The dual-specific active site of 7,8-diaminopelargonic acid synthase and the effect of the R391A mutation.
7,8-diaminopelargonic acid (DAPA) synthase (EC 2.6.1.62) is a pyridoxal phosphate (PLP)-dependent transaminase that catalyzes the transfer of the alpha-amino group from S-adenosyl-L-methionine (SAM) to 7-keto-8-aminopelargonic acid (KAPA) to form DAPA in the antepenultimate step in the biosynthesis of biotin. The wild-type enzyme has a steady-state kcat value of 0.013 s(-1), and the K(m) values for SAM and KAPA are 150 and <2 microM, respectively. The k(max) and apparent K(m) values for the half-reaction of the PLP form of the enzyme with SAM are 0.016 s(-1) and 300 microM, respectively, while those for the reaction with DAPA are 0.79 s(-1) and 1 microM. The R391A mutant enzyme exhibits near wild-type kinetic parameters in the reaction with SAM, while the apparent K(m) for DAPA is increased 180-fold. The 2.1 A crystal structure of the R391A mutant enzyme shows that the mutation does not significantly alter the structure. These results indicate that the conserved arginine residue is not required for binding the alpha-amino acid SAM, but it is important for recognition of DAPA.
Eliot
Andrew C
AC
Department of Chemistry, University of California-Berkeley, Berkeley, California 94720-3206, USA.
Sandmark
Jenny
J
Schneider
Gunter
G
Kirsch
Jack F
JF
D000409:Alanine / Q000737:chemistry / Q000235:genetics*; D000599:Amino Acids, Diamino / Q000737:chemistry; D001120:Arginine / Q000737:chemistry / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D003460:Crystallization; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007097:Imines / Q000737:chemistry; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D012436:S-Adenosylmethionine / Q000737:chemistry; D013053:Spectrophotometry; D013237:Stereoisomerism; D013379:Substrate Specificity / Q000235:genetics; D000637:Transaminases / Q000737:chemistry* / Q000235:genetics*
D000599:Amino Acids, Diamino; D029968:Escherichia coli Proteins; D007097:Imines; C004720:7,8-diaminopelargonic acid; D012436:S-Adenosylmethionine; D001120:Arginine; C020029:7,8-diaminopelargonic acid aminotransferase; D000637:Transaminases; D000409:Alanine