Please use this identifier to cite or link to this item: http://scholarbank.nus.edu.sg/handle/10635/27664
Title: Solubility studies, rational amino acid replacements and structural analyses of Streptomyces jumonjinensis isopenicillin N synthase
Authors: WONG SIEW PENG, ESTHER
Keywords: isopenicillin N synthase, Streptomyces jumonjinensis, site-directed mutagenesis, solubility, hydrophobic contacts, charge-charge interactions
Issue Date: 23-Feb-2004
Source: WONG SIEW PENG, ESTHER (2004-02-23). Solubility studies, rational amino acid replacements and structural analyses of Streptomyces jumonjinensis isopenicillin N synthase. ScholarBank@NUS Repository.
Abstract: Isopenicillin N synthase (IPNS) is a crucial enzyme catalyzing the formation of the first bioactive penicillin in the beta-lactam biosynthetic pathway. Of the IPNS isozymes isolated so far, Streptomyces jumonjinensis IPNS (sjIPNS) is inherently insoluble when expressed in Escherichia coli. To investigate how an enzymea??s molecular sequence influences its soluble expression, IPNS genes were cloned from S. jumonjinensis (M36687), S. fimbriatus (AF320779), S. clavuligerus (M19421) and Nocordia lactamdurans (X57310) for this study. Seven structural determinants that enhanced the solubility of sjIPNS were elucidated by computational analyses and supported by experimentation. Specific replacements of six solvent exposed loop residues, Ser19, Asp112, Glu120, Arg242, Thr308 and Thr309, and one buried alpha-helix residue, Tyr160, of sjIPNS successfully increased its soluble expression by ~6-8 folds. These mutations were purported to have replaced unfavorable interactions by either optimization of the hydrophobic contacts (Ser19Phe, Thr308Ala, Thr309Val or Thr309Leu) or optimization of charge-charge interactions (Asp112Glu, Glu120Gly and Arg242Thr). Furthermore, manipulation of various expression conditions including lowering the cultivation temperature to 18A?C and fusing sjIPNS to highly soluble E. coli NusA protein also resulted in ~3-8 folds improvement in the levels of soluble, active sjIPNS expressed. Obtaining solubly expressed active enzymes may thus be achievable by manipulating their molecular sequences and folding structures optimally.
URI: http://scholarbank.nus.edu.sg/handle/10635/27664
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