New function for Escherichia coli xanthosine phophorylase (xapA): genetic and biochemical evidences on its participation in NAD+ salvage from nicotinamide

BMC Microbiology

Jian-Zhong Shao¹  Li-Xin Xiang¹  Shi-Hua Hu¹  Guan Zhu²  Cen-Cen Sun¹  Wei-Ren Dong¹ 

[1] Key Laboratory for Cell and Gene Engineering of Zhejiang Province, Hangzhou 310058, People’s Republic of China;Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, 4467 TAMU, College Station, TX, USA

关键词: Pyridine nucleotide cycles;    Salvage pathway;    Nicotinamide riboside;    Purine nucleoside phosphorylase;   

Others  :  1141996
DOI  :  10.1186/1471-2180-14-29

 received in 2013-12-11, accepted in 2014-02-03,  发布年份 2014

【 摘 要 】

Background

In an effort to reconstitute the NAD⁺ synthetic pathway in Escherichia coli (E. coli), we produced a set of gene knockout mutants with deficiencies in previously well-defined NAD⁺de novo and salvage pathways. Unexpectedly, the mutant deficient in NAD⁺de novo and salvage pathway I could grow in M9/nicotinamide medium, which was contradictory to the proposed classic NAD⁺ metabolism of E. coli. Such E. coli mutagenesis assay suggested the presence of an undefined machinery to feed nicotinamide into the NAD⁺ biosynthesis. We wanted to verify whether xanthosine phophorylase (xapA) contributed to a new NAD⁺ salvage pathway from nicotinamide.

Results

Additional knockout of xapA further slowed down the bacterial growth in M9/nicotinamide medium, whereas the complementation of xapA restored the growth phenotype. To further validate the new function of xapA, we cloned and expressed E. coli xapA as a recombinant soluble protein. Biochemical assay confirmed that xapA was capable of using nicotinamide as a substrate for nicotinamide riboside formation.

Conclusions

Both the genetic and biochemical evidences indicated that xapA could convert nicotinamide to nicotinamide riboside in E. coli, albeit with relatively weak activity, indicating that xapA may contribute to a second NAD⁺ salvage pathway from nicotinamide. We speculate that this xapA-mediated NAD⁺ salvage pathway might be significant in some bacteria lacking NAD⁺de novo and NAD⁺ salvage pathway I or II, to not only use nicotinamide riboside, but also nicotinamide as precursors to synthesize NAD⁺. However, this speculation needs to be experimentally tested.

【 授权许可】

   
2014 Dong et al.; licensee BioMed Central Ltd.

【 图 表 】

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