The missing link in the fungal D-galacturonate pathway -: Identification of the L-threo-3-deoxy-hexulosonate aldolase

被引:42
作者
Hilditch, Satu
Berghall, Suvi
Kalkkinen, Nisse
Penttilae, Merja
Richard, Peter
机构
[1] VTT Tech Res Ctr, Espoo 02044, Finland
[2] Univ Helsinki, Inst Biotechnol, Helsinki 00014, Finland
关键词
D O I
10.1074/jbc.M704401200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The fungal path for the catabolism of D-galacturonate is only partially known. It is however distinctly different to the well-known bacterial path. The known elements of the fungal path are D-galacturonate reductase converting D-galacturonate to L-galactonate and L-galactonate dehydratase converting L-galactonate to L-threo-3-deoxy-hexulosonate (2-keto-3-deoxy-L-galactonate). Here we describe the missing link in this pathway, an aldolase converting L-threo-3-deoxy-hexulosonate to pyruvate and L-glyceraldehyde. Fungal enzymes converting L-glyceraldehyde to glycerol have been described previously. The L-threo-3deoxy-hexulosonate aldolase activity was induced in the mold Hypocrea jecorina ( Trichoderma reesei) during growth on D-galacturonate. The enzyme was purified from this mold and a partial amino acid sequence obtained. This sequence was then used to identify the corresponding gene from the H. jecorina genome. The deletion of the gene resulted in a strain unable to grow on D-galacturonate and accumulating L-threo-3-deoxy-hexulosonate. The open reading frame was cloned from cDNA and functionally expressed in the yeast Saccharomyces cerevisiae. A histidine-tagged protein was expressed, purified, and characterized. The enzyme catalyzed reaction was reversible. With L-threo-3-deoxy-hexulosonate as substrate the K-m was 3.5 mM and with pyruvate and L- glyceraldehyde the K-m were 0.5 and 1.2 mM, respectively.
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页码:26195 / 26201
页数:7
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