Biochemical characterization of the Arabidopsis biotin synthase reaction. The importance of mitochondria in biotin synthesis

Abstract

Biotin synthase, encoded by the bio2 gene in Arabidopsis, catalyzes the final step in the biotin biosynthetic pathway. The development of radiochemical and biological detection methods allowed the first detection and accurate quantification of a plant biotin synthase activity, using protein extracts from bacteria overexpressing the Arabidopsis Bio2 protein. Under optimized conditions, the turnover number of the reaction was >2 h(-1) with this in vitro system. Purified Bio2 protein was not efficient by itself in supporting biotin synthesis. However, heterologous interactions between the plant Bio2 protein and bacterial accessory proteins yielded a functional biotin synthase complex. Biotin synthase in this heterologous system obeyed Michaelis-Menten kinetics with respect to dethiobiotin (K(m) = 30 microM) and exhibited a kinetic cooperativity with respect to S-adenosyl-methionine (Hill coefficient = 1.9; K(0.5) = 39 microM), an obligatory cofactor of the reaction. In vitro inhibition of biotin synthase activity by acidomycin, a structural analog of biotin, showed that biotin synthase reaction was the specific target of this inhibitor of biotin synthesis. It is important that combination experiments using purified Bio2 protein and extracts from pea (Pisum sativum) leaf or potato (Solanum tuberosum) organelles showed that only mitochondrial fractions could elicit biotin formation in the plant-reconstituted system. Our data demonstrated that one or more unidentified factors from mitochondrial matrix (pea and potato) and from mitochondrial membranes (pea), in addition to the Bio2 protein, are obligatory for the conversion of dethiobiotin to biotin, highlighting the importance of mitochondria in plant biotin synthesis.

Scheme 1

Figure 1

Detection of biotin synthase activity in the in vitro heterologous system. A, Biotin synthase activity was measured by the conversion of [³H]DTB to [³H]biotin (“Materials and Methods”). Substrate and product were separated by thin-layer chromatography (TLC) and detected by phosphorimaging Analysis. Lane 1, DTB and biotin standards; lane 2, assay with 1.5 mg of protein extract from untransformed BL21 E. coli strain; lane 3, assay with 1.5 mg of protein extract from BL21 E. coli cells overproducing Bio2 from Arabidopsis. Reaction mixtures were incubated for 2 h. B, Quantitative analysis of radioactive spots in A, using ImageQuant software (Molecular Dynamics, Sunnyvale, CA), by the area quantitation method with peak finder.

Figure 2

Optimization of the biotin synthase reaction medium from the in vitro heterologous system. Biotin synthase activity was measured by the conversion of [³H]DTB to [³H]biotin. Biotin formed was determined by TLC analysis and phosphorimaging quantitation (“Materials and Methods”). Complete reaction medium consisted of 1.5 mg of protein extract from BL21 E. coli cells overproducing Bio2 from Arabidopsis, 10 mm dithiothreitol (DTT), 0.5 mm Fe(NH4)₂(SO4)₂, 1 mm NADPH, 10 mm KCl, 0.2 mm Ado-Met, 5 mm Fru-1,6-bisP, 0.5 mm l-Cys, 0.1 mm thiamin pyrophosphate (TPP), 20 mm l-Asn, and 150 μm [³H]DTB in 50 mm Tris-HCl, pH 8 [lane BL21(DE3)/Bio2]. The effect of omissions, as indicated, was then investigated. An assay with complete reaction medium except for substitution of the protein extract from Bio2-overproducing strain by a protein extract from untransformed bacteria, was run as negative control [lane BL21(DE3)]. The data are from a representative experiment repeated three times. Fe²⁺ = Fe(NH4)₂(SO4)₂.

Figure 3

Biotin synthase reaction time course in the in vitro heterologous system. Biotin synthase activity was assayed under optimized assay conditions, in the presence of 0.5, 1, and 1.5 mg of protein extract from BL21 E. coli cells overproducing Bio2 from Arabidopsis, respectively, in a total volume of 100 μL. Biotin produced was determined by TLC analysis and phosphorimaging quantitation (conversion of [³H]DTB to [³H]biotin).

Figure 4

Effect of DTB and Ado-Met on the activity of biotin synthase in a protein extract from E. coli cells overproducing Bio2 from Arabidopsis (in vitro heterologous system). Biotin synthase activity was measured under optimized conditions by varying the concentrations of one of the substrates (DTB or Ado-Met) while keeping the other substrates and cofactors at saturating levels (see “Materials and Methods”). Biotin produced was determined by a turbidimetric microbiological method using Lactobacillus plantarum. From the curves, apparent K_m value for d-DTB (A) and K_0.5 for Ado-Met (B) were determined. The lines represent nonlinear regressions to the Michaelis-Menten equation (A) or the Hill equation (B), using KaleidaGraph, as described in “Materials and Methods.”

Figure 5

Effect of acidomycin on the activity of biotin synthase in a protein extract from E. coli cells overproducing Bio2 from Arabidopsis (in vitro heterologous system). Biotin synthase activity was measured under optimized conditions with 1.5 mg of protein extract. Reaction mixtures were incubated for 2 h. A, Biotin formed was determined by TLC analysis and phosphorimaging quantitation (conversion of [³H]DTB to [³H]biotin). Lane 1, DTB and biotin standards; lane 2, assay in the absence of acidomycin; lane 3, assay in the presence of 400 μm acidomycin. B, Effect of increasing concentrations of acidomycin on biotin synthase activity. Biotin produced was determined by a turbidimetric microbiological method using L. plantarum.

Figure 6

Activation of purified recombinant biotin synthase by plant organelle extracts (plant-reconstituted system). A, Biotin synthase activity was measured under optimized conditions with 0.5 nmol purified Bio2 protein from Arabidopsis alone (none), or in the presence of 0.5 mg of protein extracts from isolated pea (Pisum sativum) leaf organelle (chloroplasts and mitochondria) subfractions, added separately or in combination, as indicated on the figure. B, Biotin synthase activity was measured under optimized conditions with 0.5 nmol purified Bio2 protein from Arabidopsis, in the presence of 0.5 mg of protein extracts from isolated potato (Solanum tuberosum) tuber mitochondria subfractions, added separately or in combination, as indicated in the figure. Biotin produced was determined by a turbidimetric microbiological method using L. plantarum. The error bars represent sds from three independent measurements, and when not shown fall within the column. St, Stroma; Mb, membranes; Ma, matrix).