Journal
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Volume 425, Issue 1, Pages 65-76Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.abb.2004.02.015
Keywords
monoterpene synthases; monoterpene cyclases; terpene cyclase structure-function; geranyl diphosphate cyclization; turpentine biosynthesis; limonene; pinene; Abies grandis; grand fir
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Funding
- NIGMS NIH HHS [GM 31354] Funding Source: Medline
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(-)-(4S)-limonene synthase (LS) and (-)-(4S)-limonene/(-)-(1S, 5S)-alpha-pinene synthase (LPS) from grand fir (Abies grandis) exhibit nearly 91% sequence identity (93% similarity) at the amino acid level, yet produce very different mixtures of monoterpene olefins. To elucidate critical amino acids involved in determining monoterpene product distribution, a combination of domain swapping and reciprocal site-directed mutagenesis was carried out between these two enzymes. Exchange of the predicted helix D through F region in LS gave rise to an LPS-like product outcome, whereas reciprocal substitutions of four amino acids in LPS (two in the predicted helix D and two in the predicted helix F) altered the product distribution to that intermediate between LS and LPS, and resulted in a 5-fold increase in relative velocity. These results, in conjunction with modeling of the two enzymes, suggest that amino acids in the predicted D through F helix regions are critical for product determination. (C) 2004 Elsevier Inc. All rights reserved.
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