para
-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 ‘Hydroquinone (HQ) pathway’ in Gram-negative degraders, whereas via ‘Benzenetriol (BT) pathway’ in Gram-positive ones. ‘HQ pathway’ is initiated by a single-component PNP 4-monooxygenase and ‘BT pathway’ by a two-component PNP 2-monooxygenase. Their rigio-selectivity intrigues us 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
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 group D flavin-dependent monooxygenases with an 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 towards FAD, hence positioning the substrate properly. A variant N450A was found with improved activity for 4NC and 2C4NP, probably because of the reduced steric hindrance. PnpA1 shows obvious difference in substrate selectivity with its close homologues TcpA and TftD, which may be determined by Thr296 and loop 449–454. Above all, our study allows the structural comparison between the two types of PNP monooxygenases. An explanation that accounts for their regio-selectivity was proposed: the different PNP binding manner determines their choice of
ortho
- or
para
-hydroxylation on PNP.
IMPORTANCE
Single-component PNP monoxygenases hydroxylate PNP at 4-position while two-component ones at 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 towards 4NC and 2C4NP was obtained. Moreover, through comparing 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 molecular level. And these results will also be of further aid in enzyme rational design in bioremediation and biosynthesis.
Severity of the Streptomycin Resistance and Streptomycin Dependence Phenotypes of Ribosomal Protein S12 of Thermus thermophilus Depends on the Identity of Highly Conserved Amino Acid Residues