Crystal Structures of Native and Inactivated cis-3-Chloroacrylic Acid Dehalogenase

Abstract

The bacterial degradation pathways for the nematocide 1,3-dichloropropene rely on hydrolytic dehalogenation reactions catalyzed by cis- and trans-3-chloroacrylic acid dehalogenases (cis-CaaD and CaaD, respectively). X-ray crystal structures of native cis-CaaD and cis-CaaD inactivated by (R)-oxirane-2-carboxylate were elucidated. They locate four known catalytic residues (Pro-1, Arg-70, Arg-73, and Glu-114) and two previously unknown, potential catalytic residues (His-28 and Tyr-103′). The Y103F and H28A mutants of these latter two residues displayed reductions in cis-CaaD activity confirming their importance in catalysis. The structure of the inactivated enzyme shows covalent modification of the Pro-1 nitrogen atom by (R)-2-hydroxypropanoate at the C3 position. The interactions in the complex implicate Arg-70 or a water molecule bound to Arg-70 as the proton donor for the epoxide ring-opening reaction and Arg-73 and His-28 as primary binding contacts for the carboxylate group. This proposed binding mode places the (R)-enantiomer, but not the (S)-enantiomer, in position to covalently modify Pro-1. The absence of His-28 (or an equivalent) in CaaD could account for the fact that CaaD is not inactivated by either enantiomer. The cis-CaaD structures support a mechanism in which Glu-114 and Tyr-103′ activate a water molecule for addition to C3 of the substrate and His-28, Arg-70, and Arg-73 interact with the C1 carboxylate group to assist in substrate binding and polarization. Pro-1 provides a proton at C2. The involvement of His-28 and Tyr-103′ distinguishes the cis-CaaD mechanism from the otherwise parallel CaaD mechanism. The two mechanisms probably evolved independently as the result of an early gene duplication of a common ancestor. The bacterial degradation pathways for the nematocide 1,3-dichloropropene rely on hydrolytic dehalogenation reactions catalyzed by cis- and trans-3-chloroacrylic acid dehalogenases (cis-CaaD and CaaD, respectively). X-ray crystal structures of native cis-CaaD and cis-CaaD inactivated by (R)-oxirane-2-carboxylate were elucidated. They locate four known catalytic residues (Pro-1, Arg-70, Arg-73, and Glu-114) and two previously unknown, potential catalytic residues (His-28 and Tyr-103′). The Y103F and H28A mutants of these latter two residues displayed reductions in cis-CaaD activity confirming their importance in catalysis. The structure of the inactivated enzyme shows covalent modification of the Pro-1 nitrogen atom by (R)-2-hydroxypropanoate at the C3 position. The interactions in the complex implicate Arg-70 or a water molecule bound to Arg-70 as the proton donor for the epoxide ring-opening reaction and Arg-73 and His-28 as primary binding contacts for the carboxylate group. This proposed binding mode places the (R)-enantiomer, but not the (S)-enantiomer, in position to covalently modify Pro-1. The absence of His-28 (or an equivalent) in CaaD could account for the fact that CaaD is not inactivated by either enantiomer. The cis-CaaD structures support a mechanism in which Glu-114 and Tyr-103′ activate a water molecule for addition to C3 of the substrate and His-28, Arg-70, and Arg-73 interact with the C1 carboxylate group to assist in substrate binding and polarization. Pro-1 provides a proton at C2. The involvement of His-28 and Tyr-103′ distinguishes the cis-CaaD mechanism from the otherwise parallel CaaD mechanism. The two mechanisms probably evolved independently as the result of an early gene duplication of a common ancestor. The cis- and trans-3-chloroacrylic acid dehalogenases (cis-CaaD and CaaD) 4The abbreviations used are: cis-CaaD and CaaD, cis- and trans-3-chloroacrylic acid dehalogenase, respectively; Fo and Fc, observed and calculated structure factor amplitudes, respectively; MSAD, malonate semialdehyde decarboxylase; r.m.s.d., root-mean-square deviation. catalyze the cofactor-independent hydrolytic dehalogenation of, respectively, the cis- and trans-isomers of 3-chloroacrylic acid (1 and 2, Scheme 1) to produce malonate semialdehyde (5) and HCl (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google Scholar, 2Poelarends G.J. Saunier R. Janssen D.B. J. Bacteriol. 2001; 183: 4269-4277Crossref PubMed Scopus (46) Google Scholar, 3Wang S.C. Person M.D. Johnson Jr., W.H. Whitman C.P. Biochemistry. 