Directed evolution and rational approaches to improving <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis> deacetoxycephalosporin C synthase for cephalosporin production |
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Authors: | Kian-Sim Goo Chun-Song Chua Tiow-Suan Sim |
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Institution: | (1) Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Singapore, 117597, Singapore |
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Abstract: | It is approximately 60 years since the discovery of cephalosporin C in Cephalosporium acremonium. Streptomycetes have since been found to produce the structurally related cephamycin C. Studies on the biosynthetic pathways
of these two compounds revealed a common pathway including a step governed by deacetoxycephalosporin C synthase which catalyses
the ring-expansion of penicillin N to deacetoxycephalosporin C. Because of the therapeutic importance of cephalosporins, this
enzyme has been extensively studied for its ability to produce these antibiotics. Although, on the basis of earlier studies,
its substrate specificity was believed to be extremely narrow, relentless efforts in optimizing the in-vitro enzyme assay
conditions showed that it is able to convert a wide range of penicillin substrates differing in their side chains. It is a
member of 2-oxoglutarate-dependent dioxygenase protein family, which requires the iron(II) ion as a co-factor and 2-oxoglutarate
and molecular oxygen as co-substrates. It has highly conserved HXDX
n
H and RXS motifs to bind the co-factor and co-substrate, respectively. With advances in technology, the genes encoding this enzyme
from various sources have been cloned and heterologously expressed for comparative analyses and mutagenesis studies. A high
level of recombinant protein expression has also enabled crystallization of this enzyme for structure determination. This
review will summarize some of the earlier biochemical characterization and describe the mechanistic action of this enzyme
revealed by recent structural studies. This review will also discuss some of the approaches used to identify the amino acid
residues involved in binding the penicillin substrate and to modify its substrate preference for possible industrial application. |
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Keywords: | Mutational approach Industrial application Cephalosporin synthesis Penicillin Enzyme improvement |
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