Gene Cloning And Enzymatic Synthesis Of The Substrates Of Two Uroporphyrinogen Decarboxylases From Maize

Uroporphyrinogen decarboxylase (UROD) is a key enzyme in a branch pathway of plant tetrapyrroles shch as chlorophyll, heme and phytochrome. It catalyzes uroporphyrinogen I or III to coproporphyrinogen I or III by decarboxylation. In higher plant, there are two kinds of UROD. Both of them are expressed in Aribidopsis thaliana. UROD is associated with thioredoxins. However, the molecular mechanism of the regulation is not known. The UROD substrates, uroporphyrinogen I and III, are unstable. They can be synthetized enzymatically using stable compound 5-aminolevulinate as the pre-substrate. In this study, we cloned two genes encoding the mature URODs from maize and expressed them in Escherchia coli. Using the rapidly purified three recombiniant hexahistidine-tagged enzymes from E.coli, We synthetized uroporphyrinogen in vitro. The results are shown as followed:1. The cleavage sites of two UROD signal pepetides from maize were analyzed using the ChloroP 1.1. The restriction enzyme sites of two UROD gene fragments encoding the mature enzymes were analyzed using NEBcutter V 2.0. The primers for amplified maize UROD genes were designed based on the above-mentioned bioinfomatics analysis. The total RNA was extracted from the maize inbred B73 whose genome sequence has been sequenced. The RT-PCR was carried out and the products were detected approximately 1000 bp. Sequencing analyzes showed there were five mutations in the cloned gene fragment encoding mature UROD2. They were corrected respectively. There is one mutation in the cloned gene fragment encoding mature UROD1, as shown by the sequencing analysis. Since the mutated nucleotide encodes the similar characteristic amino acid residue, it is not essential to correct this mutation.2 The cloned genes encoding two UROD cut with the restriction enzymes were inserted into the different expression vectors with the same treatment respectively. No obvious expression protein bands were observed in SDS-PAGE, even with the rare tRNA codons coexpression.3 The enzymes 5-aminolevulinate dehydratase, porphobilinogen deaminase and uroporphyrinogen synthase encoded by hemB, hemC and hemD in E.coli. Three genes were amplified by PCR using the E. coli strain DH 5αgenomic DNA as the template extracted with CTAB. They were degested with restrictive enzymes, inserted into the individual expression vectors with the same desgestion, and sequenced respectively. The gene products with the hexahistidine tag at N terminus were overexpressed and purified using Ni-NTA affinity chramatography. The SDS-PAGE ananlyses revealed that the purified proteins were high homogeniety.4 The uroporphyrinogen compounds were synthesized with some purified three enzymes and 5-aminolevulinate. The oxidized uroporphyrinogen compounds displayed the red colour, suggesting that the purified three enzymes have the high activity.In conclusion, two uroporphyrinogen decarboxylases from maize for gene cloning and enzymatic synthesis of the substrates will faciliate the study of the function of UROD, of the gene expression level in maize leaves, and of their molecular regulating mechanism targeted by thioredoxin.