Exploring the Active Site of the Tungsten, Iron-Sulfur Enzyme Acetylene Hydratase

Abstract

The soluble tungsten, iron-sulfur enzyme acetylene hydratase (AH) from mesophilic Pelobacter acetylenicus is a member of the dimethyl sulfoxide (DMSO) reductase family. It stands out from its class as it catalyzes a nonredox reaction, the addition of H 2 O to acetylene (HOC'COH) to form acetaldehyde (CH 3 CHO). Caught in its active W(IV) state, the high-resolution three-dimensional structure of AH offers an excellent starting point to tackle its unique chemistry and to identify catalytic amino acid residues within the active site cavity: Asp13 close to W(IV) coordinated to two molybdopterin-guanosine-dinucleotide ligands, Lys48 which couples the [4Fe-4S] cluster to the W site, and Ile142 as part of a hydrophobic ring at the end of the substrate access channel designed to accommodate the substrate acetylene. A protocol was developed to express AH in Escherichia coli and to produce active-site variants which were characterized with regard to activity and occupancy of the tungsten and iron-sulfur centers. By this means, fusion of the N-terminal chaperone binding site of the E. coli nitrate reductase NarG to the AH gene improved the yield and activity of AH and its variants significantly. Results from site-directed mutagenesis of three key residues, Asp13, Lys48, and Ile142, document their important role in catalysis of this unusual tungsten enzyme.Molybdenum and tungsten are the only transition metals of the second (Mo) and third (W) row of the periodic table of elements with known biological functions (7). In their biologically active form, both metals are bound to the cofactor molybdopterin (Moco), which is present in all molybdenum and tungsten enzymes with the exception of nitrogenase, where molybdenum is coordinated to a large iron-sulfur cluster, MoFe 7 S 9 (9). Virtually all organisms including plants and mammals use either molybdenum or tungsten proteins in important metabolic pathways (35

Structure and Function of the Unusual Tungsten Enzymes Acetylene Hydratase and Class II Benzoyl-Coenzyme A Reductase

“…The development of a successful protocol for the heterologous expression of ACH in Escherichia coli allowed for site-directed mutagenesis studies [Ten Brink et al, 2011]. Overall, when related to its W content, the activity of the heterologously produced ACH was nearly identical to that of the native enzyme purified from P. acetylenicus .…”

“…When purified under N 2 /H 2 (94/6% v/v) atmosphere, it contains 0.4-0.5 mol W/mol enzyme and 3.7-3.9 mol Fe/mol enzyme (inductively coupled plasma MS) [Meckenstock et al, 1999;Rosner and Schink, 1995;Ten Brink et al, 2011], and the bis-WPT-guanine-dinucleotide cofactor. When isolated under N 2 /H 2 (94/6% v/v) atmosphere, ACH was silent in electron paramagnetic resonance (EPR) spectroscopic analysis; EPR spectra of the enzyme reduced with dithionite showed the typical signal of a low-potential ferredoxin-type [4Fe-4S] +1 cluster (g z = 2.048, g y = 1.939, g x = 1.920 [Meckenstock et al, 1999]).…”

“…acetylenicus is so far the only ACH that has been purified and studied by biochemical, spectroscopic, crystallographic and computational methods. The first purification of ACH was published in 1995 [Rosner and Schink, 1995], followed by a high-resolution crystal structure and heterologous expression and site-directed mutagenesis of several amino acids located at the active site [Ten Brink et al, 2011]. ACH was isolated from the soluble fraction of P .…”

Acetylene hydratase: a non-redox enzyme with tungsten and iron–sulfur centers at the active site

“…When purified in the absence of dioxygen, the enzyme did not exhibit any EPR signal, it contained 0.4-0.5 mol W and 3.7-3.9 mol Fe/mol enzyme, and 1.3-1.4 mol pyranopterin-guaninedinucleotide cofactor (MGD). The amount of MGD and W was always low but the ratio of these two constituents never fell below 2 [41,42,64]. EPR spectra of AH reduced with Na + dithionite showed the typical signal of a low potential ferredoxin-type [4Fe-4S] cluster (g z = 2.048, g y = 1.939, g x = 1.920) [42].…”

“…In 2007 the high resolution crystal structure was reported [63], and in 2011 the heterologous expression and site-directed mutagenesis of several amino acid residues at the active site was achieved [64]. AH activity is localized exclusively in the soluble fraction of the cell extract.…”