2003; 42: 8762-8773Crossref PubMed Scopus (62) Google Scholar). Both reactions may be initiated by the attack of water at C3 to form an enzyme-stabilized enediolate intermediate (3). Subsequent ketonization of 3 with protonation at C2 generates a chlorohydrin intermediate (4), which can collapse by direct expulsion of the chloride to afford 5 (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google Scholar, 4Azurmendi H.F. Wang S.C. Massiah M.A. Poelarends G.J. Whitman C.P. Mildvan A.S. Biochemistry. 2004; 43: 4082-4091Crossref PubMed Scopus (33) Google Scholar, 5de Jong R.M. Brugman W. Poelarends G.J. Whitman C.P. Dijkstra B.W. J. Biol. Chem. 2004; 279: 11546-11552Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Alternatively, ketonization of 3 can result in chloride loss and the formation of the enol intermediate, 6, which tautomerizes to afford 5. The two enzymes are found in bacterial pathways that convert the cis- and trans-isomers of 1,3-dichloropropene, used as nematocides, to acetaldehyde (7) and carbon dioxide (6Poelarends G.J. Wilkens M. Larkin M.J. van Elsas J.D. Janssen D.B. Appl. Environ. Microbiol. 1998; 64: 2931-2936Crossref PubMed Google Scholar). The cis- and trans-3-chloroacrylic acid dehalogenases have low sequence identity (∼20%) and different oligomerization states (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google Scholar, 2Poelarends G.J. Saunier R. Janssen D.B. J. Bacteriol. 2001; 183: 4269-4277Crossref PubMed Scopus (46) Google Scholar, 3Wang S.C. Person M.D. Johnson Jr., W.H. Whitman C.P. Biochemistry. 2003; 42: 8762-8773Crossref PubMed Scopus (62) Google Scholar). CaaD is a heterohexamer consisting of three 75-residue α-chains and three 70-residue β-chains, whereas cis-CaaD forms a homotrimer of three identical 149-residue polypeptide chains, which can be considered as the fusion product of a CaaD α- and β-chain (2Poelarends G.J. Saunier R. Janssen D.B. J. Bacteriol. 2001; 183: 4269-4277Crossref PubMed Scopus (46) Google Scholar, 3Wang S.C. Person M.D. Johnson Jr., W.H. Whitman C.P. Biochemistry. 2003; 42: 8762-8773Crossref PubMed Scopus (62) Google Scholar, 5de Jong R.M. Brugman W. Poelarends G.J. Whitman C.P. Dijkstra B.W. J. Biol. Chem. 2004; 279: 11546-11552Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). As a result, the two enzymes have been classified in two different families in the tautomerase superfamily, with each being related to 4-oxalocrotonate tautomerase (7Murzin A.G. Curr. Opin. Struct. Biol. 1996; 6: 386-394Crossref PubMed Scopus (224) Google Scholar, 8Whitman C.P. Arch. Biochem. Biophys. 2002; 402: 1-13Crossref PubMed Scopus (99) Google Scholar, 9Poelarends G.J. Whitman C.P. Chem. 2004; PubMed Scopus Google Scholar). the in catalytic are and of the catalytic mechanisms are a in cis-CaaD and in CaaD) is proposed to as a to activate a water molecule for attack at C3 or and the in cis-CaaD and in CaaD) is to a proton at C2. residues and Arg-73 in cis-CaaD and and in CaaD) are proposed to interact with the C1 carboxylate the substrate in the and from C3 (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google Scholar, 3Wang S.C. Person M.D. Johnson Jr., W.H. Whitman C.P. Biochemistry. 2003; 42: 8762-8773Crossref PubMed Scopus (62) Google Scholar, 4Azurmendi H.F. Wang S.C. Massiah M.A. Poelarends G.J. Whitman C.P. Mildvan A.S. Biochemistry. 2004; 43: 4082-4091Crossref PubMed Scopus (33) Google Scholar, 5de Jong R.M. Brugman W. Poelarends G.J. Whitman C.P. Dijkstra B.W. J. Biol. Chem. 2004; 279: 11546-11552Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). an a at C3 and the addition of The of that the two enzymes are related by from a common ancestor. and two the of the water molecule residues in cis-CaaD (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google Scholar). of Glu-114 to a a whereas the of CaaD a cis-CaaD is by the (R)-oxirane-2-carboxylate to covalent modification of Pro-1 G.J. Serrano H. Person M.D. Johnson Jr, W.H. Whitman C.P. Biochemistry. 2004; 43: PubMed Scopus Google Scholar). The is a of cis-CaaD and CaaD G.J. Serrano H. Person M.D. Johnson Jr, W.H. Whitman C.P. Biochemistry. 