The use of alternative substrates in the characterization of actin‐methylating and carnosine‐methylating enzymes

Abstract

Actin isolated from nearly every eukaryotic species contains approximately 1 mol 3-methylhistidine/ mol protein. His73 in actin has been shown, by protein sequencing, to be the site of methylation. The methylation occurs enzymically and post-translationally. A rabbit skeletal muscle myofibrillary fraction has previously been shown to contain a histidine methyltransferase activity that is actin specific. Detailed study of this enzyme has been hampered by lack of a suitable substrate assay. Naturally occurring actins are poor substrates for the enzyme, presumably due to prexistent methylation at His73. In this study, two potential alternative substrates have been investigated. These are a chicken p-actin expressed in Escherichia coli as a fusion protein with 80 amino acids of an influenza protein, NS1, and a synthetic peptide, Tyr-Pro-Ile-Glu-His-Gly-Ile-Ile-Thr, corresponding to residues 69 -77 of actin. Both substrates were covalently methylated at histidine residues in the presence of S-adenosylmethionine and partially purified enzyme fractions from rabbit muscle. In methylation experiments employing the fusion actin in the form of inclusion bodies, 3-methylhistidine is the major product, as is the case when soluble muscle or non-muscle actin is used. However, for the synthetic peptide, the methylated product primarily contained 1-methylhistidine and only a small amount of the isomeric 3-methylhistidine. Further investigations revealed that the peptide was recognized by carnosine N-methyltransferase, another histidine methyltransferase found in muscle tissue. Carnosine N-methyltransferase appears to copurify with the actin-methylating enzyme in preliminary fractionation experiments. Separation of the two methyltransferase activities is described.

A Novel 3-Methylhistidine Modification of Yeast Ribosomal Protein Rpl3 Is Dependent upon the YIL110W Methyltransferase

“…Another possibility is that the mammalian YIL110W/Hpm1 ortholog may modify its own Rpl3 ortholog as the amino acid sequence is highly conserved around the histidine 243 residue in yeast to mammals. Finally, it is possible that the mammalian ortholog may be the enzyme that converts the dipeptide carnosine (␤-Ala-His) to anserine (␤-Ala-1-methyl-His) (32,64). Our studies now open the door to exploring protein histidine methylation in other yeast proteins and in identifying histidine protein methyltransferases in other organisms.…”

“…Evidence has been presented for functional roles of methylation in actin polymerization and ATP hydrolysis (30) and in actin structure (4). The enzyme(s) responsible for the formation of 3-methylhistidine in actin and/or the myosin heavy chain has been partially purified (31,32), but no genes encoding protein histidine methyltransferases have been identified to date in any organism.…”

Molecular Identification of Carnosine N-Methyltransferase as Chicken Histamine N-Methyltransferase-Like Protein (HNMT-Like)

“…In the contrary, both anserine and carnosine N-methyltransferase activities have been reported in some mammals [3]. Raghavan and coworkers [11] reported a partial purification of carnosine N-methyltransferase from rabbit muscle, occurring to be an 85 kDa protein. Indeed, preliminary data from our laboratory indicate the presence of only one form of carnosine-methylating enzyme in rat muscle which exhibits molecular weight equal to 94 kDa, as estimated by the gel filtration.…”

“…On the other hand, very little is known about carnosine N-methyltransferase that catalyzes the synthesis of anserine. The enzyme has been only partially purified from various sources [10], [11] and shown to catalyze the transfer of methyl group of S-adenosyl-L-methionine to carnosine with a high substrate specificity, yielding anserine.…”

Post-translational modification and regulation of actin

“…These effects are thought to be due in part to methylated His-73 slowing the inorganic phosphate (Pi) release after ATP hydrolysis, leading to F-actin stabilization. Actin methylating enzymes have been identified [42,43], although it is unclear if they methylate His-73. Methylation also occurs in some species on a specific actin lysine residue (Lys-326; Tables 1, S2–3) and is post-translationally added to arginylated actin residues (Tables 1, S2–3; [44]).…”