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the biosynthesis of the bottromycin core scaffold (Figure 40c). The yield of the reaction was initially limited to ~20% and no amount of optimisation could improve it. It was known the SalCYP substrate existed as an equilibrium of two epimers, and eventually it was discovered that the enzyme was stereoselective it was only able to accept one of the two epimers. The exchange between the two epimers was so slow that it limited the reaction yield. A hydrolase encoded in the bottromycin biosynthetic gene cluster was found to function as an amino acid epimerase and was able to interconvert the two substrate epimers rapidly [8]. When both enzymes

were added together, the epimerase was able to supply SalCYP with the correct epimer rapidly enough to achieve near complete conversion (Figure 40d).

The characterisation of SalCYP made it possible to establish a biosynthetic route to the bottromycin core scaffold, which achieves near complete conversion of the peptide substrate to its product in two one-pot reactions. This is a much simpler route than total synthesis, which requires at least 17 steps, and has made it possible to generate a number of non-natural bottromycin variants, which will be tested for their antibiotic activity.

PRINCIPAL PUBLICATION AND AUTHORS

Characterization of the Stereoselective P450 Enzyme BotCYP Enables the In Vitro Biosynthesis of the Bottromycin Core Scaffold, S. Adam (a), L. Franz (a), M. Milhim (b), R. Bernhardt (b), O.V. Kalinina (c,d), J. Koehnke (a,e), J. Am. Chem. Soc. 142, 49, 20560-20565 (2020); https:/doi.org/10.1021/jacs.0c10361 (a) Workgroup Structural Biology of Biosynthetic Enzymes, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Centre for Infection Research (HZI), Saarland University, Saarbrücken (Germany) (b) Department of Biochemistry, Saarland University, Saarbrücken (Germany) (c) Drug Bioinformatics Group, HIPS, HZI, Saarbrücken, Germany. Medical Faculty, Saarland University, Homburg (Germany) (d) School of Chemistry, University of Glasgow, Glasgow (UK)

REFERENCES

[1] J.M. Waisvisz et al., J. Am. Chem. Soc. 79, 4520-4521 (1957). [2] H. Shimamura et al., Angew. Chem. Int. Ed. 48, 914-917 (2009). [3] L. Huo et al., Chem. Biol. 19, 1278-87 (2012). [4] Y. Hou et al., Org. Lett. 14, 5050-5053 (2012). [5] W.J.K. Crone et al., Chem. Sci. 3, 3516 (2012). [6] J.P. Gomez-Escribano et al., Chem. Sci. 3, 3522 (2012). [7] W.J.K. Crone et al., Angew. Chem. Int. Ed. Engl. 55, 9639-9643 (2016). [8] A. Sikandar et al., Nat. Chem. Biol. 16, 1013-1018 (2020).

Why does heme bind to only one member of the SOUL/HBP family of proteins?

HEBP1 and HEBP2 are two proteins that form a family called HBP/SOUL. Although both proteins are present in animals, plants and bacteria, no definitive functional role has been found. X-ray crystallography and NMR spectroscopy was used to determine the structures of these proteins to gain insight into their function.

The HBP/SOUL family represents a group of evolutionary conserved putative heme-binding proteins (HEBP) that contains a number of members from animal, plant and bacterial species. The structures of the murine form of HEBP1 (mHEBP1), or p22HBP, and the human form of HEBP2 (hHEBP2), or hSOUL, were determined in 2006 [1] and 2011 [2] respectively. Interestingly, HEBP1 has been found to bind heme, while HEBP2 has not. A survey of the functions attributed to HEBP1 and HEBP2 over the last 20 years reveal they may be involved in a wide range of cellular pathways. Interestingly, many of them are specific to higher eukaryotes, particularly mammals, and a potential link between heme release under oxidative stress and hHEBP1 may be relevant. However, no clear

physiological function can be attributed to the proteins HEBP1 and HEBP2.

HEBP1 is a small (22 kDa) protein expressed in various tissues, with highest mRNA levels seen in liver, spleen and kidney [3]. It can bind heme and other molecules found in the heme biosynthesis pathway such as protoporphyrin- IX (PPIX) and coproporphyrinogen. As cellular free heme is considered a dangerous precursor to the formation of radical oxygen species that can severely damage the cell, HEBP1 was initially thought to be involved in protecting the cell from this reactive molecule.

The solution structure of mHEBP1, determined by nuclear magnetic resonance (NMR) spectroscopy, presented a novel fold in eukaryotes: a 9-stranded twisted b-barrel flanked by two α-helices [1]. Fluorescence quenching (FQ) and NMR chemical shift perturbations were used to identify the magnitude (Kd at the nM level) and the residues (a hydrophobic pocket) responsible for the hemin and PPIX interaction with the protein. However, the orientation of the heme ring could not be determined, and NMR studies of site-specific mutants could not pinpoint the key amino acids responsible for binding. Recently, the crystal structure of heme-bound murine HEBP1 (7OON) was determined at beamline ID23, enabling the binding orientation of the heme