Trehalose-6-phosphate phosphorylase is part of a novel metabolic pathway for trehalose utilization in Lactococcus lactis
Trehalose-6-phosphate phosphorylase is part of a novel metabolic pathway for trehalose utilization in Lactococcus lactis
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DOI：
10.1074/jbc.m108279200
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发表时间：
2001-11-16
影响因子：
4.8
通讯作者：
Rådström, P
中科院分区：
文献类型：
--
作者：
Andersson, U;Levander, F;Rådström, P
乳酸乳球菌通过无机磷酸盐依赖性海藻糖-6-磷酸磷酸化酶（TrePP）的作用在一种新的分解代谢途径中分解磷酸化海藻糖。通过测量来自海藻糖培养的乳球菌的细胞提取物中的中间糖磷酸，发现TrePP催化海藻糖6-磷酸可逆转化为β-葡萄糖1-磷酸和葡萄糖6-磷酸。根据非变性PAGE和SDS-PAGE，显示TrePP是分子量为94 kDa的单体酶。反应动力学表明，该酶遵循三元复合物的机制与最佳的磷酸解在35 degreesC和pH 6.3。在pH值为6.3和7.0时，平衡常数分别为0.026和0.032，有利于海藻糖6-磷酸的形成。测定TrePP对海藻糖6-磷酸、无机磷酸、β-葡萄糖1-磷酸和葡萄糖6-磷酸的米氏常数分别为6、32、0.9和4 mm。将编码TrePP的基因命名为trePP，定位于L.乳酸菌。该操纵子包括编码β-磷酸葡萄糖变位酶的基因，以及据信编码转录调节因子的三个开放阅读框和两个海藻糖特异性磷酸转移酶系统组分。通过测定TrePP的N-末端氨基酸序列并通过其在大肠杆菌和乳杆菌中的过表达来确认TrePP的身份。乳酸菌，以及乳球菌trePP敲除突变体的构建。此外，在粪肠球菌细胞提取物中检测到TrePP和P-磷酸葡萄糖变位酶活性，表明该细菌表现出与L.乳酸菌。
Lactococcus lactis splits phosphorylated trehalose by the action of inorganic phosphate-dependent trehalose-6-phosphate phosphorylase (TrePP) in a novel catabolic pathway. TrePP was found to catalyze the reversible conversion of trehalose 6-phosphate into beta -glucose 1-phosphate and glucose 6-phosphate by measuring intermediate sugar phosphates in cell extracts from trehalose-cultivated lactococci. According to native PAGE and SDS-PAGE, TrePP was shown to be a monomeric enzyme with a molecular mass of 94 kDa. Reaction kinetics suggested that the enzyme follows a ternary complex mechanism with optimal phosphorolysis at 35 degreesC and pH 6.3. The equilibrium constants were found to be 0.026 and 0.032 at pH 6.3 and 7.0, respectively, favoring the formation of trehalose 6-phosphate. The Michaelis-Menten constants of TrePP for trehalose 6-phosphate, inorganic phosphate, beta -glucose 1-phosphate, and glucose 6-phosphate were determined to be 6, 32, 0.9, and 4 mm, respectively. The TrePP-encoding gene, designated trePP, was localized in a putative trehalose operon of L. lactis. This operon includes the gene encoding beta -phosphoglucomutase in addition to three open reading frames believed to encode a transcriptional regulator and two trehalose-specific phosphotransferase system components. The identity of trePP was confirmed by determining the N-terminal amino acid sequence of TrePP and by its overexpression in Escherichia coli and L. lactis, as well as the construction of a lactococcal trePP knockout mutant. Furthermore, both TrePP and P-phosphoglucomutase activity were detected in Enterococcus faecalis cell extract, indicating that this bacterium exhibits the same trehalose assimilation route as L. lactis.