Improving Functional Annotation in the DRE-TIM Metallolyase Superfamily through Identification of Active Site Fingerprints

Abstract

Within the DRE-TIM metallolyase superfamily, members of the Claisen-like condensation (CC-like) subgroup catalyze C-C bond-forming reactions between various α-ketoacids and acetyl-coenzyme A. These reactions are important in the metabolic pathways of many bacterial pathogens and serve as engineering scaffolds for the production of long-chain alcohol biofuels. To improve functional annotation and identify sequences that might use novel substrates in the CC-like subgroup, a combination of structural modeling and multiple-sequence alignments identified active site residues on the third, fourth, and fifth β-strands of the TIM-barrel catalytic domain that are differentially conserved within the substrate-diverse enzyme families. Using α-isopropylmalate synthase and citramalate synthase from Methanococcus jannaschii (MjIPMS and MjCMS), site-directed mutagenesis was used to test the role of each identified position in substrate selectivity. Kinetic data suggest that residues at the β3-5 and β4-7 positions play a significant role in the selection of α-ketoisovalerate over pyruvate in MjIPMS. However, complementary substitutions in MjCMS fail to alter substrate specificity, suggesting residues in these positions do not contribute to substrate selectivity in this enzyme. Analysis of the kinetic data with respect to a protein similarity network for the CC-like subgroup suggests that evolutionarily distinct forms of IPMS utilize residues at the β3-5 and β4-7 positions to affect substrate selectivity while the different versions of CMS use unique architectures. Importantly, mapping the identities of residues at the β3-5 and β4-7 positions onto the protein similarity network allows for rapid annotation of probable IPMS enzymes as well as several outlier sequences that may represent novel functions in the subgroup.

‘Democratized’ genomic enzymology web tools for functional assignment

“…Using EFI-EST, SSNs can be constructed for Pfam and/or InterPro families (and/or user-defined sets of sequences). SSNs generated by EFI-EST [16] have been used in more than 100 publications, providing examples of the types of analyses in which SSNs have been leveraged [17–29]. In many studies SSNs are used to organize protein superfamilies into isofunctional groups using node attributes, thereby focusing sequence-function analyses and functional assignment to uncharacterized orthologous groups; in other studies SS s are used to segregate protein families into groups that differ by mechanistically important sequence signature motifs.…”

Genomic Enzymology: Web Tools for Leveraging Protein Family Sequence–Function Space and Genome Context to Discover Novel Functions

“…56 (F) SSNs for members of the DRE-TIM metallolyase superfamily. 52 Figures reproduced with permission from refs (40), (43), (52), (56), (60), and (76). …”

Changing substrate specificity and iteration of amino acid chain elongation in glucosinolate biosynthesis through targeted mutagenesis ofArabidopsismethylthioalkylmalate synthase 1

“…IPMSs and MAMs both condensate 2-oxo acids with acetyl-CoA, which places them in the Claisen-like condensation (CC-like) subgroup of the DRE-TIM metallolyase superfamily [13]. Two IPMS enzymes have been crystallised from this subgroup: Mycobacterium tuberculosis (MtIPMS, [14] and Neisseria meningitidis (NmIPMS, [15].…”