The copper active site

The copper active site is the key to LPMO reactivity and highly conserved in all LPMO families. The first crystal structure of an LPMO in which the metal binding site was identified was from Cel61B from H. jecorina (T. reseei). Karkehabadi and colleagues identified a solvent exposed nickel binding site, where the metal ion was bound in a hexacoordination sphere (i.e. an octahedral geometry; Figure 12). The cation was coordinated by the δ-nitrogen and the amino group of the N-terminal histidine, the ε-nitrogen of another highly conserved histidine, the hydroxyl group of a tyrosine in addition to two water molecules (Karkehabadi et al., 2008).

Before copper was shown to be the correct metal of the active site, the importance of divalent metal ions for protein activity was probed in two studies in which Ca

²⁺

, Ni

²⁺

, Mn

²⁺

, Co

²⁺

, Mg

²⁺

and Zn

²⁺

were included in reactions mixtures (Harris et al., 2010, Vaaje-Kolstad et al., 2010). Interestingly, these metals yielded LPMO activity and the inhibition of enzymatic activity by EDTA confirmed the pivotal role of a divalent metal ion (Harris et al., 2010, Vaaje-Kolstad et al., 2010). In retrospect, it is likely that contaminating copper ions in the metal solution used in these studies may have been responsible for the LPMO activity observed.

The first solid proof that this metal binding site is a type 2 copper site was given by Quinlan

and co-workers who could not detect significant binding of the previously used metals, but

very tight binding of Cu

²⁺

in isothermal titration calorimetry (ITC) experiments. By means of

EPR experiments the type 2 copper site was identified and crystallography confirmed

coordination of the metal ion by three nitrogen atoms, provided by two histidines in a

so-called “histidine brace”. The hydroxyl group of the axial tyrosine contributed to shaping the

binding site. In the same publication the authors report methylation of the ε-nitrogen in the

N-terminal histidine which could have an impact on activity (Quinlan et al., 2011). Shortly

after the Quinlan study was published, the Marletta group reported equally convincing data

on the topic of the active site metal, showing that only copper gave LPMO activity in enzymes

reconstituted with various metals and that LPMO-Cu(II) stoichiometry measured by ICP-AES

on purified LPMOs was 1:1 (Phillips et al., 2011a). Since then, the structure and binding

affinity of the copper binding site has been studied in different LPMOs, confirming the key

observations by Quinlan et al. and Phillips et al. but also revealing variation, especially

between the different LPMO families.

30

Figure 12. Detailed view of copper coordination of a AA9 LPMO (LsAA9A) from Lentinus simils;

PDB ID 5ACG) where copper is present in its Cu(II) form. Red spheres represent water molecules.

Amino acid side chains are shown in stick representation. The axial positions of the copper coordination sphere are indicated. The equatorial positions are inhabited by the three nitrogen ligands and the second water molecule. The figure was made with PyMol (DeLano and Lam, 2005).

Copper-binding to LPMOs has been observed to be extremely tight. By means of ITC various dissociation constants (K

d

) have been determined. A K

d

of less than 1 nM at pH 5 has been estimated for TaLPMO9A and Cu(II) (Quinlan et al., 2011). For CBP21 the K

d

for Cu(II) was measured to be 55 nM at pH 6.5 (Aachmann et al., 2012). In the same study it was also reported that binding of Cu(I) was tighter compared to Cu(II) and that the K

d

for CBP21-Cu(I) was calculated to be 1 nM (Aachmann et al., 2012). In other studies authors have reported a dissociation constant ranging from 6 nM at pH 5 to 80 nM at pH 7 for BaAA10A from B. amyloliquefaciens (Hemsworth et al., 2013b) and a K

d

<1 nM for AoAA11 at pH 5 (Hemsworth et al., 2014). In a recent study, divergent copper-binding exhibiting two different K

d

s was observed, one with nanomolar and one with micromolar value. Based on their findings the authors suggest flexibility in the apo copper-binding site and that correct coordination of the copper may be steered by delivery of the copper to the LPMO by specific copper-chaperones (Chaplin et al., 2016).

A unique property of fungal LPMOs is the presence of a methyl group on the -nitrogen of

the N-terminal histidine of LPMOs expressed in fungal hosts (Quinlan et al., 2011, Li et al.,

2012, Lo Leggio et al., 2015). The role of this post-translational modification is unknown, but

it has been suggested to influence catalysis by modification of the histidine pK

a

or alteration

of the active site electronic properties (Aachmann et al., 2012, Hemsworth et al., 2013a,

Beeson et al., 2015). Kim et al. (2014) used a theoretical approach i.e. quantum mechanical

31

calculations and suggested based on their results, that this posttranslational modification has only very minor or no influence on the LPMO catalytic activity. Fungal LPMOs expressed in P. pastoris or E. coli do not show this modification, but still show activity (Kittl et al., 2012, Wu et al., 2013, Hemsworth et al., 2014, Borisova et al., 2015).

