Investigating the Catalytic Mechanisms of Bio-degrading Copper Proteins:

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Bio-degrading Cu Enzymes and Reactive Intermediates

Biological copper centers

Bio-degrading copper enzymes

Multicopper Oxidases (MCOs)

Roles of redox-active Trp/Tyr in electron transfer

Stability/Activity Tradeoffs in Thermus thermophilus HB27 Laccase

Abstract
Introduction
Methods
Results and discussion
Acknowledgment
References
Supplementary material
Introduction
Laser sample preparation

Transient absorption data collected at 510 nm and 555 nm (Trp radical absorption regions) for both the S117C mutant and the S117C-W118F mutant.

Trp/Tyr Pair Protects Tth-lac from Oxidative Damage

Abstract
Introduction
Results and discussion
Materials and methods
Funding sources
References
Supporting information

Activity assay of Tth-lac WT and the W118F mutant with promazine hydrochloride as substrate, pH 4.5. Residue distribution of Met (magenta), His (yellow), Asp (green), and active site copper coordinating residues (blue) in Tth-lac. Comparison of the activity of Tth-lac WT and Tth-lac MA (all Met, Asp and His in the Met-rich loop mutated to Ala) in the presence and absence of excess copper in activity assay media (NaOAc, pH 4,5).

Effects of excess copper concentration on the growth rate of activity for Tth-lac WT, Tth-lac MA, and Tth-lac DH.

Investigating the Factors Affecting Laccase Catalysis: Active-site Potentials &

Potential discrepancy

From this observation, it is clear that the enzyme can oxidize high potential substrates, such as ABTS, only in the presence of oxygen. We expected the Phe mutant to have much lower activity. the surface Trp was involved in the binding and oxidation of substrates with high potential. A previous study reported a burst of activity for Tth-lac WT in the presence of excess copper, and the highest degree of activity enhancement was reported with 1 mM CuSO4.

To investigate the roles of the metal-binding residues in the Met-rich loop present in Tth-lac, the 6-His tag was removed, as histidines in the tag can affect enzyme activity in the presence of excess copper. Comparison of the activity of Tth-lac WT and Tth-lac MA (all Met, Asp, and His in the Met-rich loop mutated to Ala) in the presence and absence of excess copper in the activity. Both Tth-lac WT and Tth-lac MA mutant showed almost identical activities in the absence of excess CuSO4 in the test medium (Figure 5.6).

Although the burst of activity is not observed for the Tth-lac MA mutant up to about 1 mM Cu 2+ supplementation in the assay media, the increase in activity became more pronounced at higher excess copper concentration (5 mM, 10 mM). By selecting the 5'NdeI cloning site, the pelB leader sequence was cleaved from the gene for expression in the cytoplasmic region of the cell. Text in orange at the beginning of the sequence above indicates the pelB leader sequence present in the pET-26b vector.

After the pelB leader sequence is cleaved off, the protein begins with what remains of the Nco I ATGg sequence. Protein expression can be achieved even without the ATG start codon at the Nco I site (red bold) due to the presence of another start codon (ATG) in the pelB leader sequence (orange bold). Both genes are in the pRSETB vector which is resistant to ampicillin and both plasmids contain a signal sequence from SmLPMO10A (also known as CBP21) which functions similarly to the pelB leader sequence.

Even then, the decay kinetics of Ru3+ species can be compared between the protein sample and the control sample to see how the redox activity of Ru3+ changes in the presence of LPMOs upon generation of Cu3+.

Figure 5.1. Oxidation of leucoberbelin blue (LBB) and manganese by Tth-lac, pH 4.5.

The Cu T1 -depleted (T1D) laccase and T1D-W118F

Met-rich loop mutant

Since this phenomenon is observed even for the fully metallized enzymes, the presence of additional transient metal binding sites has been speculated, and the most likely candidate is the Met-rich loop in Tth-lac. The Met-rich loop is observed as a common structural motif in several multi-copper oxidases such as CueO and McoA and a Met-rich loop also exists in Tth-lac close to the CuT1, which consists of eleven methionines and one histidine. and one aspartate (Figure 5.5). Since the idea of an additional transient metal binding site coordinated by Met, His and Asp contributing to the enzyme activity has been suggested in other copper enzymes such as CueO, it was tested whether a similar scenario also applies to Tth-lac.

