Photoaffinity Labeling and Mutational Analysis of 24‐C‐Sterol Methyltransferase Defines the AdoMet Binding Site

Abstract

Photolabeling and site-directed mutagenesis were performed on recombinant Saccharomyces cerevisiae 24-C-sterol methyltransferase (SMT) to elucidate the location and role of active site residues involved in AdoMet binding and catalysis. Bioinformatic analysis of the SMT revealed a ten amino acid segment, conserved between L124 and P133, associated with the Rossmann-like fold belonging to AdoMet-dependent methyltransferases. Irradiation of the SMT in the presence of [methyl-3H3]AdoMet directly photolabeled the protein. The specificity of photolabeling was demonstrated by inactivation experiments with structural analogs of AdoMet, including sinefungin. Trypsin digestion of the [methyl-3H3]AdoMet photolabeled Erg6p afforded a single radioactive band in SDS-PAGE gel of 4 kDa. HPLC purification of this material generated a single radioactive fraction. The corresponding 3H-AdoMet-peptide adduct was subjected to Edman sequencing and the first fifteen residues gave a sequence Gly120-Asp-Leu-Val-Leu-Asp-Val-Gly-Cys-Gly-Val-Gly-Gly-Pro-Ala134 that contained the predicted AdoMet binding site. Amino acid residues in the tryptic digest fragment considered to bind covalently with the AdoMet at Asp125, Cys128, Pro133 and Tyr153 were replaced with leucine and analyzed kinetically and by photolabeling inactivation experiments. The results indicate that one or both of Cys128 and Pro133 are covalently bound to AdoMet.

Sterol methyltransferase a target for anti-amoeba therapy: towards transition state analog and suicide substrate drug design

“…Sterol methylation carried out as the first (Δ 24(25) -substrate) C 1 -transfer reaction and second (Δ 24(28) -substrate) C 1 -transfer reaction is correlated to the corresponding 24-SMT (=SMT1) and 28-SMT (=SMT2) sequences detected in the Genebank. Substrate binding segments established in references 29 , 36 for sterol (Regions I, III and IV) and SAM (Region II) are highlighted by black and blue boxes, respectively. Conserved residues are in red.…”

“…After filtration and concentration, 22 µg total protein was loaded on 12% SDS-PAGE gel against a protein ladder (Bio-Rad) that ranged from 15 to 100 kDa. Next, five bands were excised from the gel corresponding to approximate molecular masses that range between 1 ) 15–25 kDa, 2 ) 25–35 KDa, 3 ) 35–45 kDa, 4 ) 45–55 kDa, and 5 ) 55–75 kDa, placed into a vial of 50% hydrogen peroxide, and left overnight at 60°C for digestion ( 29 ). To these vials was added scintillant and the radioactivity ( 3 H in dpm) measured by liquid scintillation counting.…”

Effect of substrate features and mutagenesis of active site tyrosine residues on the reaction course catalysed by Trypanosoma brucei sterol C-24-methyltransferase

“…Consequently, when considering similarity of substrate interactions in the Michaelis complex of fungal, plant and protozoan 24-SMTs in which a common 24(28) -olefin is produced, we independently reasoned that the central region of these enzymes is lined with aromatic and acidic amino acid residues whose positions and orientation appear to be responsible for producing the specific geometry that determines the product specificity that occurs with each 24-SMT [42, 43,45]. For the TbSMT, when the OH group of the aromatic residues located some distance away from the reactive carbocation intermediate at Tyr 177 and Try 208 are replaced with H (phenylalanine) catalytic activity decreases without a corresponding affect on the sterol composition in the mutant proteins.…”

“…To ascertain mechanistic inferences on the involvement of a cationic intermediate in catalysis by mutants prepared in the present study, we undertook modelling and docking experiments of the TbSMT to show putative interaction between protein and co-substrates sterol and AdoMet in the active site. The current modelling studies take into consideration our investigations that involved photoaffinity and chemical labelling and site-directed mutagenesis experiments in the Erg6p [27,[43][44][45]. The combination of these efforts reveal four substrate-binding segments for AdoMet and sterol referred to as Regions I-IV [45], and that tyrosine residues at positions 81, 192 and 223 (equivalent to Tyr 66 , Tyr 177 and Tyr 208 in TbSMT) represent contact amino acids for sterol-binding sites (Figure 6 and Supplementary Figure S6 at http://www.BiochemJ.org/bj/439/bj4390413add.htm).…”

A nematode sterol C4α-methyltransferase catalyzes a new methylation reaction responsible for sterol diversity

“…7A, supplemental Fig. 19) highlighting the four substrate binding segments for sterol and SAM, identified by us through chemical affinity labeling and site-directed mutagenesis of Erg6p (46)(47)(48)(49), shows a high degree of conservation in the three-dimensional structure of these enzymes, suggesting equivalence in the catalytic competence of 4-SMT and 24-SMT. The structural homology between 4-SMT and 24-SMT enzymes further suggests a common primordial ancestor early in the evolution of sterol biosynthetic pathways prior to the divergence of invertebrates from vertebrates.…”