Utilising non-canonical amino acids in the design of artificial enzymes: an exploration of Cu-enzymes, steroid carrier protein scaffolds and synthetic biology

Abstract

Artificial metalloenzymes (ArMs) merge the selectivity of enzymes and reactivity of transition metals, bringing novel reactivity into the field of biocatalysis. However, ArMs are a relatively new field and there are still many unknowns that need to be explored for the catalysts to be applied in industry.

In this work, the design of novel Cu(II) ArMs using human sterol carrier protein (SCP_2L) scaffold was investigated to study sterol carrier protein (SCP) as privileged scaffold for artificial enzyme design. The SCP_2L scaffold was modified to contain a 2,2’-bipyridine side chain to create a novel, site-selective metal binding site for Cu(II) ions and thus generating a catalytically active site in the SCP_2L protein scaffold. The 2,2’-bipyridine moiety was either bioconjugated to SCP_2L via unique cysteines residues or incorporated as unnatural amino acid (UAA) (2,2’-bipyridin-5-yl)alanine (BpyAla) using genetic code expansion (GCE). The novel Cu(II) ArMs show moderate initial selectivity in the enantioselective Friedel-Crafts alkylation reaction. The most promising catalyst was the SCP_Q111BpyAla mutant that exhibited induced product enantioselectivity of 60% e.e. The X-ray structures of the SCP_Q111CBpy_Cu and SCP_Q111BpyAla_Cu were solved, showing a different orientation of 2,2'- bipyridine in the protein environment as a result of different modification strategies. An alanine scan of nearby residues to the Cu in SCP_Q111Bpy_Cu was conducted, and the mutants were tested in the Friedel-Crafts reaction. None of the mutants were found to conclusively favour or disfavour catalysis, and current work is looking to use computational methods to help optimise the catalytic activity. The work improved the understanding of ArMs as looking at the same ligand and reaction through different modification strategies. Overall, the results demonstrate that the SCP_2L scaffold could be used as a scaffold for the design of ArMs using both genetic code expansion and bioconjugation methods.

A limitation of the SCP-2L scaffold for unnatural reactions is the relative instability to metal ions and temperature. A survey of the protein crystallographic database (PDB) revealed two known structures of steroid carrier proteins from thermophilic organisms. SCP from Thermus thermophilus (TT_SCP) was chosen as a scaffold to expand the reaction scope of the SCP-based ArMs to be able to catalyse reactions at higher temperatures and in the presence of solvents. Residues W83, L87, A96, and L102 were identified as potential sites for bioconjugation via computational analysis. To date, W83 and L102 were successfully obtained using site-directed mutagenesis and expressed in good yield. Nano differential scanning fluorimetry (NanoDSF), circular dichroism (CD) and thermal shift assay (TSA) were used to assess the chemical stability and thermostability of human SCP_2L and TT_SCP. From the initial studies, TT_SCP seems to be more stable than human SCP_2L with Tm higher than 95 °C. Initial bioconjugation studies of TT_SCP show successful attachment of phenanthroline ligand and Cu binding.

Unnatural amino acids (UAAs) are valuable additions to the genetic code for the design of new artificial enzymes. However, industrial use of this technology is unknown in part due to the need to synthesize the amino acid in large quantities. As a technology development project, a self-sustainable E. coli chemical factory was designed to produce UAA para-aminophenylalanine (pAF) using three genes of the biosynthetic pathway papABC. PapA, papB, and papC were designed to be compatible with the JUMP platform to facilitate pathway optimisation and provide a system for UAAs offering a high throughput screening of biosynthetic pathways. The selective incorporation of pAF into LmrR protein scaffold was achieved and the artificial enzyme catalysed the hydrazone formation in silico.