Characterization of 4-hydroxyphenylpyruvate dioxygenases, inhibition by herbicides and engineering for herbicide tolerance in crops.

4-Hydroxyphenylpyruvate dioxgenase (HPPD) enzymes from rat and from several plants contained only about a single inhibitor-binding active site per dimer which matched the content of iron in the purified Arabidopsis thaliana and Avena sativa enzymes. The dimeric HPPDs were about 10 fold more catalytically active than the tetrameric P. fluorescens enzyme with kcat/KmHPP values ranging from 0.8 to 2.5 s-1 µM-1. Most were also highly sensitive to herbicides with, for example, Ki values for mesotrione ranging from 25 to 100 pM. Curiously HPPDs from cool climate grasses were much less herbicide-sensitive. When likewise expressed in Nicotinia tabacum, Avena sativa HPPD, Ki value of 11 nM for mesotrione, conferred far greater tolerance to mesotrione (CallistoTM) than did any of the more sensitive HPPDs. Targeted mutagenesis of the Avena HPPD led to the discovery of 4 mutations imparting improved inherent tolerance, defined as the ratio of Ki to KmHPP, by about 16 fold without any loss of catalytic activity. The Nicotinia line with the highest expression of this quadruple mutant exhibited substantial resistance even up to a 3 kg/ha post-emergence application of mesotrione. The maximum observed expression level of heterologous plant HPPDs in tobacco was ca. 0.35% of the total soluble protein whereas the endogenous tobacco HPPD constituted only ca. 0.00075%. At such high expression even HPPDs with impaired catalytic activity could be effective. A quintuple mutant Avena sativa HPPD conferred substantial tolerance across a broad range of HPPD herbicide chemistries despite being only ca. 5 % as catalytically active as the wild type enzyme. Testing various wild type and mutant HPPDs in tobacco revealed that tolerance to field rates of herbicide generally requires about two order of magnitude increases in both inherent herbicide tolerance and expression relative to endogenous levels. This double hurdle may explain why target-site based resistance to HPPD-inhibiting herbicides has been slow to evolve in weeds.

The metal-binding features of the recombinant mussel Mytilus edulis MT-10-IV metallothionein.

In contrast with the paradigmatic mammalian metallothioneins (MTs), mollusc MT systems consist at least of a high-cadmium induced form, possibly involved in detoxification, and another isoform either constitutive or regulated by essential metals and probably associated with housekeeping metabolism. With the aim of providing a deeper characterization of the coordination features of a molluscan MT peptide of the latter kind, we have analyzed here the metal-binding abilities of the recombinant MeMT-10-IV isoform of Mytilus edulis (MeMT). Also, comparison with other MTs of this type has been undertaken. A synthetic complementary DNA was constructed, cloned and expressed into two Escherichia coli systems. Upon zinc coordination, MeMT folds in vivo into highly chiral and stable Zn(7) complexes, with an exceptional reluctance to fully substitute cadmium(II) and/or copper(I) for zinc(II). In vivo cadmium binding leads to homometallic Cd(7) complexes that structurally differ from any of the in vitro prepared Cd(7) complexes. Homometallic Cu-MeMT can only be obtained in vitro from Zn(7)-MeMT after a great molar excess of copper(I) has been added. In vivo, two different heterometallic Zn,Cu-MeMT complexes are recovered, which nicely correspond to two distinct stages of the in vitro zinc/copper replacement. These MeMT metal-binding features are consistent with a physiological role related to basal/housekeeping metal, mainly zinc, metabolism, and confirm the correspondence between the MeMT gene response pattern and the functional properties of the encoded protein.