Patent ID: 8951937
Filing Date: 2015-02-10
Classification: C12N,C12P,C12Y,G16B,Y02E

Abstract:
1. A method for identifying [2Fe-2S] dihydroxy-acid dehydratase (DHAD) enzymes comprising: (a) querying one or more amino acid sequences with a Profile Hidden Markov Model prepared using the proteins of SEQ ID NO: 164, 168, 230, 232, 298, 310, 344, and 346, wherein amino acid sequences with an E-value of less than 10 (b) analyzing the first subset of sequences of step (a) for the presence of three conserved cysteines that correspond to positions 56, 129, and 201 in SEQ ID NO: 168, whereby a second subset of sequences is identified; (c) analyzing the second subset of sequences of step (b) for the presence of signature conserved amino acids at positions corresponding to positions in SEQ ID NO: 168, wherein said amino acids are aspartic acid at position 88, arginine or asparagine at position 142, asparagine at position 208, and leucine at position 454, whereby a third subset of sequences is identified; (d) expressing a protein having an amino acid sequence identified by step (b) or step (c) in a host cell; (e) purifying the protein of step (d); (f) confirming that the protein of step (e) has DHAD activity; and (g) confirming that the protein of step (f) is a [2Fe-2S] DHAD enzyme by UV-vis and EPR spectroscopy.