Conformational heterogeneity suggests multiple substrate binding modes in CYP106A2

Nathan R Wong; Reethy Sundar; Sophia Kazanis; Jeetayu Biswas; Thomas C Pochapsky

doi:10.1016/j.jinorgbio.2023.112129

Conformational heterogeneity suggests multiple substrate binding modes in CYP106A2

J Inorg Biochem. 2023 Apr:241:112129. doi: 10.1016/j.jinorgbio.2023.112129. Epub 2023 Jan 19.

Authors

Nathan R Wong¹, Reethy Sundar¹, Sophia Kazanis², Jeetayu Biswas³, Thomas C Pochapsky⁴

Affiliations

¹ Dept. of Biochemistry, Brandeis University, 415 South St., Waltham, MA 02454, United States of America.
² Dept. of Chemistry, Brandeis University, MS 015, 415 South St., Waltham, MA 02454, United States of America; Middlesex Community College, 33 Kearney Sq., Lowell, MA 01852, United States of America.
³ Dept. of Chemistry, Brandeis University, MS 015, 415 South St., Waltham, MA 02454, United States of America; Department of Medicine, Weill-Cornell Medicine, New York, NY, United States of America.
⁴ Dept. of Biochemistry, Brandeis University, 415 South St., Waltham, MA 02454, United States of America; Dept. of Chemistry, Brandeis University, MS 015, 415 South St., Waltham, MA 02454, United States of America; Rosenstiel Center for Basic Biomedical Research, Brandeis University, 415 South St., Waltham, MA 02454, United States of America. Electronic address: pochapsk@brandeis.edu.

Abstract

CYP106A2 (cytochrome P450_meg) is a bacterial enzyme originally isolated from B. megaterium, and has been shown to hydroxylate a wide variety of substrates, including steroids. The regio- and stereochemistry of CYP106A2 hydroxylation has been shown to be dependent on a variety of factors, and hydroxylation often occurs at more than one site and/or with lack of stereospecificity for some substrates. Comprehensive backbone ¹⁵N, ¹H and ¹³C resonance assignments based on multidimensional nuclear magnetic resonance (NMR) experiments performed with uniform and selective isotopically labeled CYP106A2 samples are reported herein, and broadening and splitting of resonances assigned to regions of the enzyme shown to be affected by substrate binding in other P450 enzymes indicate that substrate binding does not reduce structural heterogeneity as has been observed previously in P450 enzymes CYP101A1 and MycG. Paramagnetic relaxation enhancement (PRE) due to proximity between substrate protons and the heme iron were measured for three different substrates, and the relatively uniform nature of the PREs support the proposal that multiple substrate binding modes are occupied at saturating substrate concentrations.

Keywords: Cytochrome P450; Enzyme conformations; Nuclear magnetic resonance; Paramagnetism; Regiochemistry; Stereochemistry; Substrate.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Bacterial Proteins / chemistry
Cytochrome P-450 Enzyme System* / metabolism
Hydroxylation
Magnetic Resonance Spectroscopy
Models, Molecular
Molecular Conformation
Steroids*
Substrate Specificity

Substances

Cytochrome P-450 Enzyme System
Steroids
Bacterial Proteins

Abstract

Publication types

MeSH terms

Substances

Grants and funding