Investigation of Metal Ion Binding in Phosphonoacetaldehyde Hydrolase Identifies Sequence Markers for Metal-Activated Enzymes of the HAD Enzyme Superfamily

Guofeng Zhang, Marc Morais, Jianying Dai, Wenhai Zhang, Debra Dunaway-Mariano, Karen N. Allen

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

The 2-haloalkanoic acid dehalogenase (HAD) family, which contains both carbon and phosphoryl transferases, is one of the largest known enzyme superfamilies. HAD members conserve an α,β-core domain that frames the four-loop active-site platform. Each loop contributes one or more catalytic groups, which function in mediating the core chemistry (i.e., group transfer). In this paper, we provide evidence that the number of carboxylate residues on loop 4 and their positions (stations) on the loop are determinants, and therefore reliable sequence markers, for metal ion activation among HAD family members. Using this predictor, we conclude that the vast majority of the HAD members utilize a metal cofactor. Analysis of the minimum requirements for metal cofactor binding was carried out using Mg(II)-activated Bacillus cereus phosphonoacetaldehyde hydrolase (phosphonatase) as an experimental model for metal-activated HAD members. Mg(II) binding occurs via ligation to the loop 1 Asp12 carboxylate and Thr14 backbone carbonyl and to the loop 4 Asp186 carboxylate. The loop 4 Asp190 forms a hydrogen bond to the Mg(II) water ligand. X-ray structure determination of the D12A mutant in the presence of the substrate phosphonoacetaldehyde showed that replacement of the loop 1 Asp, common to all HAD family members, with Ala shifts the position of Mg(II), thereby allowing innersphere coordination to Asp190 and causing a shift in the position of the substrate. Kinetic analysis of the loop 4 mutants showed that Asp186 is essential to cofactor binding while Asp190 simply enhances it. Within the phosphonatase subfamily, Asp186 is stringently conserved, while either position 185 or position 190 is used to position the second loop 4 Asp residue. Retention of a high level of catalytic activity in the G185D/D190G phosphonatase mutant demonstrated the plasticity of the metal binding loop, reflected in the variety of combinations in positioning of two or three Asp residues along the seven-residue motif of the 2700 potential HAD sequences that were examined.

Original languageEnglish (US)
Pages (from-to)4990-4997
Number of pages8
JournalBiochemistry
Volume43
Issue number17
DOIs
StatePublished - May 4 2004
Externally publishedYes

Fingerprint

phosphonoacetaldehyde hydrolase
Metal ions
Metals
Ions
Enzymes
Viperidae
Bacillus cereus
Substrates
Transferases
2-haloacid dehalogenase
Plasticity
Ligation
Hydrogen

ASJC Scopus subject areas

  • Biochemistry

Cite this

Investigation of Metal Ion Binding in Phosphonoacetaldehyde Hydrolase Identifies Sequence Markers for Metal-Activated Enzymes of the HAD Enzyme Superfamily. / Zhang, Guofeng; Morais, Marc; Dai, Jianying; Zhang, Wenhai; Dunaway-Mariano, Debra; Allen, Karen N.

In: Biochemistry, Vol. 43, No. 17, 04.05.2004, p. 4990-4997.

Research output: Contribution to journalArticle

Zhang, Guofeng ; Morais, Marc ; Dai, Jianying ; Zhang, Wenhai ; Dunaway-Mariano, Debra ; Allen, Karen N. / Investigation of Metal Ion Binding in Phosphonoacetaldehyde Hydrolase Identifies Sequence Markers for Metal-Activated Enzymes of the HAD Enzyme Superfamily. In: Biochemistry. 2004 ; Vol. 43, No. 17. pp. 4990-4997.
@article{f74e7cfb9b5e46d2916810bce51817fa,
title = "Investigation of Metal Ion Binding in Phosphonoacetaldehyde Hydrolase Identifies Sequence Markers for Metal-Activated Enzymes of the HAD Enzyme Superfamily",
abstract = "The 2-haloalkanoic acid dehalogenase (HAD) family, which contains both carbon and phosphoryl transferases, is one of the largest known enzyme superfamilies. HAD members conserve an α,β-core domain that frames the four-loop active-site platform. Each loop contributes one or more catalytic groups, which function in mediating the core chemistry (i.e., group transfer). In this paper, we provide evidence that the number of carboxylate residues on loop 4 and their positions (stations) on the loop are determinants, and therefore reliable sequence markers, for metal ion activation among HAD family members. Using this predictor, we conclude that the vast majority of the HAD members utilize a metal cofactor. Analysis of the minimum requirements for metal cofactor binding was carried out using Mg(II)-activated Bacillus cereus phosphonoacetaldehyde hydrolase (phosphonatase) as an experimental model for metal-activated HAD members. Mg(II) binding occurs via ligation to the loop 1 Asp12 carboxylate and Thr14 backbone carbonyl and to the loop 4 Asp186 carboxylate. The loop 4 Asp190 forms a hydrogen bond to the Mg(II) water ligand. X-ray structure determination of the D12A mutant in the presence of the substrate phosphonoacetaldehyde showed that replacement of the loop 1 Asp, common to all HAD family members, with Ala shifts the position of Mg(II), thereby allowing innersphere coordination to Asp190 and causing a shift in the position of the substrate. Kinetic analysis of the loop 4 mutants showed that Asp186 is essential to cofactor binding while Asp190 simply enhances it. Within the phosphonatase subfamily, Asp186 is stringently conserved, while either position 185 or position 190 is used to position the second loop 4 Asp residue. Retention of a high level of catalytic activity in the G185D/D190G phosphonatase mutant demonstrated the plasticity of the metal binding loop, reflected in the variety of combinations in positioning of two or three Asp residues along the seven-residue motif of the 2700 potential HAD sequences that were examined.",
author = "Guofeng Zhang and Marc Morais and Jianying Dai and Wenhai Zhang and Debra Dunaway-Mariano and Allen, {Karen N.}",
year = "2004",
month = "5",
day = "4",
doi = "10.1021/bi036309n",
language = "English (US)",
volume = "43",
pages = "4990--4997",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "17",

