Paul G Hamm

Chemistry 563
Professor Erb
September 18th, 2014
Paper I Summary:
Structure and Catalytic Mechanism of Yeast 4-Amino-4-deoxychorismate Lyase
J. Biol. Chem. 2013 288:22985-22992
Doi: 10.1074/jbcM113.480335 originally published online July 1, 2013
Introduction Folate and Coenzyme Q are important cofactors in many biological processes
and many severe diseases result from their deficiencies. Both Folate and Coenzyme Q can be
created in vivo from a common precursor called para-aminobenzoate (PABA). Biosynthesis
pathways of PABA have been identified in a few species and in most cases it involves two
separate steps controlled by three enzymes PabA, PabB, and PabC respectively. In step one of
PABA synthesis, PabA and PabB work together to form 4-amino-4-deoxychorismate (ADC)
synthase, which converts chorismate to ADC. Step two involves PabC, which depends on
pyridoxal 5'-phosphate (PLP) as a cofactor. PabC and PLP together work as ADC lyase and
perform the aromatization of the ADC ring into PABA. (See Figure 1)
To date, there are four known prokaryotic ADC lyases in the Protein Data Bank (PDB),
all of which have an overall dimeric fold with slightly different active sites. In this article, the
researchers describe the overall structure of yeast (Saccharomyces cerevisiae) ADC lyase which
is named Abz2 and is the first eukaryotic ADC lyase to have its structure determined. Obtaining
the crystalized form of Abz2 with cofactor PLP, the researchers performed structural
computational analysis, site directed mutagenesis, and enzymatic activity assays. They propose a
catalytic pathway for Abz2 and argue it is the first in a unique class of monomeric ADC lyases.
Experimental First the Abz2 gene was amplified and purified from the yeast genome. Using
E.coli plasmids as a vector, the gene was inserted into E.coli and the recombinant DNA was
induced to overexpress Abz2. The cells were lysed and subjected to sonication and

centrifugation. Column chromatography was then used to separate the protein. SDS gel
electrophoresis was used to assess the protein purity. Selenomethionine (SeMet) was used to
label Abz2 proteins and these SeMet forms were expressed in E.coli and purified and stored the
same way. Site-directed mutagenesis was then performed, using native Abz2 as the template, and
the mutant proteins were expressed, purified, and stored the same way.
Both native and SeMet forms of Abz2 were crystallized and their structure was studied
using X-ray crystallography. Electron density maps were created and several different programs
were used to interpret the data and predict the structure of Abz2. To test the structure of the
various Abz2 mutants, ADC lyase activity was assessed using a coupled assay by reverse phase
high performance liquid chromatography. PabB was used to generate ADC for the Abz2 proteins
to work upon. Once PabB had generated sufficient ADC, wild type and mutant Abz2 were added
to test their activity levels. As a control, the reactions were also performed without wild type or
mutant Abz2. All of the products were then centrifuged and the supernatants were purified
through column chromatography to determine PABA levels.
Results/Discussion- The structural analysis reveals that Abz2 is a monomeric protein with two
large domains. Domain I is made of eight -helices and six -strands (with a total of 227
residues) while Domain II is made of three -helices and eight -strands (with a total of 145
residues). When superimposed upon the structures of the other PabC enzymes from the PDB, it is
evident that Abz2 has a similar structure despite having low primary sequence homology.
Domain II is very similar for all of these proteins and the main difference seems to lie in Domain
I. The key difference is that Abz2 has an auxiliary subdomain in Domain I which is unique. It
was determined that the auxiliary subdomain is not needed for the formation of the active site but
is needed to maintain the stability of Abz2. The comparison of Abz2 also showed that part of the
dimeric structure of E.coli PabC was missing from the structure of Abz2. The PLP cofactor is

covalently bonded to Lys-251 in the large cleft between the two domains by a Schiff base
linkage. In addition, PLP is also held in place by eight hydrogen bonds from surrounding
residues as well as four water molecules.
The researchers attempted to determine the crystal structure of the Abz2-product complex
but crystallization failed. A computer program was then used to build a model of Abz2 with
cofactor PLP and substrate ADC. This docking model was then tested with a series of sitedirected mutagenesis in combination with activity assays. These tests revealed two residues, Arg182 and Arg-255, which played a crucial role in enzymatic activity. When either one of these was
mutated, the result was zero enzyme activity, indicating their importance in substrate binding.
From their docking model and overall knowledge of the Abz2 structure, the researchers then
propose a catalytic pathway for Abz2.
PLP is covalently bonded to Lys-251 as well as hydrogen bonded to Arg-255. ADC
attaches to PLP, causing it to disassociate from Lys-251 and creating an external aldimine. The
next two intermediates are rearranged to create a quinonoid. The creation of a quinonoid releases
a pyruvate and forms the ketamine. PABA is then released from the ketamine allowing the
regeneration of PLP to covalently bond back to Lys-251. (Throughout the pathway the hydrogen
bond between PLP and Arg-255 remains) Comparing the structure and catalytic pathway of Abz2
to other PabC enzymes shows that it can be placed in a unique class because it is monomeric
rather than dimeric and it uses arginine rather than tyrosine to fix PLP.
Analysis Overall I feel the research was well done and proper parameters were used
throughout. It was interesting to discover that when they tried to obtain crystals of the Abz2product complex their methods failed. They did not elaborate as to the cause of their failure and
it would be interesting if they at least provided one conjecture as to why they could not obtain
these crystals. They then used the HADDOCK program to create a model and it would be

interesting to know why they chose this program. I assume that it is an internationally recognized
program, and thus meets certain criteria, but if there are other programs out there, then why did
they chose this one? In the end it must be exciting for biochemists to be the first to classify a new
type of enzyme and to know they are contributing to the overall discipline by illuminating new
distinctions. I am curious to know the researchers motives though and why they deliberately
choose to study yeast cells. It would be nice if they would give a short explanation about why
they are invested in this area of research.