Team:SCUT-China/Modeling/Overview

We devote to dividing the polyketide synthases (PKSs) and catalyze according to specific mechanism. Therefore, as if we standardize the genes which encode independent domains of PKSs, we can synthesize new kinds of polyketides by means of permutation and combination of domains.

In the process of analyzing the DEBS1 which catalyze and synthesize 6-deoxyerythronolide B (6dEB)，the precursor of erythromycin, we know that each domain has independent and specific chemical reaction and function.

By preliminary analysis of chemical reaction and function of each domain, we can conclude that it is available to analyze their own kinetic mechanism, and it's significant. If we simulate the kinetic equations of chemical reactions occurring in each domain, we can finally establish kinetic model of PKSs related to all kinds of polyketides.

Aimed at the final goal of our project, we will simulate the kinetic equations of chemical reactions occurring in each domain, including AT, ACP, KS, KR, ER, DH, and TE. Then we will test these equations and analyze their feasibility.

DEBS 1 is the first part of the PKS which catalyzes and synthesizes 6dEB, the precursor of erythromycin. Its domain sequence is AT, ACP, KS, KR, ACP, KS, AT, KR, ACP. Theoretically, the kinetic model of domains which have been stacked is match with the kinetic model of DEBS 1+TE.

In modeling section, we keep pace with the members who are responsible for experiment. We stack the related equations according to the sequence of DEBS 1+TE. Meanwhile, we also simulate the kinetic equations of the whole DEBS 1+TE. Then we will compare the equations of domains which have been stacked with the equation of DEBS 1+TE, and verify the feasibility of the model.

Introduction

(1) Kinetics of single substrate enzyme catalyzed reactions

The model for enzyme action, first suggested by Brown and Henri but later established more thoroughly Michaelis and Menten, suggests the binding of free enzyme to the substrate forming a enzyme-substrate(ES) complex. This complex undergoes a transformation, releasing product and free enzyme. The free enzyme is then available for another round of binding to new substrate.

In the single substrate enzyme catalyzed reactions, the mechanism is often written as:

And the Michaelis-Menten equation is:

(2) Kinetics of multi substrate enzyme catalyzed reactions

We apply two mechanisms of kinetics of multi substrate enzyme catalyzed reactions to our modeling section, one is Theorell-Chance bi-bi (T-C bi-bi), the other is Ping-pong bi-bi.

Sequential bi-bi reaction means that all substrates must bind to the enzyme before any reaction takes place. Now we take two substrates as an example. In sequential bi-bi reaction, a ternary complex is formed when both substrates bind to the enzyme. But the complex is not steady and two products release after new covalent bonds are formed and old covalent bonds are broken in the complex.

Sequential bi-bi reaction can be random or ordered. The random sequential bi-bi means that any substrate can bind first to the enzyme and any product can release first. The ordered sequential bi-bi means that the substrates bind to the enzyme and the products release in a specific order.

T-C bi-bi reaction is a kind of the ordered sequential bi-bi reactions. Now we take two substrates as an example again. The characteristic of T-C bi-bi is that two products can release in a flash because of the extreme instability of the ternary complex. The allosteric process is not obvious.

In T-C bi-bi reactions, the mechanism is often written as:

And the equation is:

Ping-pong bi-bi reaction is a double-displacement reaction. Substrate A binds to the enzyme followed by product P release. Typically, product P is a fragment of the original substrate A. The rest of the substrate is covalently attached to the enzyme E, which we now designate as F. The substrate B binds and reacts with enzyme. The substrate B changes to a covalent adduct with the covalent fragment of A in the enzyme F to form product Q. The enzyme is finally restored to its initial form E.

In Ping-pong bi-bi reactions, the mechanism is often written as:

And the equation is:

Reactions

Although each domain has its independent function, some reactions are catalyzed by several domains together in the process of polyketide's synthesis, such as activation of propionyl groups, combination of the extended unit and polyketide chain and so on. Therefore, in analysis of reactions of their mechanism, the introduction will be started from the perspective of the equations of chemical reactions.

(1)Acyl carrier protein, ACP

ACP domain is a carrier. Its function is anchoring the polyketide chains needed to extend. The synthesis of polyketides is engaged on these carriers. The polyketide chains are activated by AT domain, and then are anchored on ACP domains. Until the catalysis of TE domain, polyketide chains are released from ACP domain and become ripe polyketides.

(2)Loading Part

The leading role of the loading part is Acyltransfer-ase, AT domain. In this part, we will introduce the function of AT domain by analyzing the chemical reactions between AT domain, ACP domain and polyketide chains.

The function of AT domain is activating the extended units, propionyl groups which will be combined with the polyketide chains.

In Loading, Proplonyl-CoA reacts with the oxhydryl on the pecific binding sites of AT domains and binds to AT domain, and then CoA-SHs are displaced. At this time, proplonyl groups have been activated. They are transferred onto the ACP domains in Loading and anchored there.

