PXD053893-1

PXD053893 is an original dataset announced via ProteomeXchange.

Title	Distinct mechanistic, conformational and substrate selectivity profiles emerging in the evolution of high catalytic activity enzymes via parallel trajectories
Description	Recent laboratory evolution studies have demonstrated that parallel evolutionary trajectories can lead to genetically distinct enzymes with high activity towards a non-preferred substrate. However, it is unknown whether such genetically distinct enzymes have convergent conformational dynamics and mechanistic features. To address this question, we used as a model the Homo sapiens kynureninase (HsKYNase), which is of great interest for cancer immunotherapy. Earlier, we reported on a highly unusual evolutionary trajectory that led to the isolation of HsKYNase_66, having a 410-fold increase in the kcat/KM for kynurenine (KYN) and reverse substrate selectivity relative to the parental wild-type enzyme. Here, by following a completely different evolutionary trajectory we generated a genetically distinct variant, HsKYNase_93D9, that exhibits KYN catalytic activity comparable to that of HsKYNase_66, but instead it is a “generalist” that accepts 3’-hydroxykynurenine (OH-KYN) with the same proficiency. Pre-steady-state kinetic analysis revealed that whereas the evolution of HsKYNase_66 was accompanied by a change in the rate-determining step of the reaction, HsKYNase_93D9 retained the same catalytic mechanism as the parental OH-KYN-preferring human enzyme. HDX-MS revealed that the conformational dynamics of the two enzymes during catalysis are markedly different and distinct from ortholog procaryotic enzymes with high KYN activity. Our work provides a mechanistic framework for understanding the relationship betweenevolutionary mechanisms and phenotypic traits of evolved generalist and specialist enzyme species.
HostingRepository	PRIDE
AnnounceDate	2024-07-29
AnnouncementXML	Submission_2024-07-29_08:07:03.279.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Sheena D'Arcy
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	Synapt MS

Revision	Datetime	Status	ChangeLog Entry
0	2024-07-11 15:51:49	ID requested
⏵ 1	2024-07-29 08:07:03	announced
2	2024-10-22 06:51:43	announced	2024-10-22: Updated project metadata.

Dataset with its publication pending

submitter keyword: kynureninase, enzyme, substrate,HDX, engineering

Sheena D'Arcy
contact affiliation	Department of Chemistry and Biochemistry, The University of Texas at Dallas
contact email	sheena.darcy@utdallas.edu
lab head
Sheena D'Arcy
contact affiliation	The University of Texas at Dallas
contact email	sheena.darcy@utdallas.edu
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2024/07/PXD053893

PRIDE project URI

• PRIDE
  1. PXD053893
     1. Label: PRIDE project
     2. Name: Distinct mechanistic, conformational and substrate selectivity profiles emerging in the evolution of high catalytic activity enzymes via parallel trajectories