Discovery of a Novel l-Lyxonate Degradation Pathway in Pseudomonas aeruginosa PAO1

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• 作者：Salehe Ghasempur ; Subramaniam Eswaramoorthy ; Brandan S. Hillerich ; Ronald D. Seidel ; Subramanyam Swaminathan ; Steven C. Almo ; John A. Gerlt
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：May 27, 2014
• 年：2014
• 卷：53
• 期：20
• 页码：3357-3366
• 全文大小：640K
• 年卷期：v.53,no.20(May 27, 2014)
• ISSN：1520-4995

文摘

The mallcaps">l-lyxonate dehydratase (LyxD) in vitro enzymatic activity and in vivo metabolic function were assigned to members of an isofunctional family within the mandelate racemase (MR) subgroup of the enolase superfamily. This study combined in vitro and in vivo data to confirm that the dehydration of mallcaps">l-lyxonate is the biological role of the members of this family. In vitro kinetic experiments revealed catalytic efficiencies of 10⁴ M^鈥? s^鈥? as previously observed for members of other families in the MR subgroup. Growth studies revealed that mallcaps">l-lyxonate is a carbon source for Pseudomonas aeruginosa PAO1; transcriptomics using qRT-PCR established that the gene encoding LyxD as well as several other conserved proximal genes were upregulated in cells grown on mallcaps">l-lyxonate. The proximal genes were shown to be involved in a pathway for the degradation of mallcaps">l-lyxonate, in which the first step is dehydration by LyxD followed by dehydration of the 2-keto-3-deoxy-mallcaps">l-lyxonate product by 2-keto-3-deoxy-mallcaps">l-lyxonate dehydratase to yield 伪-ketoglutarate semialdehyde. In the final step, 伪-ketoglutarate semialdehyde is oxidized by a dehydrogenase to 伪-ketoglutarate, an intermediate in the citric acid cycle. An X-ray structure for the LyxD from Labrenzia aggregata IAM 12614 with Mg²⁺ in the active site was determined that confirmed the expectation based on sequence alignments that LyxDs possess a conserved catalytic His-Asp dyad at the end of seventh and sixth 尾-strands of the (尾/伪)₇尾-barrel domain as well as a conserved KxR motif at the end of second 尾-strand; substitutions for His 316 or Arg 179 inactivated the enzyme. This is the first example of both the LyxD function in the enolase superfamily and a pathway for the catabolism of mallcaps">l-lyxonate.