PnpC1C2 is an enzyme from the soil bacterium Pseudomonas putida DLL-E4 that is in the pathway for the oxidative catabolism of 4-nitrophenol. PnpC1C2 oxidatively cleaves hydroquinone into γ-hydroxymuconic semialdehyde. It belongs to the type II hydroquinone dioxygenase family, a relatively uncharacterized group of mononuclear non-heme Fe(II)-dependent enzymes that catalyze oxidative ring-cleavage reactions, which includes the well-studied catechol extradiol dioxygenases as well as the structurally unrelated 2,6-dichlorohydroquinone dioxygenase (PcpA). Steady-state kinetics studies using UV/Vis spectroscopy were performed to characterize the enzyme specificity towards various substituted hydroquinones. In addition to its native substrate, PnpC1C2 was active towards a variety of monosubstituted hydroquinones. Methyl- and methoxyhydroquinone showed a moderately higher <InlineEquation ID="IEq1"> <InlineMediaObject> <ImageObject Color="BlackWhite" FileRef="775_2025_2101_Article_IEq1.gif" Format="GIF" Height="20" Rendition="HTML" Resolution="72" Type="Linedraw" Width="37" /> </InlineMediaObject> <EquationSource Format="TEX">$K_{mA}^{app}$</EquationSource> <EquationSource Format="MATHML"><math> <msubsup> <mi>K</mi> <mrow> <mi mathvariant="italic">mA</mi> </mrow> <mrow> <mi mathvariant="italic">app</mi> </mrow> </msubsup> </math></EquationSource> </InlineEquation>, and chloro- and bromohydroquinone showed a moderately lower <InlineEquation ID="IEq2"> <InlineMediaObject> <ImageObject Color="BlackWhite" FileRef="775_2025_2101_Article_IEq2.gif" Format="GIF" Height="19" Rendition="HTML" Resolution="72" Type="Linedraw" Width="30" /> </InlineMediaObject> <EquationSource Format="TEX">$k_{cat}^{app}$</EquationSource> <EquationSource Format="MATHML"><math> <msubsup> <mi>k</mi> <mrow> <mi mathvariant="italic">cat</mi> </mrow> <mrow> <mi mathvariant="italic">app</mi> </mrow> </msubsup> </math></EquationSource> </InlineEquation>, but all had a <InlineEquation ID="IEq3"> <InlineMediaObject> <ImageObject Color="BlackWhite" FileRef="775_2025_2101_Article_IEq3.gif" Format="GIF" Height="20" Rendition="HTML" Resolution="72" Type="Linedraw" Width="74" /> </InlineMediaObject> <EquationSource Format="TEX">${{k_{cat}^{app} } \mathord{\left/ {\vphantom {{k_{cat}^{app} } {K_{mA}^{app} }}} \right. \kern-0pt} {K_{mA}^{app} }}$</EquationSource> <EquationSource Format="MATHML"><math> <mrow> <msubsup> <mi>k</mi> <mrow> <mi mathvariant="italic">cat</mi> </mrow> <mrow> <mi mathvariant="italic">app</mi> </mrow> </msubsup> <mrow> <mfenced open="/"> <mphantom> <mpadded width="0pt"> <msubsup> <mi>k</mi> <mrow> <mi mathvariant="italic">cat</mi> </mrow> <mrow> <mi mathvariant="italic">app</mi> </mrow> </msubsup> <msubsup> <mi>K</mi> <mrow> <mi mathvariant="italic">mA</mi> </mrow> <mrow> <mi mathvariant="italic">app</mi> </mrow> </msubsup> </mpadded> </mphantom> </mfenced> </mrow> <msubsup> <mi>K</mi> <mrow> <mi mathvariant="italic">mA</mi> </mrow> <mrow> <mi mathvariant="italic">app</mi> </mrow> </msubsup> </mrow> </math></EquationSource> </InlineEquation> within an order of magnitude of unsubstituted hydroquinone. Likewise, only small differences in the rates of mechanism-based inactivation were observed among these substrates. Among disubstituted hydroquinones, only 2,6- and 2,5-dimethylhydroquinone showed any activity, with the latter only barely detectable. A variety of para-substituted phenols were found to be good inhibitors of PnpC1C2. NMR studies were performed to determine the regioselectivity of ring-cleavage with monosubstituted hydroquinones. All monosubstituted hydroquinones tested (methyl-, chloro-, bromo-, and methoxyhydroquinone) yielded exclusively the 1,6-cleavage product. Thus, PnpC1C2 shows notable differences in both its substrate specificity and the ring-cleavage regioselectivity compared to that of PcpA. These results provide an important basis for future comparison of structure–function correlations among the hydroquinone ring-cleaving dioxygenases. Graphical abstract

Characterization of the substrate specificity and regioselectivity of ring-cleavage of Pseudomonas putida DLL-E4 hydroquinone 1,2-dioxygenase (PnpC1C2)

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发表日期：

2025年2月17日

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Timothy E. Machonkin,
Madeleine S. Maker,
Nandin Ganjoloo,
Drew F. Conkin

摘要

PnpC1C2 is an enzyme from the soil bacterium Pseudomonas putida DLL-E4 that is in the pathway for the oxidative catabolism of 4-nitrophenol. PnpC1C2 oxidatively cleaves hydroquinone into γ-hydroxymuconic semialdehyde. It belongs to the type II hydroquinone dioxygenase family, a relatively uncharacterized group of mononuclear non-heme Fe(II)-dependent enzymes that catalyze oxidative ring-cleavage reactions, which includes the well-studied catechol extradiol dioxygenases as well as the structurally unrelated 2,6-dichlorohydroquinone dioxygenase (PcpA). Steady-state kinetics studies using UV/Vis spectroscopy were performed to characterize the enzyme specificity towards various substituted hydroquinones. In addition to its native substrate, PnpC1C2 was active towards a variety of monosubstituted hydroquinones. Methyl- and methoxyhydroquinone showed a moderately higher $K_{mA}^{app}$ $K_{mA}^{app}$ , and chloro- and bromohydroquinone showed a moderately lower $k_{cat}^{app}$ $k_{cat}^{app}$ , but all had a ${{k_{cat}^{app} } \mathord{\left/ {\vphantom {{k_{cat}^{app} } {K_{mA}^{app} }}} \right. \kern-0pt} {K_{mA}^{app} }}$ $k_{cat}^{app} /) K_{mA}^{app}$ within an order of magnitude of unsubstituted hydroquinone. Likewise, only small differences in the rates of mechanism-based inactivation were observed among these substrates. Among disubstituted hydroquinones, only 2,6- and 2,5-dimethylhydroquinone showed any activity, with the latter only barely detectable. A variety of para-substituted phenols were found to be good inhibitors of PnpC1C2. NMR studies were performed to determine the regioselectivity of ring-cleavage with monosubstituted hydroquinones. All monosubstituted hydroquinones tested (methyl-, chloro-, bromo-, and methoxyhydroquinone) yielded exclusively the 1,6-cleavage product. Thus, PnpC1C2 shows notable differences in both its substrate specificity and the ring-cleavage regioselectivity compared to that of PcpA. These results provide an important basis for future comparison of structure–function correlations among the hydroquinone ring-cleaving dioxygenases.

Graphical abstract