Pannell, Sarah Esme (2011) Structural determinants of peroxidase activities. Doctoral thesis (DPhil), University of Sussex.

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Abstract

Horseradish peroxidase (HRP) is a robust enzyme with commercial applications as an
immunodiagnostic reporter enzyme and in the catalysis of difficult chemical
transformations. The commercial enzyme is still isolated from the roots of the
horseradish plant Armoracia rusticana, and has been studied as a model haem enzyme
system since the early 1940’s. Following the development of methods to produce the
active recombinant enzyme in E.coli (Smith et al., 1990) and completion of the
crystallographic structure in 1997 (Gajhede et al., 1997) it has been possible to identify
the structural requirements for activity and to extend these activities by protein
engineering techniques. Three aspects relating to the enhancement of the ‘normal’
and ‘designed’ activities of selected variants have been explored at the structure
function level in this work.

Earlier work (Gajhede et al., 1997; White et al., 1997) highlighted residues that interact
with aromatic substrates but that also potentially occlude access to the reactive haem
edge by larger bulky substrates of potential commercial interest. The X-ray structure
of the HRP-C* A140G/F179A variant was solved to 2.0Å. A larger engineered cavity at
the haem edge was observed consistent with the ability of the variant to oxidise
luminol directly, a property not seen in the wild-type enzyme. The structure factors
for other residues in the haem access channel were not significantly affected.

The structure of the S167M HRP variant was also solved, because of interest in its
ability to form a novel sulphonium linkage to the haem vinyl group (K. Cali, DPhil
thesis, University of Sussex). The sulphur to β vinyl distance was found to be 3.15Å,
compared to 1.7Å in the natural sulphonium linkage of myeloperoxidase. This implies
that significant thermal motion in the structure is required for the haem-protein crosslink to form, accounting for the relatively slow autocatalytic modification process
observed in the presence of hydrogen peroxide.

Previous work (Ngo and Smith, Int. Pat. No. WO/2007/020428) has shown that HRP
engineered with a more open distal haem pocket (mimicking that of chloroperoxidase
or cytochrome P450s) with a weak surrogate base (provided by a Glu residue as in
chloroperoxidase) was capable of both entantioselective sulphoxidation and
epoxidation. Building on this work, an alternative variant was designed in which the
location of the weak base, provided by a Glu or Asp residue, has been varied within the active site. In particular, the HRP variant R38E:F41A:H42A (EAA) catalyses the
production of the 1 and 2 naphthol from naphthalene at a rate of 124±4 min-1,
suggesting the generation of an epoxide intermediate in the active site. The wild-type
enzyme does not catalyse this reaction or the sulphoxidation reactions described for
earlier variants. This is believed to be the first report of aromatic C-H bond activation
by an engineered plant peroxidase and is unusual in that C-H bond activation of this
type normally requires a P450-type thiolate ligated haem system. Equilibrium binding
studies show that naphthalene binds to the engineered haem cavity with an estimated
Kd of 30±2 μM. Unfortunately, crystals of HRP variants described by Ngo and Smith
(2007) and of the new EAA variant described here could not be obtained, despite many
crystallisation attempts under a wide range of conditions.

Item Type:	Thesis (Doctoral)
Schools and Departments:	School of Life Sciences > Biochemistry
Subjects:	Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry
Depositing User:	Library Cataloguing
Date Deposited:	18 Nov 2011 15:49
Last Modified:	21 Aug 2015 13:59
URI:	http://sro.sussex.ac.uk/id/eprint/7463

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