Structural and kinetic characterization of 4-hydroxy-4-methyl-2-oxoglutarate/4-carboxy-4-hydroxy-2-oxoadipate aldolase, a protocatechuate degradation enzyme evolutionarily convergent with the HpaI and DmpG pyruvate aldolases.
4-Hydroxy-4-methyl-2-oxoglutarate/4-carboxy-4-hydroxy-2-oxoadipate (HMG/CHA) aldolase from Pseudomonas putida F1 catalyzes the last step of the bacterial protocatechuate 4,5-cleavage pathway. The preferred substrates of the enzyme are 2-keto-4-hydroxy acids with a 4-carboxylate substitution. The enzyme also exhibits oxaloacetate decarboxylation and pyruvate α-proton exchange activity. Sodium oxalate is a competitive inhibitor of the aldolase reaction. The pH dependence of k(cat)/K(m) and k(cat) for the enzyme is consistent with a single deprotonation with pK(a) values of 8.0 ± 0.1 and 7.0 ± 0.1 for free enzyme and enzyme substrate complex, respectively. The 1.8 Å x-ray structure shows a four-layered α-β-β-α sandwich structure with the active site at the interface of two adjacent subunits of a hexamer; this fold resembles the RNase E inhibitor, RraA, but is novel for an aldolase. The catalytic site contains a magnesium ion ligated by Asp-124 as well as three water molecules bound by Asp-102 and Glu-199'. A pyruvate molecule binds the magnesium ion through both carboxylate and keto oxygen atoms, completing the octahedral geometry. The carbonyl oxygen also forms hydrogen bonds with the guanadinium group of Arg-123, which site-directed mutagenesis confirms is essential for catalysis. A mechanism for HMG/CHA aldolase is proposed on the basis of the structure, kinetics, and previously established features of other aldolase mechanisms.
Wang
Weijun
W
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Mazurkewich
Scott
S
Kimber
Matthew S
MS
Seah
Stephen Y K
SY
D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003460:Crystallization; D018360:Crystallography, X-Ray; D019143:Evolution, Molecular; D062385:Hydroxybenzoates / Q000378:metabolism*; D007700:Kinetics; D062907:Oxaloacetic Acid / Q000378:metabolism; D007652:Oxo-Acid-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016958:Pseudomonas putida / Q000201:enzymology*; D011773:Pyruvates / Q000378:metabolism; D013379:Substrate Specificity
D062385:Hydroxybenzoates; D011773:Pyruvates; D062907:Oxaloacetic Acid; C009091:protocatechuic acid; C470744:4-carboxy-4-hydroxy-2-oxoadipate aldolase; D000446:Aldehyde-Lyases; D007652:Oxo-Acid-Lyases; C021412:4-hydroxy-4-methyl-2-oxoglutarate aldolase