这些突变LpxA酶中的许多在标准测定条件下显示出严重降低的比活性。对于所有突变体，活性的降低对应于降低的k（cat）/K-m，K-UDP-GlcNAc值。除H125 A（在任何测定条件下均未观察到活性）外，k（cat）/K-m，K-UDP-GlcNAc的降低主要反映了K-m，K-UDP-GlcNAc的增加。（125）E.因此，大肠杆菌LpxA可能起催化残基的作用，也可能起一般碱基的作用。LpxA不催化可测量的UDP-3-O-（R-3-羟基肉豆蔻酰基）-GlcNAc水解或UDP-GlcNAc/UDP-3-O-（R-3-羟基肉豆蔻酰基）-GlcNAc交换，这与酰基酶中间体的乒乓机制相反。
UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes the reversible transfer of an R-3-hydroxyacyl chain from R-3-hydroxyacyl-acyl carrier protein to the glucosamine 3-OH of UDP-GlcNAc in the first step of lipid A biosynthesis. Lipid A is required for the growth and virulence of most Gram-negative bacteria, making its biosynthetic enzymes intriguing targets for the development of new antibacterial agents. LpxA is a member of a large family of left-handed beta-helical proteins, many of which are acyl- or acetyltransferases. We now demonstrate that histidine-, lysine-, and arginine-specific reagents effectively inhibit LpxA of Escherichia coli, whereas serine- and cysteine-specific reagents do not. Using this information in conjunction with multiple sequence alignments, we constructed site-directed alanine substitution mutations of conserved histidine, lysine, and arginine residues. Many of these mutant LpxA enzymes show severely decreased specific activities under standard assay conditions. The decrease in activity corresponds to decreased k(cat)/K-m,K-UDP-GlcNAc values for all the mutants. With the exception of H125A, in which no activity is seen under any assay condition, the decrease in k(cat)/K-m,K-UDP-GlcNAc mainly reflects an increased K-m,K-UDP-GlcNAc. His(125) of E. coli LpxA may therefore function as a catalytic: residue, possibly as a general base. LpxA does not catalyze measurable UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc hydrolysis or UDP-GlcNAc/UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc exchange, arguing against a ping-pong mechanism with an acyl-enzyme intermediate.