The sialate-pyruvate lyase from pig kidney. Elucidation of the primary structure and expression of recombinant enzyme activity.
The first complete primary structure of a mammalian sialate-pyruvate lyase, namely of the enzyme from porcine kidney, was elucidated by a combination of different PCR techniques followed by sequencing of the resulting fragments. The primers used were either deduced from four porcine lyase peptides or from an alignment of human and mouse expressed sequence tags (ESTs), which were found to be homologous to already known microbial lyase sequences, and cDNA alone or after ligation with a plasmid vector served as a template. The lyase primary structure consists of 319 amino acids with a calculated protein molecular mass of approximately 35 kDa, which fits well to the value determined for the native enzyme. The porcine lyase sequence made it possible to assemble several ESTs from mouse and man in order to obtain the complete putative lyase genes. The three mammalian sequences reveal a high degree of homology both on the nucleotide (83% of the nucleotides are identical between all three sequences) and on the amino-acid level (72% of the amino acids are identical between all three sequences), and thus form a tightly related group. In contrast, the identity between the lyase primary structures from pig kidney and the microbial enzyme from Clostridium perfringens is much less pronounced (25%). Thirty-one amino acids were found to be absolutely conserved in all lyase sequences. Among them are two amino acids (lysine 173 and tyrosine 143 in the porcine lyase) that are most important for the catalytic reaction. After expression cloning, recombinant enzyme activity was expressed in Escherichia coli BL21(DE3)pLysS, which confirms the identity of the cloned sequence and verifies one of the putative human and murine sequences. After SDS/PAGE of a cell extract of the expression clone, a band of 35kDa was stained on the gel.
Traving
C
C
Biochemisches Institut, Christian-Albrechts-Universität, Olshausenstrasse, Kiel, Germany.
Bruse
P
P
Wächter
A
A
Schauer
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D020224:Expressed Sequence Tags; D007668:Kidney / Q000201:enzymology*; D008969:Molecular Sequence Data; D007652:Oxo-Acid-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013552:Swine
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D007652:Oxo-Acid-Lyases; C443820:sialate-pyruvate lyase