9c4l
From Proteopedia
Crystal structure of mutant NonPro1 Tautomerase Superfamily Member NJ7-V1P in complex with 3-bromopropiolate inhibitor
Structural highlights
FunctionPublication Abstract from PubMedPro1 is a critical catalytic residue in the characterized activities of tautomerase superfamily (TSF) members. Only a handful of members ( approximately 346) lack Pro1 in a sequence similarity network (SSN) that consists of over 11,000 members. Most (294 members) are in the malonate semialdehyde decarboxylase (MSAD)-like subgroup, but the ones characterized thus far have little or no MSAD activity. Moreover, there is little to no activity with other TSF substrates. Five non-Pro1 members were selected randomly for kinetic [using phenylenolpyruvate (PP) and 2-hydroxymuconate (2HM)], mutagenic, inhibition, and crystallographic analysis. Using PP, k(cat)/K(m) values ( approximately 10(1)-10(2) M(-1) s(-1)) could be estimated for three native proteins whereas using 2HM, a k(cat)/K(m) value could only be estimated for one native protein ( approximately 10(3) M(-1) s(-1)). The k(cat) and K(m) values could not be determined. However, changing the N-terminal residue to a proline gave a significant improvement in k(cat)/K(m) values for all mutant enzymes using PP or 2HM. For PP, the k(cat)/K(m) values ranged from 10(3)-10(5) M(-1) s(-1) and for 2HM, the k(cat)/K(m) values ranged from 10(2)-10(4) M(-1) s(-1). In addition, it was now possible to measure k(cat) and K(m) values for all mutant proteins using PP and one mutant protein using 2HM. Incubation of the Pro1 mutants with 3-bromopropiolate (3BP) results in covalent modification of the prolyl nitrogen of Pro1 by a 3-oxopropanoate adduct. Crystallographic analysis of two mutant enzymes (NJ7V1P and 8U6S1P) modified by the 3-oxopropanoate adduct identified binding ligands and suggest a mechanism for the tautomerase activity involving Pro1, Arg71, Tyr124, and the backbone amide of Phe68. Conversion of Inactive Non-Pro1 Tautomerase Superfamily Members into Active Tautomerases: Analysis of the Pro1 Mutants.,Lancaster EB, Hardtke HA, Melkonian TR, Venkat Ramani M, Johnson WH Jr, Baas BJ, Zhang YJ, Whitman CP Biochemistry. 2025 Feb 18;64(4):812-822. doi: 10.1021/acs.biochem.4c00338. Epub , 2025 Feb 6. PMID:39914393[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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