Zhang Hua, Zhou Ying, Bao Hongbo, Liu Hung-wen
Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712, USA.
Biochemistry. 2006 Jul 4;45(26):8163-73. doi: 10.1021/bi060446d.
Vi antigen, the virulence factor of Salmonella typhi, has been used clinically as a molecular vaccine. TviB and TviC are two enzymes involved in the formation of Vi antigen, a linear polymer consisting of alpha-1,4-linked N-acetylgalactosaminuronate. Protein sequence analysis suggests that TviB is a dehydrogenase and TviC is an epimerase. Both enzymes are expected to be NAD(+) dependent. In order to verify their functions, TviB and TviC were cloned, expressed in Escherichia coli, and characterized. The C-terminal His(6)-tagged TviB protein, purified from soluble cell fractions in the presence of 10 mM DTT, shows UDP-N-acetylglucosamine 6-dehydrogenase activity and is capable of catalyzing the conversion of UDP-N-acetylglucosamine (UDP-GlcNAc) to UDP-N-acetylglucosaminuronic acid (UDP-GlcNAcA) with a k(cat) value of 15.5 +/- 1.0 min(-)(1). The K(m) values of TviB for UDP-GlcNAc and NAD(+) are 77 +/- 9 microM and 276 +/- 52 microM, respectively. TviC, purified as C-terminal hexahistidine-tagged protein, shows UDP-GlcNAcA 4-epimerase and UDP-N-acetylgalactosamine (UDP-GalNAc) 4-epimerase activities. The K(m) values of TviC for UDP-GlcNAcA and UDP-N-acetylgalactosaminuronic acid (UDP-GalNAcA) are 20 +/- 1 microM and 42 +/- 2 microM, respectively. The k(cat) value for the conversion of UDP-GlcNAcA to UDP-GalNAcA is 56.8 +/- 0.5 min(-)(1), while that for the reverse reaction is 39.1 +/- 0.6 min(-)(1). These results show that the biosynthesis of Vi antigen is initiated by the TviB-catalyzed oxidation of UDP-GlcNAc to UDP-GalNAc, followed by the TviC-catalyzed epimerization at C-4 to form UDP-GalNAcA, which serves as the building block for the formation of Vi polymer. These results set the stage for future in vitro biosynthesis of Vi antigen. These enzymes may also be drug targets to inhibit Vi antigen production.
Vi抗原是伤寒沙门氏菌的毒力因子，已在临床上用作分子疫苗。TviB和TviC是参与Vi抗原形成的两种酶，Vi抗原是一种由α-1,4-连接的N-乙酰半乳糖胺糖醛酸组成的线性聚合物。蛋白质序列分析表明，TviB是一种脱氢酶，TviC是一种表异构酶。预计这两种酶都依赖于NAD(+)。为了验证它们的功能，克隆了TviB和TviC，在大肠杆菌中表达并进行了特性鉴定。从存在10 mM DTT的可溶性细胞组分中纯化的C端带有His(6)标签的TviB蛋白显示出UDP-N-乙酰葡糖胺6-脱氢酶活性，能够催化UDP-N-乙酰葡糖胺（UDP-GlcNAc）转化为UDP-N-乙酰葡糖胺糖醛酸（UDP-GlcNAcA），催化常数k(cat)值为15.5±1.0 min⁻¹。TviB对UDP-GlcNAc和NAD(+)的米氏常数K(m)分别为77±9 μM和276±52 μM。纯化的C端带有六组氨酸标签的TviC蛋白显示出UDP-GlcNAcA 4-表异构酶和UDP-N-乙酰半乳糖胺（UDP-GalNAc）4-表异构酶活性。TviC对UDP-GlcNAcA和UDP-N-乙酰半乳糖胺糖醛酸（UDP-GalNAcA）的K(m)值分别为20±1 μM和42±2 μM。UDP-GlcNAcA转化为UDP-GalNAcA的k(cat)值为56.8±0.5 min⁻¹，而逆反应的k(cat)值为39.1±0.6 min⁻¹。这些结果表明，Vi抗原的生物合成首先由TviB催化UDP-GlcNAc氧化为UDP-GalNAc，然后由TviC催化在C-4位进行表异构化形成UDP-GalNAcA，UDP-GalNAcA作为Vi聚合物形成的构件。这些结果为未来Vi抗原的体外生物合成奠定了基础。这些酶也可能是抑制Vi抗原产生的药物靶点。
