Biotechnol Lett scDAOCS in controlling substrate entry and/or product release. This prediction is supported by the activity pr<strong>of</strong>iles <strong>of</strong> the mutants in this study. Among the 19 R308 mutants, R308L, R308V, R308I and R308T showed 3.46- to 7.62-times increased specific activity for penicillin G. The four mutated amino acids (leucine, valine, isoleucine and threonine) have similar short aliphatic sidechains; unlike the positivecharged guanidine containing arginine (Fig. 2). Another R308 mutation to aliphatic alanine (R308A) also increased the activity <strong>of</strong> mutant to convert 6-APA, penicillin G and amoxicillin as determined by spectrophotometric assay. These results confirm the important role <strong>of</strong> R308 in substrate pathway control and suggest that the decreased polarity at this position would be beneficial for penicillin G selection. Fig. 2 Structures <strong>of</strong> the WT and mutated amino acids at position 308 Fig. 3 Chemical structures <strong>of</strong> penicillin analogs 123
Biotechnol Lett Mutations to polar or charged residues generated enzymes with activities similar to WT enzyme. Although large aromatic amino acids, such as phenylalanine and tyrosine, may interact well with the side chain <strong>of</strong> penicillin G, mutations to these amino acids did not generate improved enzymes (Table 2). This maybe due to spacial hindrance caused by their bulky side chains, which could impede substrate entry or product release. Taken together, we conclude that R308 is a critical residue for substrate selection, both side chain polarity and size at this position will influence final enzyme activity. Among six penicillin analogs tested (Fig. 3), penicillin G has the least sidechain polarity, followed by penicillin V which had a phenoxymethyl sidechain. Other analogs, such as ampicillin, amoxicillin and carbenicillin, have charged amino or carboxyl groups. 6-APA has no sidechain, but an amino group. It is notable that the four hydrophobic mutants <strong>of</strong> R308 increased the catalytic activity <strong>of</strong> the enzyme to phenyl-containing penicillin analogs, but had little effect on 6-APA, again suggesting the interaction between the substrate and the enzyme is determined by both polarity and size <strong>of</strong> the substrate-product path. The apparent kinetic parameters <strong>of</strong> WT acDAOC/ DACS, R308I and R308L were determined via nonlinear curve fit; both mutants showed significantly increased k cat and K m (Table 3). These mutations <strong>of</strong>fered appropriate polarity and size for penicillin G selection, and may facilitate substrate entry and/or product release. The large increment in k cat is the direct result <strong>of</strong> accelerated product release, while K m increase maybe the result <strong>of</strong> altered substrate entry rate and secondary conformational changes induced by R308 mutations, since although R308 is far from substrate binding pocket (about 11 Å), it is close to the ferrous ligand H184 (about 5 Å) and two other key residues M181 and M306 in the substrate binding pocket (about 8 and 7 Å, respectively). The overall result is that improved k cat is compromised by simultaneously increased K m . Acknowledgments We wish to thank Pr<strong>of</strong>essor A. L. Demain for kindly providing E. coli Ess strain. This work was supported by the Ministry <strong>of</strong> Science and Technology <strong>of</strong> China (Grant 2007BAI26B02), the Knowledge Innovation Fund (Grant KSCX2-YW-G-018) from Chinese Academy <strong>of</strong> Sciences and the Open Funding Project <strong>of</strong> the National Key Laboratory <strong>of</strong> Biochemical Engineering (Grant KF-001). References Baldwin JE, Crabbe MJ (1987) A spectrophotometric assay for <strong>deacetoxy</strong>cephalosporin C synthase. FEBS Lett 214: 357–361 Chin HS, Sim TS (2002) C-terminus modification <strong>of</strong> Streptomyces clavuligerus <strong>deacetoxy</strong>cephalosporin C synthase improves catalysis with an expanded substrate specificity. 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