Methionine residues lining the substrate pathway in prolyl oligopeptidase from <i>Pleurotus eryngii</i> play an important role in substrate recognition

<p>Family S9 prolyl oligopeptidases (POPs) are of interest as pharmacological targets. We recently found that an S9 POP from <i>Pleurotus eryngii</i> showed altered substrate specificity following H₂O₂ treatment. Oxidation of Met203 on the non-catalytic β-propeller domain resulted in decreased activity toward non-aromatic aminoacyl-para-nitroanilides (pNAs) while maintaining its activity toward aromatic aminoacyl-pNAs. Given that the other Met residues should also be oxidized by H₂O₂ treatment, we constructed mutants in which all the Met residues were substituted with other amino acids. Analysis of the mutants showed that Met570 in the catalytic domain is another potent residue for the altered substrate specificity following oxidation. Met203 and Met570 lie on the surfaces of two different domains and form part of a funnel from the surface to the active center. Our findings indicate that the funnel forms the substrate pathway and plays a role in substrate recognition.</p> <p>Possible substrate entry route of prolyl oligopeptidase.</p

SDS-PAGE analysis of R18 and R43.

<p>Lane 1: protein standard; Lane 2: R18; Lane 3: R43. Powdered enzyme (100 µg) was dissolved in distilled water and loaded onto each lane.</p

FA production from corn bran by <i>Streptomyces</i> FAEs.

<p>FA production from corn bran by R18 and R43 (A). Combination effect of xylanase (STX-I) and α-L-arabinofuranosidase (STX-IV) on FA production from corn bran by treatment with R18 and R43 (B). Effect of pretreatment by STX-I and STX-IV on FA production from corn bran by treatment with R18 and R43 (C) The pretreatment of STX-1 and STX-IV was performed during 8 h, 12 h and 16 h. Bars indicate the averages of three independent experiments. Error bars represent standard deviations.</p

Application of Two Newly Identified and Characterized Feruloyl Esterases from <i>Streptomyces</i> sp. in the Enzymatic Production of Ferulic Acid from Agricultural Biomass

<div><p>Ferulic acid (FA), a component of hemicellulose in plant cell walls, is a phenolic acid with several potential applications based on its antioxidant properties. Recent studies have shown that feruloyl esterase (FAE) is a key bacterial enzyme involved in FA production from agricultural biomass. In this study, we screened a library of 43 esterases from <i>Streptomyces</i> species and identified two enzymes, R18 and R43, that have FAE activity toward ethyl ferulate. In addition, we characterized their enzyme properties in detail. R18 and R43 showed esterase activity toward other hydroxycinnamic acid esters as well, such as methyl <i>p</i>-coumarate, methyl caffeate, and methyl sinapinate. The amino acid sequences of R18 and R43 were neither similar to each other, nor to other FAEs. We found that R18 and R43 individually showed the ability to produce FA from corn bran; however, combination with other <i>Streptomyces</i> enzymes, namely xylanase and α-l-arabinofuranosidase, increased FA production from biomass such as corn bran, defatted rice bran, and wheat bran. These results suggest that R18 and R43 are effective FAEs for the enzymatic production of FA from biomass.</p></div

LC-MS plots of defatted rice bran digested by <i>Streptomyces</i> FAEs.

<p>Arrows indicate estimated di-FAs (m/z = 385).</p

Effect of metal ion/effectors in R18 and R43.

<p>Effect of metal ion/effectors in R18 and R43.</p

Screening of feruloyl esterases from a <i>Streptomyces</i> esterase library.

<p>Screening of feruloyl esterases from a <i>Streptomyces</i> esterase library.</p

FA production from biomass by <i>Streptomyces</i> FAEs.

<p>Bars indicate the averages of three independent experiments. Error bars represent standard deviations.</p

Substrate specificity and esterase activity on R18 and R43.

<p>Average from three independent experiments is shown. Error bars represent standard deviations.</p

Purification and characterization of 4-<i>N</i>-trimethylamino-1-butanol dehydrogenase from <i>Fusarium merismoides</i> var. <i>acetilereum</i>

<p>From investigation of 60 filamentous fungi, we identified <i>Fusarium merismoides</i> var. <i>acetilereum</i>, which uses 4-<i>N</i>-trimethylamino-1-butanol (TMA-butanol) as the sole source of carbon and nitrogen. The fungus produced NAD⁺-dependent TMA-butanol dehydrogenase (DH) when it was cultivated in medium containing TMA-butanol. The enzyme showed molecular mass of 40 kDa by SDS–PAGE and 160 kDa by gel filtration, suggesting that it is a homotetramer. TMA-butanol DH is stable at pH 7.5–9.0. It exhibits moderate stability with respect to temperature (up to 30 °C). Additionally, it has optimum activity at 45 °C and at pH 9.5. The enzyme has broad specificity to various alkyl alcohols and amino alkyl alcohols, and the carbon chains of which are longer than butanol. Moreover, the activity is strongly inhibited by oxidizing agents, carbonyl and thiol modulators, and chelating agents. This report is the first study examining TMA-butanol DH from eukaryotic microbes.</p> <p>Having broad substrate specificity, 4-<i>N</i>-trimethylamino-1-butanol dehydrogenase (TMA-butanol DH) from <i>Fusarium merismoides</i> var. <i>acetilereum</i> was purified and characterized.</p