Elsevier

Analytical Biochemistry

Volume 298, Issue 2, 15 November 2001, Pages 314-321
Analytical Biochemistry

Regular Article
Distribution of B6 Vitamers in Escherichia coli as Determined by Enzymatic Assay

https://doi.org/10.1006/abio.2001.5401Get rights and content

Abstract

An enzymatic method for determination of B6 vitamers is presented. In this method pyridoxal 5′-phosphate is used to activate aposerine hydroxymethyltransferase to form the catalytically active holoenzyme. The active serine hydroxymethyltransferase, and two other enzymes that form a metabolic cycle, convert serine to glycine and CO2 with the concomitant production of two equivalents of NADPH. The rate of the cycle is directly proportional to the amount of active holoserine hydroxymethyltransferase, which is a measure of the amount of pyridoxal 5′-phosphate in the original sample. The cycle operates about 50 times per minute giving a 100-fold enhancement of NADPH production with respect to original pyridoxal 5′-phosphate content. Other B6 vitamers are converted to pyridoxal 5′-phosphate by a preincubation with a combination of pyridoxal kinase and pyridoxine 5′-phosphate oxidase. A complete analysis of B6 vitamers can be completed in less than 1 h and the assay is linear in the 2- to 50-pmol range of pyridoxal 5′-phosphate. The method is applied to the determination of the B6 vitamer pools in extracts of Escherichia coli. The results show that the pool of pyridoxal 5′-phosphate that is not bound to proteins is large enough to account for product inhibition of both pyridoxal kinase and pyridoxine 5′-phosphate oxidase.

References (29)

Cited by (23)

  • Allosteric feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia coli

    2019, Journal of Biological Chemistry
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    PLP product inhibition of E. coli PNPOx has been reported to take place with a KI of 8 μm (13) and attributed to PLP binding at the active site; however, data supporting the competitive nature of product inhibition with respect to the PNP substrate have not been presented. This product inhibition is probably an important regulatory mechanism of PLP biosynthesis in E. coli cells, whose free PLP concentration in vivo has been estimated around 120 μm (14). PLP has been proposed to bind with high affinity also at a secondary “tight binding site,” spatially distinct from the active site.

  • Vitamin B6 metabolism in microbes and approaches for fermentative production

    2017, Biotechnology Advances
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    Control of vitamin B6 homeostasis might also be achieved at the level of enzyme activity (di Salvo et al., 2011). It has been observed that the PNP oxidase PdxH and PN/PM/PL kinase PdxK from E. coli are modulated by PLP (White and Dempsey, 1970; Zhao and Winkler, 1995; Yang et al., 1996; Yang and Schirch, 2000; Fu et al., 2001; Ghatge et al., 2012). PL and PLP tightly bind to the residue lys229 of PdxK, which results in inhibition of the kinase (Ghatge et al., 2012; di Salvo et al., 2015).

  • Effect of exogenous hormones on transcription levels of pyridoxal 5'-phosphate biosynthetic enzymes in the silkworm (Bombyx mori)

    2016, Comparative Biochemistry and Physiology Part - B: Biochemistry and Molecular Biology
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    Currently, our understanding of PLP dynamic regulation is superficial and known studies were mainly focused on the regulation at the enzyme structure level (Di Salvo et al., 2011; Choi et al., 1983; Choi et al., 1987; Zhao and Winkler, 1995; Laine-Cessac et al., 1997; Di Salvo et al., 2003; Safo et al., 2001, 2006; Kastner et al., 2007). Although regulations in enzyme synthesis level have been reported (Ngo et al., 1998; Fu et al., 2001), the molecular mechanism will be the main direction of research in the future. Results of our study show that transcription levels of PL kinase and PNP oxidase are regulated by development in silkworm.

  • On the mechanism of Escherichia coli pyridoxal kinase inhibition by pyridoxal and pyridoxal 5′-phosphate

    2015, Biochimica et Biophysica Acta - Proteins and Proteomics
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    On the other hand, the less specific ePLK1, which is also capable to phosphorylate pyridoxine and pyridoxamine, would be inactivated when either PL or PLP (which may also come from pyridoxine 5′-phosphate and pyridoxamine 5′-phosphate through the action of PNP oxidase and phosphatases) reaches a critical cellular level. Measurements of B6 vitamers in E. coli grown to stationary phase in minimal medium indicated that free PL and PLP contained in the cells are about 10 μM and 120 μM, respectively [27]. In these conditions, ePLK1 is expected to be fully inactivated by PLP, while PL might not play a significant role.

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