Dihydropteroate synthase (H2Pte synthase) is the target of the sulfur‐based antimalarial drugs, which are frequently used in synergistic combination with inhibitors of dihydrofolate reductase (H2folate reductase) to combat chloroquine‐resistant malaria. We have isolated the H2Pte synthase coding sequence of the most pathogenic human parasite Plasmodium falciparum. It forms part of a longer coding sequence, located on chromosome 8, that also specifies 6‐hydroxymethyl‐7,8‐dihydropterin pyrophosphokinase (CH2OH‐H2pterinPP kinase) at its 5′ proximal end. This domain is unusually large, with two long insertions relative to other CH2OH‐H2pterinPP kinase molecules. To investigate a possible genetic basis for clinical resistance to sulfa drugs, we sequenced the complete H2Pte synthase domains from eleven isolates of P. falciparum with diverse geographical origins and levels of sulfadoxine resistance. Overall, point mutations in five positions were observed, affecting four codons. Parasite lines exhibiting high‐level resistance were found to carry either a double mutation, altering both Ser436 and Ala613, or a single mutation affecting Ala581. The mutations at positions 436 and 581 have the same location relative to each of two degenerate repeated amino acid motifs that are conserved across all other known H2Pte synthase molecules. The amino acid alteration at residue 613 is identically positioned relative to a different conserved motif. The fourth amino acid residue (437) affected by mutation, though adjacent to the apparently crucial residue 436, shows no obvious correlation with resistance. Although these mutations have no exact counterparts in any other organism, that at position 581 falls within a region of three amino acids where H2Pte synthase is modified in various ways in a number of sulfonamide‐resistant pathogenic bacteria. Copy‐number analysis indicated that there was no amplification of the H2Pte synthase domain in resistant parasite lines of P. falciparum, compared to sensitive lines.

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The cpb genes of Leishmania mexicana encode stageregulated, cathepsin L-like cysteine proteinases that are leishmanial virulence factors. Field inversion gel electrophoresis and genomic mapping indicate that there are 19 cpb genes arranged in a tandem array. Five genes from the array have been sequenced and their expression analyzed. The first two genes, cpb1 and cpb2, differ significantly from the remaining 17 copies (cpb3-cpb19) in that: 1) they are expressed predominantly in metacyclic promastigotes (the form in the insect vector which is infective to mammalian macrophages) rather than amastigotes (the form that parasitizes mammals); 2) they encode enzymes with a truncation in the COOHterminal extension, an unusual feature of these cysteine proteinases of trypanosomatids. Transfection of cpb1 into a cpb null mutant resulted in expression of an active enzyme that was shown by immunogold labeling with anti-CPB antibodies to be targeted to large lysosomes. This demonstrates that the 100-amino acid COOH-terminal extension is not essential for the activation or activity of the enzyme or for its correct intracellular trafficking. Transfection into the cpb null mutant of different copies of cpb and analysis of the phenotype of the lines showed that individual isoenzymes differ in their substrate preferences and ability to restore the loss of virulence associated with the null mutant. Comparison of the predicted amino acid sequences of the isoenzymes implicates five residues located in the mature domain (Asn 18 , Asp 60 , Asn 61 , Ser 64 , and Tyr 84 ) with differences in the activities of the encoded isoenzymes. The results suggest that the individual isoenzymes have distinct roles in the parasite's interaction with its host. This complexity reflects the adaptation of cathepsin Llike cysteine proteinases to diverse functions in parasitic protozoa.

Genome sequence analyses predict many proteins that are structurally related to proteases but lack catalytic residues, thus making functional assignment difficult. We show that one of these proteins (ACN-1), a unique multi-domain angiotensin-converting enzyme (ACE)-like protein from Caenorhabditis elegans, is essential for larval development and adult morphogenesis. Green fluorescent protein-tagged ACN-1 is expressed in hypodermal cells, the developing vulva, and the ray papillae of the male tail. The hypodermal expression of acn-1 appears to be controlled by nhr-23 and nhr-25, two nuclear hormone receptors known to regulate molting in C. elegans. acn-1(RNAi) causes arrest of larval development because of a molting defect, a protruding vulva in adult hermaphrodites, severely disrupted alae, and an incomplete seam syncytium. Adult males also have multiple tail defects. The failure of the larval seam cells to undergo normal cell fusion is the likely reason for the severe disruption of the adult alae. We propose that alteration of the ancestral ACE during evolution, by loss of the metallopeptidase active site and the addition of new protein modules, has provided opportunities for novel molecular interactions important for post-embryonic development in nematodes.The large number of protease genes in animal genomes reflects the widespread importance of proteolysis to animal physiology and development. The rich functional diversity of proteases can be attributed to the variety of catalytic mechanisms and protein structures, some of which are of a modular design, offering different levels of structural and functional complexity (1). The MEROPS data base (merops.sanger.ac.uk; release 6.3) (2) catalogs 551, 553, 563, 366, and 367 proteases for human, mouse, Drosophila melanogaster, Anopheles gambiae, and Caenorhabditis elegans, respectively. The majority of these enzymes have not been studied at the physiological and biochemical level, but it has been possible to classify most of them into families based on statistically significant similarities in primary protein structure (2).Surprisingly, a significant proportion (12-25%) of the total number of the predicted protease-like genes in the human, mouse, D. melanogaster, A. gambiae, and C. elegans genomes code for proteins that lack one or more catalytic residues and are therefore classified as "non-peptidase" family members. Some of these genes may be nonfunctional pseudogenes, but many have probably lost the catalytic activity of an ancestral protein while acquiring new functions. These non-peptidase proteins are structurally related to proteases distributed across all classes, but homologues of metallopeptidases are particularly well represented. One such protein is UNC-71, a C. elegans member of the ADAMs family, which lacks a zinc binding site and yet has acquired important roles in development (3). Our attention has recently been drawn to the fact that several invertebrate members of the angiotensin-converting enzyme (ACE) 1 (EC 3.4.15.1, peptidyl dipeptidase A) famil...