All Met, Asp and His residues in the loop were replaced by Ala which is a non-coordinating metal residue to compare the activity with WT in the presence and absence of excess copper in the media. To elucidate the reason for this observation, another mutant (Tth-lac DH) which had only Asp and His in the loop replaced by Ala was also generated and tested for its activity. Since Met can only bind Cu1+, if additional transiently bound Cu2+ ions in the presence of excess CuSO4 are responsible for the increased activity, Asp and His were the likely candidates that could provide a cavity for Cu2+ binding.

Effects of excessive copper concentration on the degree of activity enhancement for Tth-lac WT, Tth-lac MA (All Met, His and Asp in the loop mutated to Ala) and Tth-lac DH (only His and Asp mutated to Ala). Surprisingly, the activity enhancement was observed (to a slightly smaller extent) even for the Tth-lac DH mutant (Figure 5.7). While for Tth-lac WT and Tth-lac DH, the activity reached the maximum with <500 μM excess copper concentration, as previously observed by a previous study on Tth-lac [26].

Moreover, at low copper concentration (50 μM, 100 μM), the degree of activity increase for Tth-lac DH was less than that for Tth-lac WT. This may mean that Asp and His are somewhat involved in transient copper binding, but the increase in activity with excess copper can still only occur with methionines in the loop.

Figure 5.5. Distribution of Met (magenta), His (yellow), Asp (green), and active-site copper- copper-coordinating residues (blue) in Tth-lac (PDB: 2YAE [2])

CotA laccase from Bacillus Subtilis (CotA-lac)

Current efforts are focused on obtaining crystal structures of the Metrich loop mutants to investigate how the loop conformation changes for each mutant and to see if copper can bind to cavities within the loop. After gene synthesis, protein expression was performed according to the published procedure with minor modifications [30]. The TB media with 0.4% glycerol supplementation gave a higher yield of protein than the LB media, and CuSO was added to make the final concentration of 0.5–0.7 mM in the media, depending on cell density ( 0.6

The enzyme purification was done with an HP SP cationic exchange column according to the published procedure with minor modifications [34], [35]. Although the published procedures use the buffer pH of 7.6, buffers at lower pH (Tris pH 7, MES pH 6 or MOPS pH 6.5) were used to facilitate better binding of the CotA-lac to the cation exchange column. As expected, the W151F point mutation caused no change in activity and total turnover number due to the presence of a Tyr/Trp chain extending from W151 to the enzyme surface.

A W151F/Y152F double mutant is of interest as in Tth-lac (see Chapter 4 for more details), but in CotA-lac also Y51 and W72 exist along the chain after the W151/Y152 pair, and these residues could potentially participate in gap hopping. Making a quadruple mutant with all these residues mutated to Phe can cause severe structural perturbation, which will also require structural analysis. As in Tth-lac, it was of interest to test the roles of surface-exposed redox-active amino acid at the end of the Trp/Tyr chain.

Each of these residues was mutated to a redox-inactive Phe to see if any of the mutations eliminated the enzyme activity. In addition, although both Phe and Ala substitutions were tried, the enzyme did not express with all the Y27F, Y28F, Y27F-Y28F, Y27A and Y28A mutant plasmids.

Xiao, “Reaction mechanisms of CueO multicopper oxidase from Escherichia coli support its functional role as a. The first two orange 'cc' letters of the Nco I site also belong to the pelB leader sequence.). This Asp as well as Met residue from the start codon at the Nco I site (indicated in the above sequence as '/ATGgAT/') was removed via site-directed mutagenesis to ensure that the N-terminal α-amino group His (residue next in the sequence shown as 'cat') is freely available for copper binding.

Since the expression yield of these bacterial LPMOs tends to be very low (5 mg/L), it was quite difficult to isolate the pure enzyme at the beginning. By centrifugation, LPMO-bound Avicel can be separated from the rest of the periplasmic extract. Therefore, instead of using citrate buffer at pH 3.5 as suggested in the published protocol, Tris buffer at pH 7 or 8 was used for purification with anion exchange chromatography.

To briefly outline, Avicel and phosphoric acid swollen cellulose (PASC) were incubated with 1 μM LPMO in 20mM sodium phosphate at pH 6 in the presence of 2 mM ascorbic acid. The sample in an Ependorf tube was gently shaken at 150 rpm for 24 h in the incubator set at 30°C to keep Avicel and PASC suspended in the LPMO solution. The mass of the degradation products can be detected using the matrix-assisted laser desorption/ionization – time of flight (MALDI-TOF) mass spectrometry.

The mass values of the products can be compared with the expected mass values to monitor the rate of cellulose degradation [6]. Irradiation of the Ru-photosensitizer in the sample can generate Ru2+ excited states, and the cobalt quencher can produce more Ru3+ oxidizing species which can potentially be.