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NicF is the maleamate amidohydrolase of the KT2440 nicotinate degradation pathway, hydrolyzing maleamate to maleate plus ammonia.
"NicF is the maleamate (maleamic acid) amidohydrolase** of the *P. putida* KT2440 nicotinate degradation pathway, catalyzing hydrolysis of **maleamate/maleamic acid** to **maleate/maleic acid plus ammonia (NH3/NH4+)**"
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NicF acts in the maleamate pathway downstream of NicD and upstream of NicE (maleate isomerase), which converts maleate to fumarate.
"NicF acts in the **maleamate pathway** of aerobic nicotinic acid degradation, downstream of NicC/NicX/NicD and upstream of **NicE** (maleate isomerase), which converts maleate to fumarate."
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In the KT2440 scheme NicF performs the maleamate amidase step and NicE (maleate isomerase) converts maleate to fumarate.
"with **NicF** catalyzing the maleamate/maleamic acid amidase step and **NicE** (maleate cis/trans isomerase) converting maleate/maleic acid to fumarate"
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KT2440 nicF overexpressed in E. coli gave crude-extract maleamate amidohydrolase specific activity of 19.3 umol/min/mg.
"crude extracts of **19.3 μmol·min⁻¹·mg⁻¹** protein"
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The NicF activity assay coupled released ammonium to glutamate dehydrogenase and monitored NADPH oxidation at 340 nm.
"The assay quantified released ammonium by coupling NH4+ to glutamate dehydrogenase and monitoring NADPH oxidation at 340 nm."
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NicF belongs to the isochorismatase superfamily of cysteine-dependent amide/ester hydrolases with a Cys-Asp-Lys catalytic triad.
"NicF in KT2440 is predicted and supported to belong to the **isochorismatase superfamily**, which includes cysteine-dependent amide/ester hydrolases often featuring a **Cys–Asp–Lys** catalytic triad."
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For KT2440 NicF the inferred catalytic triad residues are D31, K121, C154.
"For KT2440 NicF, Jiménez et al. infer conserved residues **D31, K121, C154** consistent with the isochorismatase superfamily catalytic triad."
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NicF is a metal-independent amidase, functioning efficiently without a metal ion.
"no Zn(II)** was observed and that NicF functions efficiently **without a metal ion**"
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NicF is a soluble intracellular catabolic enzyme encoded within a chromosomal metabolic gene cluster.
"NicF is presented as a **soluble intracellular catabolic enzyme** encoded within a chromosomal metabolic gene cluster"