Abstract
Candida albicans produces a single myristoyl-CoA:protein N-myristoyltransferase (Nmt) that is essential for its viability. An ADP-ribosylation factor (Arf) is included among the few cellular protein substrates of this enzyme. An octapeptide (GLYASKLS-NH2) derived from a N-terminal Arf sequence was used as the starting point to identify elements critical for recognition by the acyltransferases's peptide-binding site. In vitro kinetic studies, employing purified Nmt and a panel of peptides with single Ala substitutions at each position of GLYASKLS-NH2, established that its Gly1, Ser5, and Lys6 residues play predominant roles in binding. ALYASKLS-NH2 was found to be an inhibitor competitive for peptide (Ki = 15.3 +/- 6.4 microM) and noncompetitive for myristoyl-CoA (Ki = 31.2 +/- 0.7 microM). A survey of 26 derivatives of this inhibitor, representing (i) a complete alanine scan, (ii) progressive C-terminal truncations, and (iii) manipulation of the physical-chemical properties of its residues 1, 5, and 6, confirmed the important stereochemical requirements for the N-terminal amine, the beta-hydroxyl of Ser5, and the epsilon-amino group of Lys6. Remarkably, replacement of the the N-terminal tetrapeptide of ALYASKLS-NH2 with an 11-aminoundecanoyl group produced a competitive inhibitor, 11-aminoundecanoyl-SKLS-NH2, that was 38-fold more potent (Ki = 0.40 +/- 0.03 microM) than the starting octapeptide. Removing the primary amine (undecanoyl-SKLS-NH2), or replacing it with a methyl group (dodecanoyl-SKLS-NH2), resulted in 26- and 34-fold increases in IC50, confirming the important contribution of the amine to recognition. Removal of LeuSer from the C terminus (11-aminoundecanoyl-SK-NH2) yielded a competitive dipeptide inhibitor with a Ki (11.7 +/- 0.4 microM) equivalent to that of the starting octapeptide, ALYASKLS-NH2. Substitution of Ser with homoserine, cis-4-hydroxyproline, or tyrosine reduces potency by 3-70-fold, emphasizing the requirement for proper presentation of the hydroxyl group in the dipeptide inhibitor. Substituting D- for L-Lys decreases its inhibitory activity >100-fold, while deletion of the epsilon-amino group (Nle) or masking its charge (epsilon-N-acetyl-lysine) produces 4-7-fold attenuations. L-His, but not its D-isomer, can fully substitute for L-Lys, producing a competitive dipeptide inhibitor with similar potency (Ki = 11.9 +/- 1.0 microM). 11-Aminoundecanoyl-SK-NH2 and 11-aminoundecanoyl-SH-NH2 establish that a simple alkyl backbone can maintain an appropriate distance between three elements critical for recognition by the fungal enzyme's peptide-binding site: a simple omega-terminal amino group, a beta-hydroxyl, and an epsilon-amino group or an imidazole. These compounds contain one peptide bond and two chiral centers, suggesting that it may be feasible to incorporate these elements of recognition, or functionally equivalent mimics, into a fully de-peptidized Nmt inhibitor.
Highlights
Itive dipeptide inhibitor with similar potency (Ki ؍ 11.9 ؎ 1.0 M). 11-Aminoundecanoyl-SK-NH2 and 11-aminoundecanoyl-SH-NH2 establish that a simple alkyl backbone can maintain an appropriate distance between three elements critical for recognition by the fungal enzyme’s peptide-binding site: a simple -terminal amino group, a -hydroxyl, and an ⑀-amino group or an imidazole
Peptide Recognition by C. albicans Nmt toylated in the NMT/nmt⌬ strain, whether it is grown in the presence or absence of myristate at 24 or 37°C
Selection of a Parental Peptide to Define Elements Required for Recognition by C. albicans Nmt—ADP-ribosylation factor (Arf) proteins are produced in a wide variety of species and many are known to be substrates for Nmts in vivo (e.g. Refs. 20 –24)
Summary
Myristoyl-CoA:protein N-myristoyltransferase; Arf, ADP-ribosylation factor; t-Boc, tert-butyloxycarbonyl; HPLC, high performance liquid chromatography. Differences in peptide substrate specificities have been noted among orthologous Nmts in vitro [4, 9, 12, 15, 16] These differences can be exploited to develop species-specific enzyme inhibitors. To design such inhibitors of C. albicans Nmt, the subsite specificity of the enzyme for peptide ligands needs to be defined. To begin this process, we have systematically replaced each amino acid residue in a known, high affinity, Arf-derived octapeptide substrate with alanine. Having identified the importance of residues 1, 5, and 6 in molecular recognition, we synthesized an additional series of peptides and de-peptidized analogs containing various substituents at these critical positions
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