Catalytically important amino-acid residues of abalone alginate lyase HdAly assessed by site-directed mutagenesis

Abstract

Abbreviations: HdAly, Haliotis discus hannai alginate lyase; PL, polysaccharide lyase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.

Isolation and characterization of two alginate lyase isozymes, AkAly28 and AkAly33, from the common sea hare Aplysia kurodai

“…The N-terminal amino-acid sequences of AkAly28 and AkAly33 showed practically no identities with those of abalone and turban shell enzymes; however, the internal sequences of AkAly28 and AkAly33 showed considerably high similarity with the corresponding sequences of abalone and turban shell enzymes ( Table 2). The K95, which was predicted as a key residue for the catalytic action of HdAly (Yamamoto et al 2008), was conserved in a lysylendopeptidyl fragment (L-4) of AkAly33. The importance of this lysine residue was also reported in the Chlorella virus PL-14 enzyme (Ogura et al, 2009).…”

“…In addition, the amino-acid sequences of two lysylendopeptidyl fragments, MPGLFGGEDGDGAYK (L-1) and WNSVSEEVHINTVGK (L-2), showed 47% identity to the residues 96-111 and 170-184, respectively, of both HdAly and SP2. In these sequences, 13 residues 97-102 and 178-181 are known as the highly conserved regions among PL-14 enzymes Yamamoto et al, 2008).…”

“…The sequence YDVYFENFGFGIGGK of a lysylendopeptidyl fragment L-4 showed 73% identity to the residues 80-95 in the catalytic domains of HdAly and HdAlex. The K95 was predicted as a key residue for the catalytic action of HdAly (Yamamoto et al 2008). …”

Total Biosynthesis of Diterpene Aphidicolin, a Specific Inhibitor of DNA Polymerase α: Heterologous Expression of Four Biosynthetic Genes inAspergillus oryzae

“…The hosts used in this study were A. oryzae NSAR1, a quadruple auxotrophic mutant (niaD À , sC À , ÁargB, adeA À ) 22) for fungal expression, and S. cerevisiae BY4743 pep4Á prb1Á trp1Á (MATa/MAT, his3Á1/his3Á1, leu2Á0/leu2Á0, lys2Á0/þ, met15Á0/þ, ura3Á0/ura3Á0, pep4Á/pep4Á, prb1Á/ prb1Á, trp1Á/trp1Á) was produced by disrupting the PEP4, PRB1, and TRP1 genes in the BY4743 parental strain, 23,24) according to the protocol provided by Güldener et al 25) for yeast expression. The vectors used in this study were pTAex3, 5,6) pUSA, 26) pAdeA 27) and pPTRI 28) for fungal expression, and pLTex321sMHTRP and pLTex321sV5H 29) for yeast expression. The pLTex321sMHTRP vector was constructed by respectively replacing the V5-epitope tag and URA3 marker in pLTex321sV5H with a c-Myc-epitope tag and TRP1 marker.…”

Heat-stability and primary structure of the major alginate lyase isozyme LbAly35 from Littorina brevicula

“…Three dimensional structure of vAL-1 [34] solved by the X-ray diffraction method has provided important information for us to understand structure-function relationship of PL-14 molluscan alginate lyases [30]. The amino-acid residues crucially important for the catalytic action of vAL-1 were completely conserved in the putative β-strands and loops of abalone HdAly and sea hare AkAly30 [30,35]. Such comparative studies between molluscan alginate lyases and Chlorella virus enzyme have enriched information about molecular diversity and/or resemblance of PL-14 enzymes.…”