Structure/function studies on a S-adenosyl-L-methionine-dependent uroporphyrinogen III C methyltransferase (SUMT), a key regulatory enzyme of tetrapyrrole biosynthesis.

The crystallographic structure of the Pseudomonas denitrificans S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferase (SUMT), which is encoded by the cobA gene, has been solved by molecular replacement to 2.7A resolution. SUMT is a branchpoint enzyme that plays a key role in the biosynthesis of modified tetrapyrroles by controlling flux to compounds such as vitamin B(12) and sirohaem, and catalysing the transformation of uroporphyrinogen III into precorrin-2. The overall topology of the enzyme is similar to that of the SUMT module of sirohaem synthase (CysG) and the cobalt-precorrin-4 methyltransferase CbiF and, as with the latter structures, SUMT has the product S-adenosyl-L-homocysteine bound in the crystal. The roles of a number of residues within the SUMT structure are discussed with respect to their conservation either across the broader family of cobalamin biosynthetic methyltransferases or within the sub-group of SUMT members. The D47N, L49A, F106A, T130A, Y183A and M184A variants of SUMT were generated by mutagenesis of the cobA gene, and tested for SAM binding and enzymatic activity. Of these variants, only D47N and L49A bound the co-substrate S-adenosyl-L-methionine. Consequently, all the mutants were severely restricted in their capacity to synthesise precorrin-2, although both the D47N and L49A variants produced significant quantities of precorrin-1, the monomethylated derivative of uroporphyrinogen III. The activity of these variants is interpreted with respect to the structure of the enzyme.

cis-peptide bond mimetic tetrazole analogs of the insect kinins identify the active conformation.

The insect kinin neuropeptides have been implicated in the regulation of water balance, digestive organ contraction, and energy mobilization in a number of insect species. A previous solution conformation study of an active, restricted-conformation cyclic analog, identified two possible turn conformations as the likely active conformation adopted by the insect kinins at the receptor site. These were a cisPro type VI beta-turn over C-terminal pentapeptide core residues 1-4 and a transPro type I-like beta-turn over core residues 2-5, present in a ratio of 60:40. Synthesis and evaluation of the diuretic activity of insect kinin analogs incorporating a tetrazole moiety, which mimics a cis peptide bond, identifies the active conformation as the former. The discovery of a receptor interaction model can lead to the development of potent agonist and antagonist analogs of the insect kinins. Indeed, in this study a tetrazole analog with D stereochemistry has been shown to demonstrate partial antagonism of the diuretic activity of natural insect kinins, providing a lead for more potent and effective antagonists of this critical neuropeptide family. The future development of mimetic agonists and antagonists of insect kinin neuropeptides will provide important tools to neuroendocrinologists studying the mechanisms by which they operate and to researchers developing new, environmentally friendly pest insect control strategies.