Thiocyanate Hydrolase Is a Cobalt-Containing Metalloenzyme with a Cysteine-Sulfinic Acid Ligand

Abstract

Thiocyanate hydrolase (SCNase) purified from Thiobacillus thioparus THI115 hydrolyzes thiocyanate to carbonyl sulfide and ammonia. DNA sequences of the cloned genes revealed the close relation of SCNase to nitrile hydratase (NHase). The consensus sequences for coordination of the metal ion found in NHases were also conserved in the gamma subunit of SCNase. Here, we showed that the SCNase contained one cobalt atom per alphabetagamma heterotrimer. UV-vis absorption spectrum suggested that the cobalt exists as a non-corrin ion. Reduced SCNase showed an ESR signal characteristic of low-spin Co2+, which closely resembled that of the Co-type NHases. Mass spectrometry for the peptide fragment containing the metal-binding motif of the SCNase gamma subunit indicated that the cysteine residue at position 131 was post-translationally oxidized to a cysteine-sulfinic acid. From these results, we concluded that SCNases and NHases form a novel non-corrin and/or non-heme protein family having post-translationally modified cysteine ligands.

Experimental and Computational Study of the Thermodynamic Properties of Trivalent Cobalt Schiff Base Complexes with Cyclic Amines

“…To further confirm the bonding pattern in these complexes, 1 H NMR spectra of the cobalt(III) complexes were recorded in DMSO-d 6 using TMS as an internal standard and show that the cobalt in these complexes is low spin, the data was collected in Table 1. The 1 H NMR spectra of the ligands show OH and NH protons signal at δ 12.39-12.45 and 10.93-11.57 ppm.…”

“…Synthetic oxygen carriers are of great interest as models to mimic oxygen caring metalloenzymes 1 and oxygenases, such as hemoglobin and cytochrome P-450, 2 which play important roles in the catalytic oxygenation mechanism of organic substrates. 3 The Schiff base ligands and their complexes represent one of the most widely utilized classes of ligand in synthetic oxygen carrier compound.…”

Cloning and expression of a gene encoding a novel thermostable thiocyanate-degrading enzyme from a mesophilic alphaproteobacteria strain THI201

“…The sequence homology analysis found no significant homology to other thermostable enzymes or previously purified SCNases or NHases. In addition, the characteristic cobalt-binding motif, V-C1-X-L-C2-S-C3, present in previously characterized SCNases and NHases (Nagashima et al, 1998;Miyanaga et al, 2001;Katayama et al, 2006), was absent in the SCNase here. In contrast, addition of 1 mM Co 2+ in the crude extract of THI201 decreased the original enzyme activity by 26 %.…”

“…thioparus THI115 contains a non-corrin cobalt active centre and has two post-translationally modified cysteine ligands, i.e. cysteine sulfenic acid (or cysteine sulfenate) and cysteine sulfininate; the enzyme requires co-expression of an activator protein, P15K, for its functional expression (Kataoka et al, 2006;Katayama et al, 2006;Arakawa et al, 2007). The presence of a similar enzyme in the halophilic chemolithoautotroph Thiohalophilus (Th.)…”

Phytotoxicity of Thiocyanate to Rice Seedlings

“…An endogenous source of thiocyanate has been observed in tissues of cyanogenic plants during cyanide detoxification (Boening and Chew 1999;Bezsudnova et al 2007). The organisms carrying thiocyanatedegrading ability are considered to be distributed widely (Katayama et al 2006). We therefore have good reasons to propose that plants would be able to metabolize thiocyanate in vivo.…”