A novel thermophilic hemoprotein scaffold for rational design of biocatalysts

Abstract

Hemoproteins are commonly found in nature, and involved in many important cellular processes such as oxygen transport, electron transfer, and catalysis. Rational design of hemoproteins can not only inspire novel biocatalysts but will also lead to a better understanding of structure-function relationships in native hemoproteins. Here, the heme nitric oxide/oxygen-binding protein from Caldanaerobacter subterraneus subsp. tengcongensis (TtH-NOX) is used as a novel scaffold for oxidation biocatalyst design. We show that signaling protein TtH-NOX can be reengineered to catalyze HO decomposition and oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) by HO. In addition, the role of the distal tyrosine (Tyr140) in catalysis is investigated. The mutation of Tyr140 to alanine hinders the catalysis of the oxidation reactions. On the other hand, the mutation of Tyr140 to histidine, which is commonly observed in peroxidases, leads to a significant increase of the catalytic activity. Taken together, these results show that, while the distal histidine plays an important role in hemoprotein reactions with HO, it is not always essential for oxidation activity. We show that TtH-NOX protein can be used as an alternative scaffold for the design of novel biocatalysts with desired reactivity or functionality. H-NOX proteins are homologous to the nitric oxide sensor soluble guanylate cyclase. Here, we show that the gas sensor protein TtH-NOX shows limited capacity for catalysis of redox reactions and it can be used as a novel scaffold in biocatalysis design.

Functional characterization of a novel CYP119 variant to explore its biocatalytic potential

“…During the reaction, an increase at 470 nm can be observed (Figure 1). This band can be attributed to tetraguaicol formation 38 . For WT CYP119, no significant increase in 470 nm was observed (Figure 1); therefore, WT CYP119 does not catalyze guaiacol oxidation.…”

“…A decrease in 734 nm was observed after this time; this is thought to be due to oxidation of the ABTS •+ radical by another electron and production of azodication (ABTS 2+ ). This reaction is shown in Equation (3): 38 $$\begin{equation}ABTS{\rm{ }} \to {\rm{ }}ABT{S^ + } + {\rm{ }}{e^ - } \to {\rm{ }}ABT{S^{2 + }} + {\rm{ }}{e^ - }\end{equation}$$…”

“…The reactions were followed at 1‐min intervals from 950 to 350 nm for 45 min. The yield of the ABTS +• radical cation formation by WT and DM CYP119 was assessed by using the extinction coefficient of ABTS +• at 734 nm ( Ɛ 734nm = 1.5 × 10 4 M −1 cm −1 ) 30,38 …”

Heme-Based Gas Sensors in Nature and Their Chemical and Biotechnological Applications

“…Their peroxidase activities were investigated using the ABTS (2,2 -azino-bis(3ethylbenzothiazoline-6-sulphonic acid)) substrate along with H 2 O 2 , where the WT protein showed moderate activity but the Y140H mutant was significantly better (70% higher) in degrading ABTS. Despite this protein showed a much lower turnover compared to other known catalytic proteins, the authors claimed there is a comparable efficiency in oxidizing substrates as in some native catalysts (e.g., but using different substrates, TtH-NOX k cat = 0.06 s −1 for ABTS versus cytochrome P450BM-3 k cat < 0.008 s −1 ) [137]. This study did not provide appealing catalytical results, but it highlighted the opportunities ahead, taking advantage of the thermostability, easy production and structural robustness of that heme-based gas sensor protein.…”

“…Aiming to explore and enhance potential catalytic properties of the heme-based gas sensor Tt-H-NOX, its heme domain was studied and subjected to site-direct mutagenesis at the distal tyrosine ligand [137]. Interestingly, this protein showed capacity to decompose H 2 O 2 , which was strongly enhanced in the Y140H mutant and lowered in the Y140A mutant.…”