Discovery and Characterization of Antibody Probes of Module 2 of the 6-Deoxyerythronolide B Synthase

被引：3

作者：

Guzman, Katarina M. ^{[1
]}

Cogan, Dillon P. ^{[2
]}

Brodsky, Krystal L. ^{[2
]}

Soohoo, Alexander M. ^{[1
]}

Li, Xiuyuan ^{[2
]}

Sevillano, Natalia ^{[3
]}

Mathews, Irimpan I. ^{[4
]}

Nguyen, Khanh P. ^{[2
]}

Craik, Charles S. ^{[3
]}

Khosla, Chaitan ^{[1
,2
,5
]}

机构：

[1] Stanford Univ, Dept Chem Engn, Stanford, CA 94305 USA

[2] Stanford Univ, Dept Chem, Stanford, CA 94305 USA

[3] Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94158 USA

[4] Stanford Univ, SLAC Natl Accelerator Lab, Stanford Synchrotron Radiat Lightsource, Menlo Pk, CA 94025 USA

[5] Stanford Univ, Sarafan ChEM H, Stanford, CA 94305 USA

来源：

BIOCHEMISTRY | 2023年 / 62卷 / 11期

基金：

美国国家卫生研究院; 美国国家科学基金会;

关键词：

MECHANISTIC ANALYSIS; POLYKETIDE; DOMAINS; KETOSYNTHASE; SPECIFICITY; RECOGNITION;

D O I：

10.1021/acs.biochem.3c00156

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Fragment antigen-binding domains of antibodies (F(ab)s)are powerful probes of structure-function relationships ofassembly line polyketide synthases (PKSs). We report the discoveryand characterization of F(ab)s interrogating the structureand function of the ketosynthase-acyltransferase (KS-AT) core of Module2 of the 6-deoxyerythronolide B synthase (DEBS). Two F(ab)s (AC2 and BB1) were identified to potently inhibit the catalyticactivity of Module 2. Both AC2 and BB1 were found to modulate ACP-mediatedreactions catalyzed by this module, albeit by distinct mechanisms.AC2 primarily affects the rate (k (cat)),whereas BB1 increases the K (M) of an ACP-mediatedreaction. A third F-ab, AA5, binds to the KS-AT fragmentof DEBS Module 2 without altering either parameter; it is phenotypicallyreminiscent of a previously characterized F-ab, 1B2, shownto principally recognize the N-terminal helical docking domain ofDEBS Module 3. Crystal structures of AA5 and 1B2 bound to the KS-ATfragment of Module 2 were solved to 2.70 and 2.65 angstrom resolution,respectively, and revealed entirely distinct recognition featuresof the two antibodies. The new tools and insights reported here pavethe way toward advancing our understanding of the structure-functionrelationships of DEBS Module 2, arguably the most well-studied moduleof an assembly line PKS.

引用

页码：1589 / 1593

页数：5