Naslov
Amino acid residues important for catalytic activity of Streptomyces rimosus lipase

Autori
Bielen, Ana ; Vujaklija, Dušica

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Book of Abstracts of EMBO Young Scientist Forum / - , 2009

Skup
EMBO Young Scientist Forum

Mjesto i datum
Zagreb, Hrvatska, 15.06.2009. - 17.06.2009

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
Streptomyces rimosus lipase; amino acid residues; catalytic activity

Sažetak
Extracellular lipase from Streptomyces rimosus – SrL (Q93MW7) belongs to the GDSL family of lipolytic enzymes. Recently, SrL and many other GDSL enzymes were further classified as SGNH hydrolases due to the presence of four invariant and functionally important residues: serine (S), glycine (G), asparagine (N), and histidine (H) placed in the conserved motifs I, II, III and V, respectively. Catalytic triad consists of serine, histidine and often aspartic acid that is positioned three residues upstream of histidine. Glycine and asparagine, together with catalytic serine, serve as proton donors to the oxyanion hole. Although SGNH enzymes have a diverse range of hydrolytic functions and could be used in many industrial applications, there is still much to be learned about their structural characteristics and enzymatic mechanisms. The aim of this work was to identify amino acid residues essential for catalytic activity of SrL. The structure of this enzyme has not been solved. Based on the protein sequence alignment, it was predicted that catalytic triad was formed of Ser10 and His216, and Asp83 or Asn213 as the third residue. Gly54 and Asn82 were predicted to be involved in forming an oxyanion hole. Site-directed mutagenesis has been applied to obtain enzymes with following mutations: Ser10Ala, His216Ala, Asp83Ala, Asn213Ala, Asn213Asp, Gly54Ala and Asn82Ala. Purified proteins were analyzed by CD spectroscopy and it was shown that none of the mutations caused significant changes in secondary structure. Thus, we have concluded that differences in specific activity of mutated enzymes could be assigned to the specific mutation of particular amino acid residue. The activities of wild type enzyme and mutants toward standard lipase substrate, p-nitrophenyl palmitate (pNPP), were measured. It was shown that Ser10Ala and His216Ala have almost completely lost pNPP hydrolyzing activity (>2500-fold lower activity was observed), thus confirming their crucial role in catalysis. Further, Gly54Ala and Asn82Ala mutants had residual activity of 58% and 2%, respectively. Asp83 was ruled out as a third catalytic triad residue since Asp83Ala mutant showed activity similar to the wild type lipase. Our results pointed out that the third catalytic residue is Asn213, since Asn213Ala mutant displayed 57% of the wild type activity. This is interesting since the catalytic triad usually consists of Ser, His and an acidic residue. When this asparagine was replaced by aspartate (Asn213Asp), the activity increased to 76% but it still did not reach the full activity of the wild type.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

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