7DBW

PnpA1, the oxygenase component of a two-component para-nitrophenol hydroxylase from Rhodococcus imtechensis RKJ300

Summary for 7DBW

• Entry DOI: 10.2210/pdb7dbw/pdb
• Descriptor: 4-hydroxyphenylacetate 3-hydroxylase (2 entities in total)
• Functional Keywords: para-nitrophenol, flavin-dependent two-component monooyxgenase, hydroxylase, biodegradation, oxidoreductase
• Biological source: Rhodococcus imtechensis RKJ300 = JCM 13270
• Total number of polymer chains: 3
• Total formula weight: 179417.31

Authors

Guo, Y.,Zheng, J.T.,Zhou, N.Y. (deposition date: 2020-10-22, release date: 2020-11-04, Last modification date: 2023-11-29)

Primary citation

Guo, Y.,Li, D.F.,Zheng, J.,Xu, Y.,Zhou, N.Y.
Single-Component and Two-Component para -Nitrophenol Monooxygenases: Structural Basis for Their Catalytic Difference.
Appl.Environ.Microbiol., 87:e0117121-e0117121, 2021

PubMed Abstract: -Nitrophenol (PNP) is a hydrolytic product of organophosphate insecticides, such as parathion and methylparathion, in soil. Aerobic microbial degradation of PNP has been classically shown to proceed via the "hydroquinone (HQ) pathway" in Gram-negative degraders, whereas it proceeds via the "benzenetriol (BT) pathway" in Gram-positive ones. The "HQ pathway" is initiated by a single-component PNP 4-monooxygenase and the "BT pathway" by a two-component PNP 2-monooxygenase. Their regioselectivity intrigued us enough to investigate their catalytic difference through structural study. PnpA1 is the oxygenase component of the two-component PNP 2-monooxygenase from Gram-positive Rhodococcus imtechensis strain RKJ300. It also catalyzes the hydroxylation of 4-nitrocatechol (4NC) and 2-chloro-4-nitrophenol (2C4NP). However, the mechanisms are unknown. Here, PnpA1 was structurally determined to be a member of the group D flavin-dependent monooxygenases with an acyl coenzyme A (acyl-CoA) dehydrogenase fold. The crystal structure and site-directed mutagenesis underlined the direct involvement of Arg100 and His293 in catalysis. The bulky side chain of Val292 was proposed to push the substrate toward flavin adenine dinucleotide (FAD), hence positioning the substrate properly. An N450A variant was found with improved activity for 4NC and 2C4NP-probably because of the reduced steric hindrance. PnpA1 shows an obvious difference in substrate selectivity with its close homologues TcpA and TftD, which may be caused by the unique Thr296 and a different conformation in the loop from positions 449 to 454 (loop 449-454). Above all, our study allows structural comparison between the two types of PNP monooxygenases. An explanation that accounts for their regioselectivity was proposed: the different PNP binding manners determine their choice of - or -hydroxylation on PNP. Single-component PNP monoxygenases hydroxylate PNP at the 4 position, while two-component ones do so at the 2 position. However, their catalytic and structural differences remain elusive. The structure of single-component PNP 4-monooxygenase has previously been determined. In this study, to illustrate their catalytic difference, we resolved the crystal structure of PnpA1, a typical two-component PNP 2-monooxygenase. The roles of several key amino acid residues in substrate binding and catalysis were revealed, and a variant with improved activities toward 4NC and 2C4NP was obtained. Moreover, through comparison of the two types of PNP monooxygenases, a hypothesis was proposed to account for their catalytic difference, which gives us a better understanding of these two similar reactions at the molecular level. In addition, these results will also be of further aid in rational design of enzymes in bioremediation and biosynthesis.

PubMed: 34469195
DOI: 10.1128/AEM.01171-21

Experimental method

X-RAY DIFFRACTION (2.5 Å)