}

TY - JOUR

T1 - Investigation of Metal Ion Binding in Phosphonoacetaldehyde Hydrolase Identifies Sequence Markers for Metal-Activated Enzymes of the HAD Enzyme Superfamily

AU - Zhang, Guofeng

AU - Morais, Marc

AU - Dai, Jianying

AU - Zhang, Wenhai

AU - Dunaway-Mariano, Debra

AU - Allen, Karen N.

PY - 2004/5/4

Y1 - 2004/5/4

N2 - The 2-haloalkanoic acid dehalogenase (HAD) family, which contains both carbon and phosphoryl transferases, is one of the largest known enzyme superfamilies. HAD members conserve an α,β-core domain that frames the four-loop active-site platform. Each loop contributes one or more catalytic groups, which function in mediating the core chemistry (i.e., group transfer). In this paper, we provide evidence that the number of carboxylate residues on loop 4 and their positions (stations) on the loop are determinants, and therefore reliable sequence markers, for metal ion activation among HAD family members. Using this predictor, we conclude that the vast majority of the HAD members utilize a metal cofactor. Analysis of the minimum requirements for metal cofactor binding was carried out using Mg(II)-activated Bacillus cereus phosphonoacetaldehyde hydrolase (phosphonatase) as an experimental model for metal-activated HAD members. Mg(II) binding occurs via ligation to the loop 1 Asp12 carboxylate and Thr14 backbone carbonyl and to the loop 4 Asp186 carboxylate. The loop 4 Asp190 forms a hydrogen bond to the Mg(II) water ligand. X-ray structure determination of the D12A mutant in the presence of the substrate phosphonoacetaldehyde showed that replacement of the loop 1 Asp, common to all HAD family members, with Ala shifts the position of Mg(II), thereby allowing innersphere coordination to Asp190 and causing a shift in the position of the substrate. Kinetic analysis of the loop 4 mutants showed that Asp186 is essential to cofactor binding while Asp190 simply enhances it. Within the phosphonatase subfamily, Asp186 is stringently conserved, while either position 185 or position 190 is used to position the second loop 4 Asp residue. Retention of a high level of catalytic activity in the G185D/D190G phosphonatase mutant demonstrated the plasticity of the metal binding loop, reflected in the variety of combinations in positioning of two or three Asp residues along the seven-residue motif of the 2700 potential HAD sequences that were examined.

AB - The 2-haloalkanoic acid dehalogenase (HAD) family, which contains both carbon and phosphoryl transferases, is one of the largest known enzyme superfamilies. HAD members conserve an α,β-core domain that frames the four-loop active-site platform. Each loop contributes one or more catalytic groups, which function in mediating the core chemistry (i.e., group transfer). In this paper, we provide evidence that the number of carboxylate residues on loop 4 and their positions (stations) on the loop are determinants, and therefore reliable sequence markers, for metal ion activation among HAD family members. Using this predictor, we conclude that the vast majority of the HAD members utilize a metal cofactor. Analysis of the minimum requirements for metal cofactor binding was carried out using Mg(II)-activated Bacillus cereus phosphonoacetaldehyde hydrolase (phosphonatase) as an experimental model for metal-activated HAD members. Mg(II) binding occurs via ligation to the loop 1 Asp12 carboxylate and Thr14 backbone carbonyl and to the loop 4 Asp186 carboxylate. The loop 4 Asp190 forms a hydrogen bond to the Mg(II) water ligand. X-ray structure determination of the D12A mutant in the presence of the substrate phosphonoacetaldehyde showed that replacement of the loop 1 Asp, common to all HAD family members, with Ala shifts the position of Mg(II), thereby allowing innersphere coordination to Asp190 and causing a shift in the position of the substrate. Kinetic analysis of the loop 4 mutants showed that Asp186 is essential to cofactor binding while Asp190 simply enhances it. Within the phosphonatase subfamily, Asp186 is stringently conserved, while either position 185 or position 190 is used to position the second loop 4 Asp residue. Retention of a high level of catalytic activity in the G185D/D190G phosphonatase mutant demonstrated the plasticity of the metal binding loop, reflected in the variety of combinations in positioning of two or three Asp residues along the seven-residue motif of the 2700 potential HAD sequences that were examined.

UR - http://www.scopus.com/inward/record.url?scp=2142646427&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=2142646427&partnerID=8YFLogxK

U2 - 10.1021/bi036309n

DO - 10.1021/bi036309n

M3 - Article

VL - 43

SP - 4990

EP - 4997

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 17

ER -