Structure, recombinant expression and mutagenesis studies of the catalase with oxidase activity fromScytalidium thermophilum

Abstract

Scytalidium thermophilum produces a catalase with phenol oxidase activity (CATPO) that catalyses the decomposition of hydrogen peroxide into oxygen and water and also oxidizes various phenolic compounds. A codon-optimized catpo gene was cloned and expressed in Escherichia coli. The crystal structures of native and recombinant S. thermophilum CATPO and two variants, H82N and V123F, were determined at resolutions of 2.7, 1.4, 1.5 and 1.9 Å, respectively. The structure of CATPO reveals a homotetramer with 698 residues per subunit and with strong structural similarity to Penicillium vitale catalase. The haem component is cis-hydroxychlorin γ-spirolactone, which is rotated 180° with respect to small-subunit catalases. The haem-binding pocket contains two highly conserved water molecules on the distal side. The H82N mutation resulted in conversion of the native d-type haem to a b-type haem. Kinetic studies of the H82N and V123F mutants indicate that both activities are likely to be associated with the haem centre and suggest that the secondary oxidase activity may be a general feature of catalases in the absence of hydrogen peroxide.

Role of Active Site Residues on Catalytic Activity of Catalase with Oxidase Activity from Scytalidium Thermophilum

“…For example, the Scytalidium thermophilum catalase (CATPO) has been shown to oxidize o-and some p-diphenolic compounds in the absence of hydrogen peroxide (Ögel et al, 2006;Sutay Kocabas et al, 2008;Yuzugullu et al, 2013). This and other studies have led to the proposal that this secondary oxidative activity may be a general characteristic of catalases.…”

“…Single-point mutations were introduced into the catpo coding region by a QuikChange approach with mutagenic primer pairs using the approach described by Stratagene but with KOD DNA polymerase (Novagen) for 25 cycles before DpnI digestion (Yuzugullu et al, 2013). The PCR primers containing the desired mutations were purchased from Sacem and Sentegen and are listed in Table 1.…”

“…pET28a-CATPO was freshly transformed into E. coli BL21 Star (DE3) cells and a single colony was inoculated into 10 ml LB medium supplemented with 50 mg ml -1 kanamycin and incubated overnight at 310 K with shaking (200 rev min -1 ) (Yuzugullu et al, 2013). This overnight culture was used to inoculate 1 l LB with 50 mg ml -1 kanamycin in a 2.4 l conical flask and was grown at 310 K (200 rev min -1 ) to an OD600 of 0.6-0.8.…”

“…Although catalases have been studied for many years, a peroxide independent oxidative activity of catalases has been recently recognized (Vetrano et al, 2005;Sutay Kocabas et al, 2008;Yuzugullu et al, 2013). For example, the Scytalidium thermophilum catalase (CATPO) has been shown to oxidize o-and some p-diphenolic compounds in the absence of hydrogen peroxide (Ögel et al, 2006;Sutay Kocabas et al, 2008;Yuzugullu et al, 2013).…”

Unprecedented access of phenolic substrates to the heme active site of a catalase: Substrate binding and peroxidase-like reactivity ofBacillus pumiluscatalase monitored by X-ray crystallography and EPR spectroscopy

“…The putative expression of a single catalase‐peroxidase in a soil bacterium was also unusual in comparison with other Bacillus species, which usually encode one or more heme catalases . While both bovine liver catalase (BLC) and the large subunit Scytalidium thermophilum catalase have been reported to support oxidase activities, the only previously reported oxidase activity of catalase‐peroxidases was that of a low level NADH oxidase for Burkholderia pseudomallei KatG . Furthermore, there has been one report of a polyphenol peroxidase activity associated with BLC …”