PMID- 33716126
OWN - NLM
STAT- MEDLINE
DCOM- 20210812
LR  - 20210812
IS  - 1096-0309 (Electronic)
IS  - 0003-2697 (Linking)
VI  - 622
DP  - 2021 Jun 1
TI  - A direct spectropolarimetric assay of arabinose 5-phosphate isomerase.
PG  - 114116
LID - S0003-2697(21)00017-8 [pii]
LID - 10.1016/j.ab.2021.114116 [doi]
AB  - Arabinose 5-phosphate isomerase (API) catalyzes the reversible isomerization of 
      Ribulose 5-phosphate (Ru5P) to Arabinose 5-Phosphate (Ar5P) for the production of 
      3-deoxy-2-octulosonic acid 8-phosphate (KDO), a component of bacterial 
      lipopolysaccharide (LPS) of gram-negative bacteria. API is an attractive target 
      for therapeutic development against gram-negative bacterial pathogens. The 
      current assay method of API activity utilizes a general reaction for keto sugar 
      determination in a secondary, 3-h color development reaction with 25 N sulfuric 
      acid which poses hazard to both personnel and instrumentation. We therefore aimed 
      to develop a more user friendly assay of the enzyme. Since Ru5P absorbs in the UV 
      region and contains at least 2 chiral centers, it can be expected to display 
      circular dichroism (CD). A wavelength scan revealed indeed Ru5P displays a 
      pronounced negative ellipticity of 30,560 mDeg M(-1)cm(-1) at 279 nm in Tris 
      buffer pH 9.1 but Ar5P does not have any CD. API enzymatic reactions were 
      monitored directly and continuously in real time by following the disappearance 
      of CD from the Ru5P substrate, or by the appearance of CD from Ar5P substrate. 
      The CD signal at this wavelength was not affected by absorption of the enzyme 
      protein or of small molecules, or turbidity of the solution. Common additives in 
      protein and enzyme reaction mixtures such as detergents, metals, and 5% 
      dimethylsulfoxide did not interfere with the CD signal. Assay reactions of 
      1-3 min consistently yielded reproducible results. Introduction of accessories in 
      a spectropolarimeter will easily adapt this assay to high throughput format for 
      screening thousands of small molecules as inhibitor candidates of API.
CI  - Copyright (c) 2021 Elsevier Inc. All rights reserved.
FAU - Kijek, Todd M
AU  - Kijek TM
AD  - Systems and Structural Biology Division, United States Army Medical Research 
      Institute of Infectious Diseases, Fort Detrick, MD, 21702, USA.
FAU - Bozue, Joel A
AU  - Bozue JA
AD  - Bacteriology Division, United States Army Medical Research Institute of 
      Infectious Diseases, Fort Detrick, MD, 21702, USA.
FAU - Panchal, Rekha G
AU  - Panchal RG
AD  - Systems and Structural Biology Division, United States Army Medical Research 
      Institute of Infectious Diseases, Fort Detrick, MD, 21702, USA.
FAU - Litosh, Vladislav A
AU  - Litosh VA
AD  - Medical Countermeasures Division, United States Army Medical Research Institute 
      of Infectious Diseases, Fort Detrick, MD, 21702, USA.
FAU - Woodard, Ronald W
AU  - Woodard RW
AD  - Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109-1065, USA.
FAU - Ahmed, S Ashraf
AU  - Ahmed SA
AD  - Systems and Structural Biology Division, United States Army Medical Research 
      Institute of Infectious Diseases, Fort Detrick, MD, 21702, USA. Electronic 
      address: syed.a.ahmed.civ@mail.mil.
LA  - eng
PT  - Journal Article
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20210312
PL  - United States
TA  - Anal Biochem
JT  - Analytical biochemistry
JID - 0370535
RN  - 0 (3-deoxyoctulosonate 8-phosphate)
RN  - 0 (Bacterial Proteins)
RN  - 0 (Lipopolysaccharides)
RN  - 0 (Pentosephosphates)
RN  - 0 (Ribulosephosphates)
RN  - 0 (Sugar Acids)
RN  - 0 (Sugar Phosphates)
RN  - 1069-03-0 (2-keto-3-deoxyoctonate)
RN  - 13137-52-5 (arabinose 5-phosphate)
RN  - 4151-19-3 (ribulose 5-phosphate)
RN  - EC 5.3.1.- (Aldose-Ketose Isomerases)
RN  - EC 5.3.1.13 (arabinose-5-phosphate isomerase)
SB  - IM
MH  - Aldose-Ketose Isomerases/*analysis
MH  - Bacterial Proteins/metabolism
MH  - Catalysis
MH  - Circular Dichroism/*methods
MH  - Enzyme Assays/*methods
MH  - Francisella tularensis/metabolism
MH  - Lipopolysaccharides/metabolism
MH  - Pentosephosphates/metabolism
MH  - Ribulosephosphates/analysis/metabolism
MH  - Substrate Specificity
MH  - Sugar Acids/metabolism
MH  - Sugar Phosphates/metabolism
OTO - NOTNLM
OT  - Arabinose 5-phosphate isomerase
OT  - Assay method
OT  - Circular dichroism
OT  - Francisella tularensis
OT  - KDO
OT  - KdsD
OT  - Spectropolarimeter
EDAT- 2021/03/16 06:00
MHDA- 2021/08/13 06:00
CRDT- 2021/03/15 06:09
PHST- 2020/10/13 00:00 [received]
PHST- 2020/12/20 00:00 [revised]
PHST- 2021/01/18 00:00 [accepted]
PHST- 2021/03/16 06:00 [pubmed]
PHST- 2021/08/13 06:00 [medline]
PHST- 2021/03/15 06:09 [entrez]
AID - S0003-2697(21)00017-8 [pii]
AID - 10.1016/j.ab.2021.114116 [doi]
PST - ppublish
SO  - Anal Biochem. 2021 Jun 1;622:114116. doi: 10.1016/j.ab.2021.114116. Epub 2021 Mar 
      12.