Figure 13. Stick representations of the T-shaped coordination of the copper active sites of LPMOs highlighting conserved residues close to the copper (orange sphere). Non-protein ligands are not shown. The figure shows an AA9 (cyan, TaAA9A, PDB ID 2YET), AA10 (gray, CBP21, PDB ID 2BEM), AA11 (magenta, AoLOMO11, PDB ID 4MAI) and an AA13 (yellow, AoAA13, PDB ID 4OPB). The distances between the atoms in the histidine brace are given in Å. It should be noted that all enzymes except CBP21 were crystallized in the presence of copper. The figure was made with PyMol (DeLano and Lam, 2005).

Even though there is some variation in the non-protein ligands of LPMOs with a Cu(II) bound,

the T-shaped histidine brace formed by the copper and its nitrogen ligands is conserved in all

LPMO families when the copper is in the reduced state. However, the surroundings of both

axial positions show some variation (Figure 13). In the buried axial position, the hydroxyl

group of the conserved axial ligand tyrosine, which is present in AA9s, AA11s and AA13s,

contributes to the octahedral coordination of the Cu(II) ion (Quinlan et al., 2011, Harris et al.,

2010, Wu et al., 2013). In AA10s, a phenylalanine usually takes this position although some

AA10s have a tyrosine, similar to the fungal LPMOs. In the solvent exposed axial position,

most AA10s have a highly conserved alanine, which restricts access and may prevent binding

of dioxygen at this position as pointed out by Hemsworth et al. (2013b). Notably, a C1/C4

oxidizing AA10 shows a displacement of the conserved alanine possibly allowing ligand

binding at the solvent exposed axial position (Forsberg et al., 2014a). Similar to AA10s, a

restricted access to the solvent exposed axial position also occurs in some AA9s. Borisova et

al. (2015) and Forsberg et al. (2014), both carried out structural comparisons showing that a

32

hydroxyl group of a conserved tyrosine restricts access to the solvent exposed axial position in strict C1 oxidizing AA9s. In strictly C4 oxidizing AA9s this axial position appears to be unrestricted and C1/C4 oxidizing AA9s exhibit an intermediate architecture (Borisova et al., 2015). AA11s possess features from both, AA9s and AA10s. Next to the axial tyrosine as present in AA9s they also feature the axial alanine like in AA10s (Figure 13; (Hemsworth et al., 2014)).

The photoreduction of Cu(II) to Cu(I) is a common event in crystallography and has been observed several times (Hemsworth et al., 2013b, Hemsworth et al., 2014, Lo Leggio et al., 2015). The structural changes that accompany this reduction have been investigated by Gudmundsson and colleagues. A continuous shift from a trigonal bipyramidal copper coordination to a T-shaped copper coordination i.e. no copper-bound water molecules, was observed with increasing doses of X-ray radiation in studies of EfCBM33A, the only AA10 from E. faecalis (Gudmundsson et al., 2014). Other structures of AA10s (e.g. BaAA10A (Hemsworth et al., 2013b) and JdLPMO10A (Mekasha et al., 2016)) show no other ligands bound to the copper than the three nitrogens, indicating that the copper ion is in Cu(I) state, resulting from X-ray photoreduction.

As already mentioned, EPR analyses of the cellulose-active TaAA9A loaded with Cu(II) clearly identified the type 2 copper center (Quinlan et al., 2011). In a later study of an AA10, also loaded with Cu(II), a copper coordination geometry was observed that lies between type 1 and type 2 in the Peisach-Blumberg classification [(Peisach and Blumberg, 1974)]

(Hemsworth et al., 2013b). In 2014, Forsberg and colleagues compared the structures and EPR spectra of chitin- and cellulose-active AA10s and showed that the cellulose-active proteins possess a type 2 copper center whereas the chitin-active LPMOs fall between the Peisach-Blumberg type 1 and type 2 classifications (Figure 14) (Forsberg et al., 2014b).

However, a general prediction of substrate specificity by EPR is not possible, since two

chitin-active LPMOs, AoLPMO11 and CjLPMO10A, both display a type 2 copper site (Hemsworth

et al., 2014, Forsberg et al., 2016).

33

Figure 14. Typical LPMO EPR signatures. A: Type-2 copper B: mixed. Adapted with permission

from [ Forsberg, Z. 2014. Comparative study of two chitin-active and two cellulose-active AA10-type

lytic polysaccharide monooxygenases. Biochemistry, 53, 1647-1656]. Copyright (2014) American

Chemical Society.

In document Biochemical investigation of catalysis by lytic polysaccharide monooxygenases (sider 43-47)