Finding of an isoleucine derivative of a recombinant protein for pharmaceutical use.
Protein modification generally occurs by addition to the amino acid side-chains of protein at the post-translational stage, for example, by enzymatic or chemical reactions after polypeptide synthesis. Recently, the recombinant hirudin analog CX-397, a potent thrombin inhibitor, was found to contain methylated Ile residues when it was overproduced by Escherichia coli in the absence of amino acids in the culture medium. The Ile derivatives, deduced to be beta-methylnorleucine [betaMeNle; (2S, 3S)-2-amino-3-methylhexanoic acid] by systematic chromatographic analysis, do not appear to be normal post-translational modifications of the protein because Ile has no functional group in its side-chain. We, therefore, propose that betaMeNle is biosynthesized by E. coli, activated by E. coli isoleucyl-tRNA synthetase (IleRS), then incorporated into the overproduced recombinant hirudin analog. The biosynthesis of betaMeNle in E. coli is thought to occur as follows: alpha-ketovalerate is synthesized from alpha-ketobutyrate by three Leu biosynthetic enzymes, alpha-isopropylmalate synthase (IPMS) (EC 4.1.3.12), alpha-isopropylmalate isomerase (ISOM) (EC 4.2.1.33) and beta-isopropylmalate dehydrogenase (IPMD) (EC 1.1.1.85), which have broad substrate specificities. alpha-Ketovalerate is then converted to alpha-keto-beta-methylcaproate by three Ile and Val biosynthetic enzymes, acetohydroxy acid synthase (AS) (EC 4.1.3.18), acetohydroxy acid isomeroreductase (IR) (EC 1.1.1.86) and dihydroxy acid dehydratase (DH) (EC 4.2.1.9). Finally, this is converted to betaMeNle by branched-chain amino acid transaminase (EC 2.6.1.42), one of the Ile and Val biosynthetic enzymes.
Muramatsu
Ryo
R
Pharmaceuticals and Biotechnology Laboratory, Japan Energy Corporation, 3-17-35 Niizo-Minami, Toda, Saitama 335-8502, Japan.
Misawa
Satoru
S
Hayashi
Hideya
H
D001426:Bacterial Proteins / Q000096:biosynthesis*; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000378:metabolism; D006629:Hirudins / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000737:chemistry; D007532:Isoleucine / Q000737:chemistry / Q000378:metabolism*; D007533:Isoleucine-tRNA Ligase / Q000378:metabolism; D009646:Norleucine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry; D004364:Pharmaceutical Preparations / Q000737:chemistry; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry; D013917:Thrombin / Q000037:antagonists & inhibitors
D001426:Bacterial Proteins; C100322:CX 397; D006629:Hirudins; D004364:Pharmaceutical Preparations; D011994:Recombinant Proteins; D007532:Isoleucine; C032957:beta-methylnorleucine; D009646:Norleucine; D013917:Thrombin; D007533:Isoleucine-tRNA Ligase