2004; 43: PubMed Scopus Google the for the dehalogenation reaction and the of the crystal structures of the native cis-CaaD and cis-CaaD inactivated by were They the of cis-CaaD and the of Arg-70, Arg-73, and The of His-28 and Tyr-103′ in of, respectively, the substrate and water molecule for by the are by can be that His-28 a in for of Pro-1 with is a of the substrate of the The support the that cis-CaaD and CaaD form two that the duplication of a 4-oxalocrotonate and were from or used for the of M. 1998; Scopus Google Scholar). The for the of the enzymes and used in the and the used for and cis-CaaD and the have been (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google Scholar, G.J. Serrano H. Person M.D. Johnson Jr, W.H. Whitman C.P. Biochemistry. 2004; 43: PubMed Scopus Google Scholar). The and the were from for and were by for and were as J. Scholar). The cis-CaaD and the were to as by to a (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google Scholar, G.J. Serrano H. Person M.D. Johnson Jr, W.H. Whitman C.P. Biochemistry. 2004; 43: PubMed Scopus Google Scholar). by on PubMed Scopus Google Scholar). The were with were by the of J. Google Scholar). The native of the were by on a the and of covalently inactivated by the enzyme with a of for the to to of inactivated cis-CaaD were from consisting of of and and of in a to on a from a The were and W. PubMed Scopus Google Scholar). The to the group with a and The of of which the residues were not in the were with the in J. PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar). the of of an and a from the CaaD Jong R.M. Brugman W. Poelarends G.J. Whitman C.P. Dijkstra B.W. J. Biol. Chem. 2004; 279: 11546-11552Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). residues were to for the and and were calculated the and The the position and of a in the of the by a of and an of The at were not but observed from of the 42: Scopus Google calculated from the from used in a and the in the Biol. 2001; PubMed Scopus Google Scholar). This the of from of the to in the of the modification The from for the acid and of the in the of of and were with and in The used to the for were of The structure and J. Struct. Biol. PubMed Scopus Google Scholar). for the covalent were on and in and were the M. Biol. 1998; PubMed Scopus Google Scholar). The of the with J. Appl. Google Scholar). of the the and of the is in and is the observed and the on of the used in the on of the from the is the observed and the on of the used in the on of the from the in a and of of native cis-CaaD were from consisting of of and as a and of a to at the at the The were and W. PubMed Scopus Google Scholar). The crystal to the group with a The three of residues of the were not in the were with the in J. PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar). from the of a cis-CaaD inactivated by with the of the covalent and were calculated and The the position and of three in the of the by J. PubMed Scopus Google Scholar, Biol. PubMed Scopus Google a of and an of were the in Biol. PubMed Scopus Google Scholar). The structure as for the inactivated of the and of the is in of and in the of cis-CaaD and CaaD, respectively, the J. Struct. Biol. PubMed Scopus Google Scholar). and of the and of three were by R.M. PubMed Scopus Google the (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google as the The and were used as the and The an and the a the H28A the were and the Y103F the were and the the were and The used to the are The the the the and the in either the and or the The and the were used to the or for of the The were to that the been the the enzymes were in and to by the for (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google Scholar). The from to of of were from for The for the three and the cis-CaaD are at a of that the structures of the are and that not as a result of the of the of the three cis-CaaD mutants were an in the in the of at the of at The were as S.C. Person M.D. Johnson Jr., W.H. Whitman C.P. Biochemistry. 2003; 42: 8762-8773Crossref PubMed Scopus (62) Google Scholar). The observed were for for and for that the the and not by the and of cis-CaaD activity at by the at to the of for and or and (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google Scholar). were on a The are a of the an of in to and in an The of the reaction to enzyme in to the reaction The reaction at or in the and the of product by of the to and the (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google Scholar). were in on a as previously (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google Scholar). of the native cis-CaaD crystal structure to by and to and of and The three of a that is by the of a of parallel each the two of the parallel to each in the of the each is of two that are of the tautomerase (7Murzin A.G. Curr. Opin. Struct. Biol. 1996; 6: 386-394Crossref PubMed Scopus (224) Google Scholar, 8Whitman C.P. Arch. Biochem. Biophys. 