Crystal structures of the wild-type, P1A mutant, and inactivated malonate semialdehyde decarboxylase: a structural basis for the decarboxylase and hydratase activities.
Malonate semialdehyde decarboxylase (MSAD) from Pseudomonas pavonaceae 170 is a tautomerase superfamily member that converts malonate semialdehyde to acetaldehyde by a mechanism utilizing Pro-1 and Arg-75. Pro-1 and Arg-75 have also been implicated in the hydratase activity of MSAD in which 2-oxo-3-pentynoate is processed to acetopyruvate. Crystal structures of MSAD (1.8 A resolution), the P1A mutant of MSAD (2.7 A resolution), and MSAD inactivated by 3-chloropropiolate (1.6 A resolution), a mechanism-based inhibitor activated by the hydratase activity of MSAD, have been determined. A comparison of the P1A-MSAD and MSAD structures reveals little geometric alteration, indicating that Pro-1 plays an important catalytic role but not a critical structural role. The structures of wild-type MSAD and MSAD covalently modified at Pro-1 by 3-oxopropanoate, the adduct resulting from the incubation of MSAD and 3-chloropropiolate, implicate Asp-37 as the residue that activates a water molecule for attack at C-3 of 3-chloropropiolate to initiate a Michael addition of water. The interactions of Arg-73 and Arg-75 with the C-1 carboxylate group of the adduct suggest these residues polarize the alpha,beta-unsaturated acid and facilitate the addition of water. On the basis of these structures, a mechanism for the inactivation of MSAD by 3-chloropropiolate can be formulated along with mechanisms for the decarboxylase and hydratase activities. The results also provide additional evidence supporting the hypothesis that MSAD and trans-3-chloroacrylic acid dehalogenase, a tautomerase superfamily member preceding MSAD in the trans-1,3-dichloropropene degradation pathway, diverged from a common ancestor but retained the key elements for the conjugate addition of water.
Almrud
Jeffrey J
JJ
Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin, Texas 78712-1074, USA.
Poelarends
Gerrit J
GJ
Johnson
William H
WH
Serrano
Hector
H
Hackert
Marvin L
ML
Whitman
Christian P
CP
D001120:Arginine / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007535:Isomerases / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000737:chemistry / Q000235:genetics; D011549:Pseudomonas / Q000201:enzymology*
D001120:Arginine; D011392:Proline; D002262:Carboxy-Lyases; C071850:malonate semialdehyde decarboxylase; D006836:Hydro-Lyases; D007535:Isomerases