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J. Biol. Chem., Vol. 281, Issue 40, 29633-29640, October 6, 2006

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Substrate Specificity of the Adenylation Enzyme SgcC1 Involved in the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027^*

Steven G. Van Lanen¹, Shuangjun Lin, Pieter C. Dorrestein², Neil L. Kelleher, and Ben Shen^¶||3

From the Division of Pharmaceutical Sciences, ^¶University of Wisconsin National Cooperative Drug Discovery Group, ^||Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53705 and Department of Chemistry, University of Illinois, Urbana, Illinois 61801

C-1027 is an enediyne antitumor antibiotic composed of a chromophore with four distinct chemical moieties, including an (S)-3-chloro-4,5-dihydroxy--phenylalanine moiety that is derived from L--tyrosine. SgcC4, a novel aminomutase requiring no added co-factor that catalyzes the formation of the first intermediate (S)--tyrosine and subsequently SgcC1 homologous to adenylation domains of nonribosomal peptide synthetases, was identified as specific for the SgcC4 product and did not recognize any -amino acids. To definitively establish the substrate for SgcC1, a full kinetic characterization of the enzyme was performed using amino acid-dependent ATP-[³²P]PP_i exchange assay to monitor amino acid activation and electrospray ionization-Fourier transform mass spectroscopy to follow the loading of the activated -amino acid substrate to the peptidyl carrier protein SgcC2. The data establish (S)--tyrosine as the preferred substrate, although SgcC1 shows promiscuous activity toward aromatic -amino acids such as -phenylalanine, 3-chloro--tyrosine, and 3-hydroxy--tyrosine, but all were <50-fold efficient. A putative active site mutant P571A adjacent to the invariant aspartic acid residue of all -amino acid-specific adenylation domains known to date was prepared as a preliminary attempt to probe the substrate specificity of SgcC1; however the mutation resulted in a loss of activity with all substrates except (S)--tyrosine, which was 142-fold less efficient relative to the wild-type enzyme. In total, SgcC1 is now confirmed to catalyze the second step in the biosynthesis of the (S)-3-chloro-4,5-dihydroxy--phenylalanine moiety of C-1027, presenting downstream enzymes with an (S)--tyrosyl-S-SgcC2 thioester substrate, and represents the first -amino acid-specific adenylation enzyme characterized biochemically.

Received for publication, June 20, 2006 , and in revised form, August 1, 2006.

^* This work is supported in part by National Institutes of Health Grants GM067725 (to N. L. K.) and CA78747 (to B. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of a National Institutes of Health postdoctoral fellowship (CA1059845).

² Recipient of a National Institutes of Health postdoctoral fellowship (GM073323).

³ Recipient of a National Institutes of Health Independent Scientist Award AI51687. To whom correspondence should be addressed: Div. of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin-Madison, 777 Highland Ave., Madison, WI 53705. Tel.: 608-263-2673; Fax: 608-262-5245; E-mail: bshen{at}pharmacy.wisc.edu.

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