2002; 402: 1-13Crossref PubMed Scopus (99) Google Scholar, 9Poelarends G.J. Whitman C.P. Chem. 2004; PubMed Scopus Google Scholar). The three in the with root-mean-square of for of a of of the three forms an by of the in the crystal the are by the of the and are in residues that interact with each and with three water in the of the interactions are by two structures the of the and the two and of three with the in the of a on of the The Pro-1 nitrogen with of the of a or a that is bound in the Both were in the The nitrogen atom with of the The three of the have and interactions with two Arg-70 and Arg-73, a His-28, and The nitrogen atom with of the of the catalytic which is and from a and residues from different of The atom of Glu-114 with the group of from a The carboxylate of Glu-114 with the of that the of Glu-114 is As Glu-114 as the catalytic in the reaction catalyzed by a protonation of the could be an of the low at which the native enzyme of cis-CaaD with of the known families as of the tautomerase (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google Scholar, 2Poelarends G.J. Saunier R. Janssen D.B. J. Bacteriol. 2001; 183: 4269-4277Crossref PubMed Scopus (46) Google Scholar, A.G. Curr. Opin. Struct. Biol. 1996; 6: 386-394Crossref PubMed Scopus (224) Google Scholar, 8Whitman C.P. Arch. Biochem. Biophys. 2002; 402: 1-13Crossref PubMed Scopus (99) Google Scholar, 9Poelarends G.J. Whitman C.P. Chem. 2004; PubMed Scopus Google Scholar, Wang S.C. R.M. Johnson Jr., W.H. Murzin M. Whitman C.P. Biochemistry. 2002; PubMed Scopus Google Scholar, G.J. Johnson Jr., W.H. Murzin Whitman C.P. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The four by their are the J. Bacteriol. PubMed Google 4-oxalocrotonate tautomerase Wang S.C. R.M. Johnson Jr., W.H. Murzin M. Whitman C.P. Biochemistry. 2002; PubMed Scopus Google Scholar, M. J. Bacteriol. PubMed Google factor H. PubMed Scopus Google and malonate semialdehyde families G.J. Johnson Jr., W.H. Murzin Whitman C.P. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). cis-CaaD been classified in a tautomerase the cis-CaaD of the absence of sequence identity with known of the four families (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google Scholar). The CaaD, to the 4-oxalocrotonate tautomerase and MSAD, cis-CaaD is a from two which in G.J. D.B. Biochemistry. 1996; PubMed Scopus Google Scholar, Johnson Jr, W.H. R.M. Whitman C.P. Biochemistry. PubMed Scopus Google Scholar, Poelarends G.J. Johnson Jr., W.H. Serrano H. Whitman C.P. Biochemistry. PubMed Scopus Google Scholar). shows that the structures of the four enzymes with of for of a of are in the four and the that the of the The low of the found in the that the and and the the the of cis-CaaD and are the in a of and with an of shows that the two in and the α- and of CaaD with of for of a of CaaD and cis-CaaD to different families of the tautomerase superfamily, cis-CaaD and CaaD are of cis-CaaD by previously been that cis-CaaD is inactivated by the G.J. Serrano H. Person M.D. Johnson Jr, W.H. Whitman C.P. Biochemistry. 2004; 43: PubMed Scopus Google Scholar). the of is a with a of G.J. Serrano H. Person M.D. Johnson Jr, W.H. Whitman C.P. Biochemistry. 2004; 43: PubMed Scopus Google Scholar). of cis-CaaD inactivated by that the of modification on the enzyme is and that the in for the inactivated cis-CaaD is with covalent modification by a The crystal structure of the inactivated cis-CaaD to by the the for in the CaaD and to and of and The of the inactivated cis-CaaD a covalent the Pro-1 nitrogen and C3 of the and an of the of the covalent from Pro-1 with an that is with a at the a the nitrogen and the C3 atom of the The nitrogen is of the of that the nitrogen is and at C3 of by Pro-1 places a group at C2 G.J. Serrano H. Person M.D. Johnson Jr, W.H. Whitman C.P. Biochemistry. 