Filling the information gap: Biochemical and proteomics approaches to sequence identification of orphan enzymes

Approximately one quarter of all enzyme activities have no associated sequence data in any major sequence resource. These “orphan” enzyme activities are significant impediments to genome annotation because existing protein sequences are the primary link between prior research and modern annotation efforts. To address this issue, we previously conducted an extensive database and literature review that resulted in the assignment of sequence information to numerous artifactual orphans. We are now pursuing biochemical and proteomics approaches to identify sequences associated with validated orphans. We determined protein sequences of two commercially available orphans, D‐fructose dehydrogenase from G. industrius (E.C. 1.1.99.11 ) and maltose epimerase from Lactobacillus sp. (E.C. 5.1.3.21 ). Enzymes were resolved by gel electrophoresis, digested with trypsin, and analyzed by MALDI TOF‐TOF and ESI‐QTOF hybrid mass spectrometry. Sequence identification of both enzymes was accomplished by matching peptide fragments against the annotated genomes of related organisms. Ongoing efforts are aimed at sequence assignment of non‐commercially available orphans through modernization of historical methods for biochemical purification and analysis. This work is supported by the National Institute of General Medical Sciences 5R01GM086755‐02.