Uml2 is a novel CalB-type lipase of Ustilago maydis with phospholipase A activity.
CalB of Pseudozyma aphidis (formerly named Candida antarctica) is one of the most widely applied enzymes in industrial biocatalysis. Here, we describe a protein with 66 % sequence identity to CalB, designated Ustilago maydis lipase 2 (Uml2), which was identified as the product of gene um01422 of the corn smut fungus U. maydis. Sequence analysis of Uml2 revealed the presence of a typical lipase catalytic triad, Ser-His-Asp with Ser125 located in a Thr-Xaa-Ser-Xaa-Gly pentapeptide. Deletion of the uml2 gene in U. maydis diminished the ability of cells to hydrolyse fatty acids from tributyrin or Tween 20/80 substrates, thus demonstrating that Uml2 functions as a lipase that may contribute to nutrition of this fungal pathogen. Uml2 was heterologously produced in Pichia pastoris and recombinant N-glycosylated Uml2 protein was purified from the culture medium. Purified Uml2 released short- and long-chain fatty acids from p-nitrophenyl esters and Tween 20/80 substrates. Furthermore, phosphatidylcholine substrates containing long-chain saturated or unsaturated fatty acids were effectively hydrolysed. Both esterase and phospholipase A activity of Uml2 depended on the Ser125 catalytic residue. These results indicate that Uml2, in contrast to CalB, exhibits not only esterase and lipase activity but also phospholipase A activity. Thus, by genome mining, we identified a novel CalB-like lipase with different substrate specificities.
Institut für Molekulare Mykologie, Heinrich-Heine-Universität, Universitätsstr. 1/26.12, 40225 Düsseldorf, Germany.
Christoph
C
Buerth
Filip
F
Kovacic
Janpeter
J
Stock
Marius
M
Terfrüchte
Susanne
S
Wilhelm
Karl-Erich
KE
Jaeger
Michael
M
Feldbrügge
Kerstin
K
Schipper
Joachim F
JF
Ernst
Denis
D
Tielker
D020816:Amino Acid Motifs; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D017353:Gene Deletion; D015870:Gene Expression; D010740:Phospholipases / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D014588:Ustilago / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D010740:Phospholipases