2004; 43: PubMed Scopus Google Scholar). the shows with an atom to the carbon atom of the that is at from a water molecule bound to with a carboxylate the of His-28 and the of these is that Pro-1 the C3 position of to result in the of (R)-2-hydroxypropanoate to the The of cis-CaaD by (R)-2-hydroxypropanoate carboxylate group a position as the in the native structure and interactions and two with the group of Arg-73 and with the nitrogen of forms a with the of The group of the with the water molecule bound to the of Arg-70 and the group of The catalytic Glu-114 a with the group of Tyr-103′ and is with to position in the native structure of As a result, the carboxylate atom of Glu-114 not interact with the group of but with a water molecule at the position of in the native is of the residues that are not in the of the inactivated cis-CaaD the of the inactivated enzyme is to of the enzyme by or to a and the at which the inactivated enzyme Glu-114 is not and in a position to activate the water molecule the cis-CaaD and CaaD cis-CaaD shows two with that of CaaD inactivated by Jong R.M. Brugman W. Poelarends G.J. Whitman C.P. Dijkstra B.W. J. Biol. Chem. 2004; 279: 11546-11552Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The is the of a His-28, in the binding of the carboxylate group of the covalent His-28 is in the four in the cis-CaaD (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google Scholar). CaaD, the position is by a the of cis-CaaD a that with the is not and is in two of the four cis-CaaD (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google to Glu-114 and the Pro-1 nitrogen that may have a catalytic Glu-114 in the of the catalytic water of Tyr-103′ an on enzyme activity the of His-28 and Tyr-103′ in but not in CaaD, could account for the substrate for the of of the structures of cis-CaaD and CaaD an for their different substrate the carboxylate group of the substrate is bound by the that the of the substrate in the is the of the enzyme CaaD, the carboxylate group of the substrate is bound by the with the of the substrate the the of an in cis-CaaD in in the of the substrate in the with to The the of their and the of cis-CaaD being whereas the of CaaD is in of the residues for is Tyr-103′ in CaaD, a the of the substrate to and The of the Tyr-103′ in cis-CaaD the binding of the group of a a by the of and could the binding of the of a His-28 and Tyr-103′ in cis-CaaD to be two of the of the The different of the may be for the different of CaaD and cis-CaaD in Scheme (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google Scholar, 3Wang S.C. Person M.D. Johnson Jr., W.H. Whitman C.P. Biochemistry. 2003; 42: 8762-8773Crossref PubMed Scopus (62) Google Scholar). Both enzymes convert to but the of CaaD is that of cis-CaaD for The is to a with a (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google Scholar, 3Wang S.C. Person M.D. Johnson Jr., W.H. Whitman C.P. Biochemistry. 2003; 42: 8762-8773Crossref PubMed Scopus (62) Google Scholar). The molecule is by the of CaaD is by the of as by the different to CaaD in an to that proposed for 2, the molecule be the proposed binding the two residues of CaaD interact with the as as or carboxylate of to cis-CaaD in a mode to that proposed for the molecule be the an not be a and may binding and catalysis. The of Tyr-103′ may with the binding of of His-28 and the importance of Tyr-103′ and His-28 for the dehalogenation the and mutants of cis-CaaD were and of their and with the and that His-28 is for catalytic the H28A activity in the Glu-114 is Tyr-103′ as by their but is as as the is as by the that the activity in the can be that the three residues His-28, and Glu-114 are for the dehalogenation of for cis-CaaD and were in at are activity for the H28A or the The were in at are activity for the H28A or the in a the importance of the residues to (Pro-1, His-28, Arg-70, Arg-73, and Glu-114) to cis-CaaD the reactions catalyzed by the and and the were by at or and the of product Both the and reactions were at whereas the reaction at the reaction a of the reaction and the reaction product at The reaction the in product The which is with the and (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google that Arg-70, and Arg-73 are the catalytic residues in substrate and protonation at C2 may be for the dehalogenation reaction the of of of cis-CaaD by previously been that of cis-CaaD by Arg-70, and Arg-73 G.J. Serrano H. Person M.D. Johnson Jr, W.H. Whitman C.P. Biochemistry. 