Photoaffinity Labeling and Mutational Analysis of 24-C-sterol Methyltransferase Defines the AdoMet Binding Site

Overview

Journal Lipids

Specialty Biochemistry

Date 2008 Jun 20

PMID 18563465

Citations 6

Authors

Pruthvi Jayasimha

W David Nes

Affiliations

Soon will be listed here.

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.

Citing Articles

Druggable Sterol Metabolizing Enzymes in Infectious Diseases: Cell Targets to Therapeutic Leads.

Nes W, Chaudhuri M, Leaver D Biomolecules. 2024; 14(3).

PMID: 38540670 PMC: 10968281. DOI: 10.3390/biom14030249.

De novo phytosterol synthesis in animals.

Michellod D, Bien T, Birgel D, Violette M, Kleiner M, Fearn S Science. 2023; 380(6644):520-526.

PMID: 37141360 PMC: 11139496. DOI: 10.1126/science.add7830.

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

Zhou W, Fisher P, Vanderloop B, Shen Y, Shi H, Maldonado A J Lipid Res. 2019; 61(2):192-204.

PMID: 31548366 PMC: 6997595. DOI: 10.1194/jlr.RA119000317.

Combined Strategies to Improve the Expression of Recombinant Sterol C24-Methyltransferase from Leishmania braziliensis in E. coli.

Freitas H, Pires A, Castilho M Mol Biotechnol. 2018; 60(4):271-278.

PMID: 29488127 DOI: 10.1007/s12033-018-0069-4.

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

Kidane M, Vanderloop B, Zhou W, Thomas C, Ramos E, Singha U J Lipid Res. 2017; 58(12):2310-2323.

PMID: 29042405 PMC: 5711494. DOI: 10.1194/jlr.M079418.

References

Nes W, McCourt B, Marshall J, Ma J, Dennis A, Lopez M . Site-Directed Mutagenesis of the Sterol Methyl Transferase Active Site from Saccharomyces cerevisiae Results in Formation of Novel 24-Ethyl Sterols. J Org Chem. 2001; 64(5):1535-1542. DOI: 10.1021/jo9819943. View

Nes W, Jayasimha P, Zhou W, Kanagasabai R, Jin C, Jaradat T . Sterol methyltransferase: functional analysis of highly conserved residues by site-directed mutagenesis. Biochemistry. 2004; 43(2):569-76. DOI: 10.1021/bi035257z. View

Roberts C, McLeod R, Rice D, Ginger M, Chance M, Goad L . Fatty acid and sterol metabolism: potential antimicrobial targets in apicomplexan and trypanosomatid parasitic protozoa. Mol Biochem Parasitol. 2003; 126(2):129-42. DOI: 10.1016/s0166-6851(02)00280-3. View

Schubert H, Blumenthal R, Cheng X . Many paths to methyltransfer: a chronicle of convergence. Trends Biochem Sci. 2003; 28(6):329-35. PMC: 2758044. DOI: 10.1016/S0968-0004(03)00090-2. View

Shields D, Altarejos J, Wang X, Agellon L, Vance D . Molecular dissection of the S-adenosylmethionine-binding site of phosphatidylethanolamine N-methyltransferase. J Biol Chem. 2003; 278(37):35826-36. DOI: 10.1074/jbc.M306308200. View

Nes W, Song Z, Dennis A, Zhou W, Nam J, Miller M . Biosynthesis of phytosterols. Kinetic mechanism for the enzymatic C-methylation of sterols. J Biol Chem. 2003; 278(36):34505-16. DOI: 10.1074/jbc.M303359200. View

Velkov T, Lawen A . Photoaffinity labeling of the N-methyltransferase domains of cyclosporin synthetase. Photochem Photobiol. 2003; 77(2):129-37. DOI: 10.1562/0031-8655(2003)077<0129:plotnm>2.0.co;2. View

Nes W, Marshall J, Jia Z, Jaradat T, Song Z, Jayasimha P . Active site mapping and substrate channeling in the sterol methyltransferase pathway. J Biol Chem. 2002; 277(45):42549-56. DOI: 10.1074/jbc.M204223200. View

McCammon M, Parks L . Inhibition of sterol transmethylation by S-adenosylhomocysteine analogs. J Bacteriol. 1981; 145(1):106-12. PMC: 217250. DOI: 10.1128/jb.145.1.106-112.1981. View

10.

Moreau R, Whitaker B, Hicks K . Phytosterols, phytostanols, and their conjugates in foods: structural diversity, quantitative analysis, and health-promoting uses. Prog Lipid Res. 2002; 41(6):457-500. DOI: 10.1016/s0163-7827(02)00006-1. View

11.

Song Z, Nes W . Sterol biosynthesis inhibitors: potential for transition state analogs and mechanism-based inactivators targeted at sterol methyltransferase. Lipids. 2007; 42(1):15-33. DOI: 10.1007/s11745-006-3017-1. View

12.

Nes W, Sinha A, Jayasimha P, Zhou W, Song Z, Dennis A . Probing the sterol binding site of soybean sterol methyltransferase by site-directed mutagenesis: functional analysis of conserved aromatic amino acids in Region 1. Arch Biochem Biophys. 2005; 448(1-2):23-30. DOI: 10.1016/j.abb.2005.08.022. View

13.

Malone T, Blumenthal R, Cheng X . Structure-guided analysis reveals nine sequence motifs conserved among DNA amino-methyltransferases, and suggests a catalytic mechanism for these enzymes. J Mol Biol. 1995; 253(4):618-32. DOI: 10.1006/jmbi.1995.0577. View

14.

Syed S, Kim S, Paik W . Identification of the S-adenosyl-L-methionine binding site of protein-carboxyl O-methyltransferase using 8-azido-S-adenosyl-L-methionine. Biochemistry. 1993; 32(9):2242-7. DOI: 10.1021/bi00060a016. View

15.

Som S, Friedman S . Direct photolabeling of the EcoRII methyltransferase with S-adenosyl-L-methionine. J Biol Chem. 1990; 265(8):4278-83. View

16.

Subbaramaiah K, Simms S . Photolabeling of CheR methyltransferase with S-adenosyl-L-methionine (AdoMet). Studies on the AdoMet binding site. J Biol Chem. 1992; 267(12):8636-42. View

17.

Bloch K . Sterol structure and membrane function. CRC Crit Rev Biochem. 1983; 14(1):47-92. DOI: 10.3109/10409238309102790. View

18.

Rodriguez R, Low C, Bottema C, Parks L . Multiple functions for sterols in Saccharomyces cerevisiae. Biochim Biophys Acta. 1985; 837(3):336-43. DOI: 10.1016/0005-2760(85)90057-8. View

19.

Chavan A, Ensor C, Wu P, Haley B, Tai H . Photoaffinity labeling of human placental NAD(+)-linked 15-hydroxyprostaglandin dehydrogenase with [alpha-32P]2N3NAD+. Identification of a peptide in the adenine ring binding domain. J Biol Chem. 1993; 268(22):16437-42. View

20.

Kornblatt J, Muzac I, Lim Y, Ahn J, Ibrahim R . Role of Serine 286 in cosubstrate binding and catalysis of a flavonol O-methyltransferase. Biochem Cell Biol. 2004; 82(5):531-7. DOI: 10.1139/o04-054. View