In Module 1 or Module 2, AT domains anchor the proplonyl groups of (2S)Methylmalonyl-CoA to the ACP domains in Module 1 or Module 2 by the same mechanism. The proplonyl groups anchored wait for the catalysis of KS domain in the next step.

The concrete equation of chemical reaction is :

After the analysis of equation above, we conclude that this enzyme catalyzed reaction is match with Ping-pong bi-bi mechanism. The graph of mechanism is written as:

(3)KS Part

The leading role of the KS part is Ketocaylsynthase, KS domain. Its last step is activation of propionyl groups. AT domains in Module 1 or Module 2 activate the propionyl groups in (2S)Methylmalonyl-CoA (details in Loading Part). Afterwards, KS domains combine the extended units anchored on the ACP domains belonging to the Modules which the KS domains belong to with the polyketide chains anchored on the ACP domains belonging to the former Modules (or Loadings). Then new polyketide chains form and are anchored the ACP domains belonging to the Modules which the KS domains belong to.

The function of KS domain is combining the propionyl group with acetyl group at the end of the polyketide chain by forming the C—C bond. In KS domain catalyzed reactions, polyketide chains are transferred from ACP doamains belonging to the former Module (or Loading) onto KS domains, and then occur decarboxylic reaction with the extended units anchored on the ACP domains belonging to the Modules which the KS domains belong to. As a result, the polyketide chains are elongated.

The concrete equation of chemical reaction is :

After the analysis of equation above, we conclude that this enzyme catalyzed reaction is match with Ping-pong bi-bi mechanism. The graph of mechanism is written as:

(4)KR Part

The process of KR domain catalyzed reactions is a little more complex. Its leading role is KR domain. The function of KR domain is deoxidizing the extended unit and enabling it to form the β-hydroxyl ester bond. In KR domain catalyzed reactions, the carbonyl groups of polyketide chains anchored on the ACP domains are combined with the specific binding sites of KR domains through weak chemical bonds. Then the electron cloud in the carbonyl groups is moving towards oxygen atoms so that the carbon atoms have net positive charge. At this time, NADPHs have a chance to bind to the polyketide chains and form instable ternary complexes. In order to reduce the inner energy of this ternary complexes and run towards stable, NADPHs provide hydride ions and KR domains provide protons to the complexes and convert the carbonyl groups to theβ-hydroxyl esters. Finally, the KR domain residues which have lost protons are deoxidized to the initial KR domains by other NADPHs.

The concrete equation of chemical reaction is :

Because KR domains catalyzed reactions are involved several steps, it is impossible for them to be match with any simple mechanism of enzyme catalyzed reactions. After the analysis of equation above, we define that these enzyme catalyzed reactions may be match with [T-C bi-bi + Ping-pong bi-bi] mechanism. In other words, KR domains catalyzed reactions may belong to Ping-pong bi-bi mechanism which include T-C bi-bi mechanism in its former half part.

The graph of mechanism is written as:

(5)ER Part

The leading role of ER Part is ER domain. The function of ER domain is deoxidizing the extended unit and enabling it to form the saturated methylene. The mechanism of ER domain catalyzed reactions is similar to KR domain. In ER domain catalyzed reactions, NADPHs provide hydride ions and ER domains provide protons to the complexes and deoxidize carbon-carbon double bond to form the saturated methylene.

The concrete equation of chemical reaction is :

After the analysis of equation above, we conclude that the mechanism of this enzyme catalyzed reaction is similar to KR domain.

The graph of mechanism is written as:

(6)DH Part

The leading role of DH Part is DH domain. The function of DH domain is dehydrating the extended unit and enabling it to form the α, β- enol ester bond. In DH domain catalyzed reactions, the oxhydryls in the extended units and the hydrogen atoms binding to the neighbor carbon atoms are removed by DH domains together and form . After catalysis, α, β-enol ester bonds are formed in the polyketide chains.

The concrete equation of chemical reaction is :

After the analysis of equation above, we conclude that this enzyme catalyzed reaction is match with the mechanism of single substrate enzyme catalyzed reactions.

The graph of mechanism is written as:

(7)TE Part

The leading role of TE Part is TE domain. The function of TE domain is removing the polyketide chain from PKS. In TE domain catalyzed reactions, polyketide chains anchored on the ACP domains react with the oxhydryl on the pecific binding sites of TE domains. Then the polyketide chains are transferred from ACP domains to TE domains. However, polyketide chains are instable when binding to TE domains and they release from TE domains, that is, release from the whole PKS. Finally, they become ripe polyketides.

The concrete equation of chemical reaction is :

After the analysis of equation above, we conclude that this enzyme catalyzed reaction is similar to the single substrate enzyme catalyzed reactions. We confirm that we can apply the mechanism of single substrate enzyme catalyzed reactions to TE domain catalyzed reactions by using King-Altman Method.

The graph of mechanism is written as: