3 research outputs found
Enzymatic properties of a novel CYP152 fatty acid decarboxylase
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Dissecting the low catalytic capability of flavin-dependent halogenases
Although flavin-dependent halogenases (FDHs) are attractive biocatalysts, their practical applications are limited because of their low catalytic efficiency. Here, we investigated the reaction mechanisms and structures of tryptophan 6-halogenase (Thal) from Streptomyces albogriseolus using stopped-flow, rapid-quench flow, quantum/mechanics molecular mechanics calculations, crystallography, and detection of intermediate (hypohalous acid [HOX]) liberation. We found that the key flavin intermediate, C4a-hydroperoxyflavin (C4aOOH-FAD), formed by Thal and other FDHs (tryptophan 7-halogenase [PrnA] and tryptophan 5-halogenase [PyrH]), can react with I-, Br-, and Cl- but not F- to form C4a-hydroxyflavin and HOX. Our experiments revealed that I- reacts with C4aOOH-FAD the fastest with the lowest energy barrier and have shown for the first time that a significant amount of the HOX formed leaks out as free HOX. This leakage is probably a major cause of low product coupling ratios in all FDHs. Site-saturation mutagenesis of Lys79 showed that changing Lys79 to any other amino acid resulted in an inactive enzyme. However, the levels of liberated HOX of these variants are all similar, implying that Lys79 probably does not form a chloramine or bromamine intermediate as previously proposed. Computational calculations revealed that Lys79 has an abnormally lower pKa compared with other Lys residues, implying that the catalytic Lys may act as a proton donor in catalysis. Analysis of new X-ray structures of Thal also explains why premixing of FDHs with reduced flavin adenine dinucleotide generally results in abolishment of C4aOOH-FAD formation. These findings reveal the hidden factors restricting FDHs capability which should be useful for future development of FDHs applications.</p
<i>p</i>‑Hydroxyphenylacetate 3‑Hydroxylase as a Biocatalyst for the Synthesis of Trihydroxyphenolic Acids
Trihydroxyphenolic acids such as
3,4,5-trihydroxycinnamic acid (3,4,5-THCA) <b>4c</b> and 2-(3,4,5-trihydroxyphenyl)Âacetic
acid (3,4,5-THPA) <b>2c</b> are strong antioxidants that are
potentially useful as medicinal agents. Our results show that <i>p</i>-hydroxyphenylacetate (HPA) 3-hydroxylase (HPAH) from <i>Acinetobacter baumannii</i> can catalyze the syntheses of 3,4,5-THPA <b>2c</b> and 3,4,5-THCA <b>4c</b> from 4-HPA <b>2a</b> and <i>p</i>-coumaric acid <b>4a</b>, respectively.
The wild-type HPAH can convert 4-HPA <b>2a</b> completely into
3,4,5-THPA <b>2c</b> within 100 min (total turnover number (TTN)
of 100). However, the wild-type enzyme cannot efficiently synthesize
3,4,5-THCA <b>4c</b>. To improve the efficiency, the oxygenase
component of HPAH (C<sub>2</sub>) was rationally engineered in order
to maximize the conversion of <i>p</i>-coumaric acid <b>4a</b> to 3,4,5-THCA <b>4c</b>. Results from site-directed
mutagenesis studies showed that Y398S is significantly more effective
than the wild-type enzyme for the synthesis of 3,4,5-THCA <b>4c</b>; it can catalyze the complete bioconversion of <i>p</i>-coumaric acid <b>4a</b> to 3,4,5-THCA <b>4c</b> within
180 min (TTN ∼ 23 at 180 min). The yield and stability of 3,4,5-THPA <b>2c</b> and 3,4,5-THCA <b>4c</b> were significantly improved
in the presence of ascorbic acid. Thermostability studies showed that
the wild-type C<sub>2</sub> was very stable and remained active after
incubation at 30, 35, and 40 °C for 24 h. Y398S was moderately
stable because its activity was retained for 24 h at 30 °C and
for 15 h at 35 °C. Transient kinetic studies using stopped-flow
spectrophotometry indicated that the key improvement in the reaction
of Y398S with <i>p</i>-coumaric acid <b>4a</b> lies
within the protein–ligand interaction. Y398S binds to <i>p</i>-coumaric acid <b>4a</b> with higher affinity than
the wild-type enzyme, resulting in a shift in equilibrium toward favoring
the productive coupling path instead of the path leading to wasteful
flavin oxidation