Crystallographic analysis of the catalytic mechanism of haloalkane dehalogenase

Verschueren, K.H.G.; Seljée, F. R.; Rozeboom, H.J.; Kalk, Kor H.; Dijkstra, B.W.

doi:10.1038/363693a0

Cited by 475 publications

(460 citation statements)

References 26 publications

Supporting

Mentioning

439

Contrasting

Unclassified

Order By: Relevance

“…In both cases, initial dehalogenation starts with a reaction catalysed by a hydrolytic haloalkane dehalogenase [1,2]. The haloalkane dehalogenases that degrade these compounds are homologous to the classical enzyme from Xanthobacter autotrophicus [3]. Even though sequence similarities are low, the overall structures are very similar and the catalytic residues are conserved [4].…”

Section: Aerobic Growth On Halogenated Aliphatic Compoundsmentioning

confidence: 99%

Microbial dehalogenation

Janssen

Oppentocht²,

Poelarends³

2001

Current Opinion in Biotechnology

159

107

View full text Add to dashboard Cite

Novel dehalogenases have been identified recently in various bacteria that utilise halogenated substrates. X-ray studies and sequence analysis have revealed insight into the molecular mechanisms of hydrolytic dehalogenases. Furthermore, genetic and biochemical studies have indicated that reductive dehalogenases are extra-cytoplasmic corrinoid-containing iron-sulphur proteins. Sequence analysis and mutagenesis studies indicate that several dehalogenases are homologous to enzymes that carry out transformations on non-halogenated substrates.

show abstract

Section: Aerobic Growth On Halogenated Aliphatic Compoundsmentioning

confidence: 99%

Microbial dehalogenation

Janssen

Oppentocht²,

Poelarends³

2001

Current Opinion in Biotechnology

159

107

View full text Add to dashboard Cite

show abstract

“…To model protein-substrate complexes, a 1,2-dichloroethane molecule (DCE) was placed into the active site of the relaxed wild-type structure and the model of the double mutant at a position that was analogous to the position of DCE in the haloalkane dehalogenase-DCE structure (Verschueren et al, 1993b; PDB entry 2dhc). The complexes were minimized by an additional 3000 steps using the conjugate gradient algorithm.…”

Section: Methodsmentioning

confidence: 99%

“…The reaction mechanism consists of four main steps (Scheme 1) as was determined by X-ray crystallographic and site-directed mutagenesis studies (Verschueren et al, 1993b;Pries et al, 1994Pries et al, , 1995. In the first step the substrate binds in the cavity and a Michaelis complex (E‚RX) is formed in which the Cl R is stabilized by interactions with the NH groups of the Trp125 and Trp175 side chains.…”

mentioning

confidence: 99%

Repositioning the Catalytic Triad Aspartic Acid of Haloalkane Dehalogenase: Effects on Stability, Kinetics, and Structure

et al. 1997

View full text Add to dashboard Cite

show abstract

“…Similar to the catalytic triad, a His⋯Asp motif is found in the active sites of serine carboxypeptidase (10), acetylcholinesterase (11), phospholipase A 2 (12), haloalkane dehalogenase (13), dienelactone hydrolase (14), a variety of zinc-dependent enzymes (15), and pancreatic ribonucleases (16,17). This His⋯Asp motif is known as the "catalytic dyad".…”

mentioning

confidence: 99%

His···Asp Catalytic Dyad of Ribonuclease A: Structure and Function of the Wild-Type, D121N, and D121A Enzymes

1998

View full text Add to dashboard Cite

The sidechains of histidine and aspartate residues form a hydrogen bond in the active sites of many enzymes. In serine proteases, the His⋯Asp hydrogen bond of the catalytic triad is known to contribute greatly to catalysis, perhaps via the formation of a low-barrier hydrogen bond. In bovine pancreatic ribonuclease A (RNase A), the His⋯Asp dyad is composed of His119 and Asp121. Previously, sitedirected mutagenesis was used to show that His119 has a fundamental role-to act as an acid during catalysis of RNA cleavage , J. Am. Chem. Soc. 116,[5467][5468]. Here, Asp121 was replaced with an asparagine or alanine residue. The crystalline structures of the two variants were determined by X-ray diffraction analysis to a resolution of 1.6 Å with an R-factor of 0.18. Replacing Asp121 with an asparagine or alanine residue does not perturb the overall conformation of the enzyme. In the structure of D121N RNase A, N δ rather than O δ of Asn121 faces His119. This alignment in the crystalline state is unlikely to exist in solution because catalysis by the D121N variant is not compromised severely. The steady-state kinetic parameters for catalysis by the wild-type and variant enzymes were determined for the cleavage of uridylyl(3′→5′)adenosine and poly(cytidylic acid), and for the hydrolysis of uridine 2′,3′-cyclic phosphate. Replacing Asp121 decreases the values of k cat /K m and k cat for cleavage by 101-fold (D121N) and 10 2 -fold (D121A). Replacing Asp121 also decreases the values of k cat /K m and k cat for hydrolysis by 10 0.5 -fold (D121N) and 10-fold (D121A), but has no other effect on the pH-rate profiles for hydrolysis. There is no evidence for the formation of a low-barrier hydrogen bond between His119 and either an aspartate or an asparagine residue at position 121. Apparently, the major role of Asp121 is to orient the proper tautomer of His119 for catalysis. Thus, the mere presence of a His⋯Asp dyad in an enzymic active site is not a mandate for its being crucial in effecting catalysis.Keywords catalytic dyad; catalytic triad; low-barrier hydrogen bond; pH-rate profile; ribonuclease A; serine protease; site-directed mutagenesis; X-ray crystallographyThe amino acid motifs available to enzymic catalysts are diverse. Still, many enzymes fall into classes wherein great similarities exist. One well-studied class is that of the serine proteases (1). This class of enzymes has an active-site motif known as the catalytic triad, which is composed of the residues Ser⋯His⋯Asp linked by hydrogen bonds (2,3). † This work was supported by Grant GM44783 (NIH). X-ray data collection and computational facilities were supported by Grant BIR-9317398 (NSF). L.W.S. was supported by postdoctoral fellowship CA69750 (NIH). D.J.Q. was supported by Cellular and Molecular Biology Training Grant GM07215 (NIH).*Author to whom correspondence should be addressed.. ‡ Present address: School of Pharmacy, University of Wisconsin -Madison, Madison, WI 53706. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2...

show abstract

Crystallographic analysis of the catalytic mechanism of haloalkane dehalogenase

Cited by 475 publications

References 26 publications

Microbial dehalogenation

Microbial dehalogenation

Repositioning the Catalytic Triad Aspartic Acid of Haloalkane Dehalogenase: Effects on Stability, Kinetics, and Structure

His···Asp Catalytic Dyad of Ribonuclease A: Structure and Function of the Wild-Type, D121N, and D121A Enzymes

Contact Info

Product

Resources

About