Bombyx mori Pyridoxal Kinase cDNA Cloning and Enzymatic Characterization

Shi, Renyi; Zhang, Jianyun; Jiang, Changjun; Huang, LongQuan

doi:10.1016/s1673-8527(07)60077-0

Cited by 7 publications

(2 citation statements)

References 18 publications

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“…Since B. mori is a large silk-secreting insect, its immense protein turnover needs the timely support of PLP. In order to understand the metabolic mechanism of VB6 in B. mori, the cDNAs encoding PLK and PNPO in B. mori larvae were cloned (Shi et al, 2007;Huang et al, 2009). In this study, the recombinant B. mori PLK was expressed in E. coli as a fusion protein with a hexa-histidine affinity tag, purified by Ni Sepharose affinity column chromatography and characterized.…”

Section: Introductionmentioning

confidence: 99%

Recombinant expression, purification and characterization of Bombyx mori (Lepidoptera: Bombycidae) pyridoxal kinase

Huang¹,

Ma²,

Zhang³

et al. 2011

Eur. J. Entomol.

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Abstract. Pyridoxal kinase (PLK; EC 2.7.1.35) is a key enzyme in the metabolism of vitamin B6 (VB6) in Bombyx mori. A fusion expressional vector pET-22b-BPLK-His was constructed using a sub-cloning technique, the recombinant B. mori PLK was then expressed in Escherichia coli, purified and characterized. Bioinformatics were used to deduce the protein structure and genomic organization of this enzyme. Using Ni Sepharose affinity column chromatography, the recombinant protein was purified to very high degree (approximately 90%). The recombinant PLK exhibits a high specific enzymatic activity (1800 nmol/min/mg of protein). The maximum catalytic activity of this enzyme was recorded over a narrow pH range (5.5-6.0) and Zn 2+ is the most effective cation for catalysis under saturating substrate concentrations. When only triethanolamine is present as the cation, K + is an activator of PLK. A double reciprocal plot of initial velocity suggests that the enzyme catalyses the reaction by means of a sequential catalytic mechanism. Under optimal conditions, the Km value for the substrates of ATP and pyridoxal are 57.9 ± 5.1 and 44.1 ± 3.9 µM. B. mori's genome contains a single copy of the PLK gene, which is 7.73 kb long and contains five exons and four introns, and is located on the eighth chromosome. The PLK may be a dimer with two identical subunits under native conditions, and it is hypothesized that each monomer contains eight -helices ( 1-8), nine -strands ( 1-9) and two segments of 310 helices.

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Section: Introductionmentioning

confidence: 99%

Recombinant expression, purification and characterization of Bombyx mori (Lepidoptera: Bombycidae) pyridoxal kinase

Huang¹,

Ma²,

Zhang³

et al. 2011

Eur. J. Entomol.

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“…The amino acid sequence of mammal PLK is highly conserved with >80% identity, while that of PLKs showed approximately 40% identity in the group of microorganisms, plants, and mammals. 6 Bioinformatics assays showed the comparison between Leishmania infantum PLK and Trypanosoma protein sequence had a low similarity, with identity percentage of 61.04 -66.11%. 7 These data indicate PLK has high variation.…”

Section: Introductionmentioning

confidence: 99%

Cloning and Characterization of Pyridoxal Kinase from Geobacillus sp. H6a

Pasri¹,

Champasri²,

Trongpanich³

2022

J. Pure Appl. Microbiol.

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Pyridoxal kinase encoded by pdxK gene, is the important key enzyme in the salvage pathway of vitamin B6 biosynthesis. The enzyme catalyzes the phosphorylation of the 5′ alcohol groups of free form vitamin B6 into their 5′-phosphate forms that requires metal ion and ATP. Pyridoxal kinase have been reported in many organisms except in the thermophilic bacterium. Therefore, this study aimed to clone, express and characterize pyridoxal kinase of Geobacillus sp. H6a isolated from the hot spring in the North of Thailand. The GhpdxK gene (810 base pairs) was inserted into pET28a(+) plasmids at restriction site of NdeI and BamHI and transformed into E.coli BL21(DE3). The expressed pyridoxal kinase of this bacterium exhibits a homodimer, in which each subunit had a molecular mass of about 32 kDa when examined by SDS-PAGE and gel filtration. The enzyme showed maximal activity at 70°C and at pH 8.0. The expressed enzyme obtained in this study was found to be more active (>50%) in the broad pH range (6.0 – 9.0) than those previously reported. This enzyme prefers Mg2+ and also accepts other cations to the less extent. Under optimal conditions, the expressed enzyme has higher affinity toward PN (20 ± 1.35 µM), while it showed the same affinity to pyridoxal (100 ± 0.76 µM) and pyridoxamine (100 ± 1.21 µM). The Km value for ATP and 4-amino-5-hydroxymethyl-2-methylpyridine were 8.99 ± 1.76 µM and 19 ± 0.85 µM, respectively. With high activity at high temperature and active in the broad pH range, it could be considered as a potential candidate for future application particularly bioconversion of vitamin B6.

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