Alcohol oxidases, including carbohydrate oxidases, have a long history of research that has generated fundamental biological understanding and biotechnological applications. Despite a long history of study, the galactose 6-oxidase/glyoxal oxidase family of mononuclear copper-radical oxidases, Auxiliary Activity Family 5 (AA5), is currently represented by only very few characterized members. Here we report the recombinant production and detailed structure–function analyses of two homologues from the phytopathogenic fungi Colletotrichum graminicola and C. gloeosporioides, CgrAlcOx and CglAlcOx, respectively, to explore the wider biocatalytic potential in AA5. EPR spectroscopy and crystallographic analysis confirm a common active-site structure vis-à-vis the archetypal galactose 6-oxidase from Fusarium graminearum. Strikingly, however, CgrAlcOx and CglAlcOx are essentially incapable of oxidizing galactose and galactosides, but instead efficiently catalyse the oxidation of diverse aliphatic alcohols. The results highlight the significant potential of prospecting the evolutionary diversity of AA5 to reveal novel enzyme specificities, thereby informing both biology and applications.

Many serine hydrolases catalyze perhydrolysis -the reversible formation of per-acids from carboxylic acids and hydrogen peroxide. Recently we showed that a single amino acid substitution in the alcohol binding pocket -L29P -in Pseudomonas fluorescens (SIK WI) aryl esterase (PFE) increased the specificity constant of PFE for peracetic acid formation >100-fold [Bernhardt et al. Angew. Chem. Intl. Ed. 2005, 44, 2742. In this paper, we extend this work to address the three following questions. First, what is the molecular basis of the increase in perhydrolysis activity? We previously proposed that the L29P substitution creates a hydrogen bond between the enzyme and hydrogen peroxide in the transition state. Here we report two x-ray structures of L29P PFE that support this proposal. Both structures show a main chain carbonyl oxygen closer to the activesite serine as expected. One structure further shows acetate in the active site in an orientation consistent with reaction by an acyl-enzyme mechanism. We also detected an acyl-enzyme intermediate in the hydrolysis of ε-caprolactone by mass spectrometry. Second, can we further increase perhydrolysis activity? We discovered that the reverse reaction -hydrolysis of peracetic acid to acetic acid and hydrogen peroxide -occurs at nearly the diffusion limited rate. Since the reverse reaction cannot increase further, neither can the forward reaction. Consistent with this prediction, two variants with additional amino acid substitutions showed two fold higher k cat , but K m also increased so the specificity constant, k cat /K m , remained similar. Third, how does the L29P substitution change the esterase activity? Ester hydrolysis decreased for most esters (75-fold for ethyl acetate), but not for methyl esters. In contrast, L29P PFE catalyzed hydrolysis of ε-caprolactone five times more efficiently than wild-type PFE. Molecular modeling suggests that moving the carbonyl group closer to the active site blocks access for larger alcohol moieties, but binds ε-caprolactone more tightly. These results are consistent with the natural function of perhydrolases being either hydrolysis of peroxycarboxylic acids or hydrolysis of lactones.* To whom correspondence should be addressed: Romas Kazlauskas: rjk@umn.edu. Fax: +1-612-625-5780. Phone: +1-612-624-5904. Joseph Schrag: joe@bri.nrc.ca. Fax: +1-514-496-5143. Phone: +1-514-496-2557. X-ray coordinates have been deposited in the Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (accession nos. 3hea and 3hi4) Supporting Information Available. The supporting information contains 1) the electrospray ionization mass spectrometry data for detection of acyl-enzyme intermediate, 2) the pH-rate profile of perhydrolysis catalyzed by mutants and wild-type PFE and 3) a discussion of previous experiments to distinguish acyl-enzyme versus a noncovalent mechanisms for PFE This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. ...

Cellulose has emerged as an attractive substrate for the production of economical, disposable, point-of-care (POC) analytical devices. Development of novel methods of (bio)activation is central to broadening the application space of cellulosic materials. Ironically, such efforts are stymied by the inherent biocompatibility and recalcitrance of cellulose fibers. Here, we have elaborated a versatile, chemo-enzymatic approach to activate cellulosic materials for CuAAC "click chemistry", to develop new fluorogenic esterase sensors. Gentle, aqueous modification conditions facilitate broad applicability to cellulose papers, gauzes, and hydrogels. Tethering of the released fluorophore to the cellulose surface prevents signal degradation due to diffusion and enables straightforward, sensitive visualization with a simple light source in resource-limited situations.