Roles of the N-terminal domain and remote substrate binding subsites in activity of the debranching barley limit dextrinase

Kallikrein 13 (KLK13) was first identified as an enzyme that is downregulated in a subset of breast tumors. This serine protease has since been implicated in a number of pathological processes including ovarian, lung and gastric cancers. Here we report the design, synthesis and deconvolution of libraries of internally quenched fluorogenic peptide substrates to determine the specificity of substrate binding subsites of KLK13 in prime and non-prime regions (according to the Schechter and Berger convention). The substrate with the consensus sequential motive ABZ-Val-Arg-Phe-Arg-ANB-NH2 demonstrated selectivity towards KLK13 and was successfully converted into an activity-based probe by the incorporation of a chloromethylketone warhead and biotin bait. The compounds described may serve as suitable tools to detect KLK13 activity in diverse biological samples, as exemplified by overexpression experiments and targeted labeling of KLK13 in cell lysates and saliva. In addition, we describe the development of selective activity-based probes targeting KLK13, to our knowledge the first tool to analyze the presence of the active enzyme in biological samples.

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Identification of Acceptor Substrate Binding Subsites +2 and +3 in the Amylomaltase fromThermus thermophilusHB8†

Biochemistry ◽

10.1021/bi602408j ◽

2007 ◽

Vol 46 (17) ◽

pp. 5261-5269 ◽

Cited By ~ 32

Author(s):

Thijs Kaper ◽

Hans Leemhuis ◽

Joost C. M. Uitdehaag ◽

Bart A. van der Veen ◽

Bauke W. Dijkstra ◽

...

Keyword(s):

Substrate Binding ◽

Acceptor Substrate ◽

Binding Subsites

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Structure of the Alkalohyperthermophilic Archaeoglobus fulgidus Lipase Contains a Unique C-Terminal Domain Essential for Long-Chain Substrate Binding

Journal of Molecular Biology ◽

10.1016/j.jmb.2009.05.017 ◽

2009 ◽

Vol 390 (4) ◽

pp. 672-685 ◽

Cited By ~ 20

Author(s):

Cammy K.-M. Chen ◽

Guan-Chiun Lee ◽

Tzu-Ping Ko ◽

Rey-Ting Guo ◽

Li-Min Huang ◽

...

Keyword(s):

Substrate Binding ◽

Archaeoglobus Fulgidus ◽

Terminal Domain ◽

Long Chain

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Structure ofMethylobacterium extorquensmalyl-CoA lyase: CoA-substrate binding correlates with domain shift

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x17001029 ◽

2017 ◽

Vol 73 (2) ◽

pp. 79-85 ◽

Cited By ~ 1

Author(s):

Javier M. González ◽

Ricardo Marti-Arbona ◽

Julian C.-H. Chen ◽

Clifford J. Unkefer

Keyword(s):

Catalytic Mechanism ◽

Substrate Binding ◽

Structural Alignment ◽

Methylobacterium Extorquens ◽

Terminal Domain ◽

Domain Motion ◽

Citrate Lyase ◽

Carbon Compounds ◽

Binding Alignment ◽

Aspartate Residue

Malyl-CoA lyase (MCL) is an Mg2+-dependent enzyme that catalyzes the reversible cleavage of (2S)-4-malyl-CoA to yield acetyl-CoA and glyoxylate. MCL enzymes, which are found in a variety of bacteria, are members of the citrate lyase-like family and are involved in the assimilation of one- and two-carbon compounds. Here, the 1.56 Å resolution X-ray crystal structure of MCL fromMethylobacterium extorquensAM1 with bound Mg2+is presented. Structural alignment with the closely relatedRhodobacter sphaeroidesmalyl-CoA lyase complexed with Mg2+, oxalate and CoA allows a detailed analysis of the domain motion of the enzyme caused by substrate binding. Alignment of the structures shows that a simple hinge motion centered on the conserved residues Phe268 and Thr269 moves the C-terminal domain by about 30° relative to the rest of the molecule. This domain motion positions a conserved aspartate residue located in the C-terminal domain in the active site of the adjacent monomer, which may serve as a general acid/base in the catalytic mechanism.

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