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2+-ion affinity chromatography, followed by anion-exchange on a monoQ column. Mutations were introduced using standard overlap-extension PCR methods (Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular cloning: A laboratory manual, 3rd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 2001). The proteins were analyzed by electrospray ionization mass spectroscopy (ESI-MS) and shown to have the expected mass (wild-type Alr, found 44 648 ± 5 Da, expected 44 644 Da; Alr Y265A, found 44 561 ± 5 Da, expected 44 566 Da; Alr Y265A/H166N, found 44 539 ± 5 Da, expected 44 543 Da).
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note
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a2 = 10.0 ± 0.3) (see Supporting Information). Reliable pH-rate data for the double mutant could not be obtained because of its low activity and instability at the pH extremes.
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0041404272
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note
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Our preliminary results indicate that Alr Y265A also promotes the retroaldol reaction of D-α-methylthreonine which has not been reported for naturally occurring threonine aldolases.
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24
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