Characterization of a new cobalt‐containing nitrile hydratase purified from urea‐induced cells of Rhodococcus rhodochrous J1

Abstract

A new cobalt-containing nitrile hydratase was purified from extracts of urea-induced cells from Rhodococcus rhodochrous J1 in seven steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. Nitrile hydratase was a 500 -530-kDa protein composed of two different subunits (a subunit 26 kDa, fl subunit 29 kDa).The enzyme contained approximately 11 -12 mol cobalt/mol enzyme. A concentrated solution of highly purified nitrile hydratase exhibited a broad absorption spectrum in the visible range, with an absorption maxima at 410 nm. The enzyme had a wide substrate specificity. Aliphatic saturated or unsaturated nitriles as well as aromatic nitriles, were substrates for the enzyme. The optimum pH of the hydratase was pH 6.5 -6.8. The enzyme was more stable than ferric nitrile hydratases. The amino-terminal sequence of each subunit of R. rhodochrous J1 enzyme was determined and compared with that of ferric nitrile hydratases. Prominent similarities were observed with the subunit. However, the amino acid sequence of the CI subunit from R. rhodochrous J1 was quite different from that of the ferric enzymes. In the present study, we purified and characterized the new cobalt-containing nitrile hydratase of R . rhodochrous J1 and the physicochemical and enzymatic properties were compared with the iron-induced and iron-containing nitrile hydratases from P. chlororaphis B23, Brevibacterium R312 and (') Rhodococcus sp. N-774.The improvement of culture conditions and the mutagenesis of P. chlororaphis B23 greatly increased the production of nitrile hydratase [4-61. As a result, P. chlororuphis B23 nitrile hydratase is used as a catalyst for the industrial production of acrylamide in Japan [7 -91. We purified and crystallized nitrile hydratase from P. MATERIALS AND METHODS Materials

Purification of a hyperactive nitrile hydratase from resting cells of Rhodococcus rhodochrous PA-34

“…αβ 2 ). However, the number of the subunits of polypeptides (α-and β-subunit) varied from 2-20 in NHases of other microorganism and the molecular mass of earlier reported active NHases varied from 54 kD to 505 kD [12,13].…”

“…These two polypeptides (subunits) of NHase were named as α-subunit and β-subunit. Usually, α-subunit is lighter in weight in comparison to β-subunit [12]. It means that the protein bands on SDS-PAGE of 25.04 kD and 30.6 kD are α-and β-subunit of NHase of R. rhodochrous PA-34, respectively.…”

Sulfur K-Edge XAS and DFT Calculations on Nitrile Hydratase: Geometric and Electronic Structure of the Non-heme Iron Active Site

“…[5][6][7] The Fe enzyme is isolated in a diamagnetic NO-bound form, activated by irradiation, and is proposed to be stabilized by the presence of butyric acid in the medium. 4 The sixth coordination site is occupied by a NO (as isolated) or an H x O molecule (after photolysis). 8,9 In a recent crystal structure of the inactive NO-bound form 3a (Figure 1a), the cysteine ligands are posttranslationally modified to RSO-(H) and RSO 2 (H) in contrast to a previously reported crystal structure showing non-oxidized CysS − coordination (Figure 1).…”

Incorporation of thiolate donation using 2,2′-dithiodibenzaldehyde: Complexes of a pentadentate N2S3 ligand with relevance to the active site of Co nitrile hydratase

“…Examples include nitrile hydratase [1][2][3][4][5][6][7][8][9][10] and the A-cluster of carbon monoxide dehydrogenase/acetyl CoA synthase [11][12][13][14][15][16][17][18][19][20][21][22][23]. Understanding the catalytic mechanisms of these enzymes can be greatly aided by the study of small-molecule analogs, or model complexes, which reproduce the active-site structure and/or the function of the enzyme.…”