2004; 43: PubMed Scopus Google Scholar). the is by that Glu-114 is not for the are with a mechanism for covalent modification the attack of Pro-1 at C2 or C3 of with the formation of a the and a proton donor or protonation of the by a proton G.J. Serrano H. Person M.D. Johnson Jr, W.H. Whitman C.P. Biochemistry. 2004; 43: PubMed Scopus Google Scholar). The crystal structure of the inactivated cis-CaaD the C3 position of as the of attack by Pro-1. The structure shows that the carboxylate group of the with the of His-28, Arg-73, and the nitrogen atom of Arg-70 The of Arg-70 a water with the and carboxylate of the that Arg-73 and His-28 are in the binding of the carboxylate group and that Arg-70 may by direct with the epoxide or by a water molecule in position to interact with the epoxide the interactions of Arg-70 and the with the covalent that to in the with the Arg-70 and the C3 atom to Pro-1 the of to cis-CaaD with interactions the carboxylate group and Arg-73 and His-28, the C3 carbon atom be from Pro-1 and Arg-70 This binding mode of Pro-1 but could result in which been observed G.J. Serrano H. Person M.D. Johnson Jr, W.H. Whitman C.P. Biochemistry. 2004; 43: PubMed Scopus Google Scholar). The proposed binding mode provides a for the fact that CaaD is not inactivated by either of CaaD His-28 (or an equivalent) in the that the carboxylate group of interact with and as observed in the crystal structure of inactivated CaaD are in binding the carboxylate of the from as the proton donor in the reaction of the epoxide a binding mode could to which been observed This His-28 as a that but not CaaD, to of the of and with (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google and the importance of His-28, Arg-70, Arg-73, and Glu-114 in the cis-CaaD catalytic mechanism The mechanism can be three of water by Glu-114 and and of substrate by His-28, Arg-70, and and protonation at C2 by Pro-1. that the of the residues in substrate and protonation are to in the of The structures of the native and inactivated enzyme that Tyr-103′ is in a position to assist Glu-114 in the of a water molecule for attack at C3 of be to but three Tyr-103′ can position the water molecule or the carboxylate group of assist in of the water molecule in Scheme or of these are with the of the Y103F but the of Glu-114 is that of Tyr-103′ in water residues cis-CaaD The structures that the in the binding and of the substrate by with the of the carboxylate group. The three residues the carboxylate which in a at in the addition of The interactions the and the carboxylate group are not but may parallel observed in the structure of the inactivated enzyme The of Arg-70 and Arg-73 are for that of His-28 is water the enzyme-stabilized enediolate intermediate 3 can two and (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google Scholar, G.J. Serrano H. Person M.D. Johnson Jr, W.H. Whitman C.P. Biochemistry. 2004; 43: PubMed Scopus Google Scholar). As in Scheme enediolate 3 can with protonation at C2 by Pro-1. The chlorohydrin intermediate can an or a to produce 5. Alternatively, enediolate 3 can with of the chloride of and protonation at C2 by Pro-1 5. The not to the but the protonation is an of with a being the of Pro-1. The of cis-CaaD a group on the enzyme with of G.J. Serrano H. Person M.D. Johnson Jr, W.H. Whitman C.P. Biochemistry. 2004; 43: PubMed Scopus Google to been found for CaaD of in CaaD the group with the of to by an not been for that of CaaD, the of Pro-1 in cis-CaaD is with a with the of the and of Glu-114 and Tyr-103′ to the on the Pro-1 nitrogen the observed of Pro-1 is in with a as a acid of the and the four catalytic residues that CaaD and cis-CaaD are related by from a common ancestor. The different structures and low sequence identity that the two enzymes can be in cis-CaaD evolved from a direct of CaaD, whereas in the CaaD and cis-CaaD evolved gene duplication of a gene the by of the two could to cis-CaaD could have from these two from the direct of by a duplication of the two by gene the two enzymes have a Alternatively, cis-CaaD have evolved from an gene duplication of the gene by gene CaaD and cis-CaaD evolved The of His-28 and Tyr-103′ in the cis-CaaD and the low sequence identity CaaD and cis-CaaD implicate the which is with previously (1Poelarends G.J. Serrano H. Person M.D. Johnson Jr, W.H. Murzin A.G. Whitman C.P. Biochemistry. 2004; 43: 759-772Crossref PubMed Scopus (39) Google Scholar). the common of CaaD and cis-CaaD probably a gene the