Facilitated Substrate Channeling in a Self‐Assembled Trifunctional Enzyme Complex

Abstract

Most cascade enzymes in metabolic pathways are spatially held together by noncovalent protein–protein interactions. The formation of a cascade enzyme complex often allows the product of one enzyme to be transferred to an adjacent enzyme where it acts as the substrate, thereby resulting in an enhanced reaction rate, because reaching equilibrium in the cytoplasm is not required; this mechanism is called substrate channeling. In nature, most intracellular enzyme complexes are dynamic so that they may be dissociated or associated, thereby resulting in forestallment of substrate competition among different pathways, regulation of metabolic fluxes, mitigation of metabolite inhibition, and circumvention of unfavorable equilibrium and kinetics. The simplest way to facilitate substrate channeling between cascade enzymes is the construction of fusion proteins, but substrate channeling in fusion proteins might not take place. The assembly of numerous enzymes and/or co-enzymes in vitro is called cascade enzyme biocatalysis and has been proposed for the implementation of complicated bioconversion that microbes and chemical catalysts cannot do, such as hydrogen production from cellulosic materials and water with high yield. Inspired by natural enzyme complexes (e.g., metabolons, which are complexes of sequential enzymes of a metabolic pathway), the construction of static rather than dynamic enzyme complexes could be an important approach to accelerating reaction rates among cascade enzymes and to avoiding the regulation of enzyme–enzyme interactions. For example, Wilner et al. linked glucose oxidase and horseradish peroxidase by DNA scaffolds of different lengths, resulting in reaction rates that were enhanced by 20–30-fold. However, DNA scaffolds may be too costly for scale-up as compared to protein scaffolds. Minteer and co-workers demonstrated that chemical cross-linking of proteins within the mitochondria of Saccharomyces cerevisiae resulted in significant increases of the power output in enzymatic fuel cells. But chemical covalent linking often impairs enzyme activity so that it may not be applied to most intracellular enzymes. Herein we demonstrate a general approach for constructing a static self-assembled enzyme complex by using the highaffinity interaction between cohesin and dockerin modules, which occur in natural extracellular complexed cellulase systems, called cellulosomes. Cohesin domains are part of the natural scaffoldin protein of the cellulosome, which is crucial to the construction of the cellulase complex by binding to enzymes carrying dockerin domains. Bayer et al. proposed to construct designed enzyme complexes by utilizing speciesspecific dockerins and cohesins, which can bind tightly in these complexes at a molar ratio of 1:1. Later, several synthetic mini-cellulosomes containing various extracellular glycoside hydrolases were constructed. However, no one attempted to construct an enzyme complex containing cascade enzymes from a metabolic pathway by using dockerins and cohesins and investigated its potential applications in cascade enzyme biocatalysis. Triosephosphate isomerase (TIM, EC 5.3.1.1), aldolase (ALD, EC 4.1.2.13), and fructose 1,6-bisphosphatase (FBP, EC3.1.3.11) are cascade enzymes in the glycolysis and gluconeogenesis pathways. TIM catalyzes the reversible conversion of glycer-aldehyde-3-phosphate (G3P) to dihydroxy-acetone phosphate (DHAP). ALD catalyzes the reversible aldol condensation of G3P and DHAP to fructose-1,6bisphosphate (F16P). FBP catalyzes the irreversible conversion of F16P to fructose-6-phosphate (F6P; Scheme 1). Previous studies reported that substrate channeling existed in dynamic metabolons of enzymes such as TIM, ALD, or FBP. Three dockerin-free proteins: Thermus thermophilus HB27 TIM (TTC0581) as well as the Thermotoga maritima ALD (TM0273) and FBP (TM1415) were expressed in E. coli and purified to homogeneity by using nickel–nitrilotriacetate (Ni–NTA) resin or a self-cleaving intein. However, a mixture of these three enzymes did not form a putative enzyme complex, as examined by affinity electrophoresis (data not shown). The synthetic static three-enzyme complex was assembled in vitro through a synthetic trifunctional scaffoldin containing a family 3 cellulose-binding module (CBM3) at the N terminus followed by three different types of cohesins from the Clostridium thermocellum ATCC 27405 CipA, Clostridium cellulovorans ATCC 35296 CbpA, and Ruminococcus flavefaciens ScaB (cohesins CTCoh, CCCoh, and RFCoh, [*] Dr. C. You, S. Myung, Y.-H. P. Zhang Biological Systems Engineering Department Virginia Tech, 304 Seitz Hall Blacksburg, VA 24061 (USA) E-mail: ypzhang@vt.edu Homepage: http://www.sugarcar.com