The missing link in the fungal D-galacturonate pathway: identification of the L-threo-3-deoxy-hexulosonate aldolase.
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-3-deoxy-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.
Hilditch
Satu
S
VTT Technical Research Centre of Finland, Tietotie 2, Espoo, 02044 VTT, Finland.
Berghäll
Suvi
S
Kalkkinen
Nisse
N
Penttilä
Merja
M
Richard
Peter
P
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D000446:Aldehyde-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D006603:Hexuronic Acids / Q000378:metabolism*; D008969:Molecular Sequence Data; D064430:NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases; D016366:Open Reading Frames / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D018076:DNA, Complementary; D005656:Fungal Proteins; D006603:Hexuronic Acids; D011994:Recombinant Proteins; C007819:galacturonic acid; D000429:Alcohol Oxidoreductases; D064430:NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases; C477073:D-galacturonate reductase; D000446:Aldehyde-Lyases