1 INTRODUCTION
Many enzymes in Enzyme Class 3 (Hydrolases) catalyze chemical group
transfer to acceptors (e.g., alcohols) other than water. Such
donor-to-acceptor transfer reactions are the basis for important
applications of hydrolytic enzymes in organic synthesis (Adlercreutz,
2017; Giordano, Ribeiro, & Giordano, 2006; Kasche, 1986; Marsden,
Mestrom, McMillan, & Hanefeld, 2020; Müller et al., in press; Seibel,
Jördening, & Buchholz, 2006; Vera, Guerrero, Aburto, Cordova, &
Illanes, 2020). Commercial-scale production of various chemicals and
ingredients (e.g., oligosaccharides and glycosides (van Rantwijk,
Woudenberg-van Oosterom, & Sheldon, 1999; Vera et al., 2020), esters
and triglycerides (Adlercreutz, 2017; Mestrom, Claessen, & Hanefeld,
2019; Norjannah, Ong, Masjuki, Juan, & Chong, 2016), β-lactam
antibiotics (Giordano et al., 2006; Kasche, 1986), phosphorylated
nucleosides (Asano, Mihara, & Yamada, 1999; Ishikawa et al., 2002;
Kato, Ooi, & Asano, 1999)) relies on hydrolase-catalyzed group
transfer. Glycoside hydrolases (Adlercreutz, 2017; Seibel et al., 2006;
Vera et al., 2020; Zeuner, Jers, Mikkelsen, & Meyer, 2014), lipases
(Adlercreutz, 2017; Subileau et al., 2017), amidases (Bruggink, Roos, &
de Vroom, 1998; Giordano et al., 2006; Gololobov et al., 1990; Sio &
Quax, 2004; Sklyarenko, El’darov, Kurochkina, & Yarotsky, 2015) and
phosphatases (Ishikawa et al., 2002; Tasnádi, Staśko, Ditrich, Hall, &
Faber, 2020; Wildberger, Pfeiffer, Brecker, & Nidetzky, 2015) are
industrial enzymes used in these synthetic processes. Mechanistically,
the enzymatic reactions proceed in two catalytic steps via a covalent
enzyme intermediate (Gololobov, Borisov, Belikov, & Švedas, 1988;
Kasche, 1986; Marsden et al., 2020; Seibel et al., 2006; Zeuner et al.,
2014). The first step involves group transfer from the donor substrate
to an active-site nucleophile of the enzyme. The enzyme intermediate
then reacts with the incoming acceptor (which is water in the canonical
hydrolysis reaction) to release the synthetic product (Gololobov et al.,
1988; Gololobov et al., 1990; Huber, Kurz, & Wallenfels, 1976; Kasche,
1986; Marsden et al., 2020; Vera et al., 2020; Youshko, Chilov,
Shcherbakova, & Švedas, 2002). Synthesis in an aqueous environment
operates in competition with hydrolysis (Adlercreutz, 2017). A minimal
kinetic mechanism for enzymatic group transfer is shown in Figure 1. The
mechanism implies that synthesis and hydrolysis product are formed in a
ratio determined by enzyme selectivity but independent of the donor
substrate used (Adlercreutz, 2017; Kasche, 1986; Marsden et al., 2020;
Youshko et al., 2002).
The industrially used glycosylation of glycerol catalyzed by sucrose
phosphorylase (SucP) (Bolivar, Luley-Goedl, Leitner, Sawangwan, &
Nidetzky, 2017; Goedl, Sawangwan, Mueller, Schwarz, & Nidetzky, 2008;
Luley-Goedl, Sawangwan, Mueller, Schwarz, & Nidetzky, 2010) represents
a number of hydrolase-promoted transfer reactions for which the reaction
scheme shown in Figure 1 is likely an oversimplification (e.g.,
(Fernandez-Lafuente, Rosell, & Guisan, 1998; Gololobov et al., 1988;
Gololobov et al., 1990; Kasche, 1986; Terreni et al., 2005; Vera,
Guerrero, Wilson, & Illanes, 2017; Youshko et al., 2002)). The SucP can
use sucrose (1 ) or G1P (2 ) as donor for
α-D-glucosyl-sn -glycerol (GG; 3 ) formation (Goedl,
Sawangwan, et al., 2008; Goedl, Schwarz, Minani, & Nidetzky, 2007;
Renirie, Pukin, van Lagen, & Franssen, 2010). However, the ratio
between GG and glucose (hydrolysis product) is ∼1.5 – 2.5-fold higher
for transfer from sucrose as compared to transfer from G1P, inconsistent
with Figure 1 (Goedl, Sawangwan, et al., 2008). This study was
conceptualized to achieve clarification. We considered that mechanistic
insight into the donor substrate dependence of the enzymatic glucosyl
transfer is fundamentally important, and can be practically significant,
in a broad field of applied bio-catalysis using hydrolase enzymes for
synthesis (Adlercreutz, 2017; Giordano et al., 2006; Kasche, 1986;
Marsden et al., 2020; van Rantwijk et al., 1999).
We studied two SucP enzymes, from Leuconostoc mesenteroides(Lm SucP) (Goedl et al., 2007; Goedl, Schwarz, Mueller, Brecker,
& Nidetzky, 2008; Luley-Goedl & Nidetzky, 2010) andBifidobacterium adolescentis (Ba SucP) (Mirza et al., 2006;
van den Broek et al., 2004). Both enzymes have been well characterized
previously (Franceus & Desmet, 2020; Goedl, Sawangwan, Wildberger, &
Nidetzky, 2010; Goedl, Schwarz, et al., 2008), and the Lm SucP is
used for commercial production of GG (Luley-Goedl et al., 2010). Guided
by kinetic framework analysis developed in here, we show that in
enzymatic reactions with G1P,
hydrolysis takes place not only from the covalent glucosyl-enzyme
intermediate (E-Glc), but additionally from a noncovalent complex of
E-Glc and G1P which unlike E-Glc cannot be intercepted by glycerol. At
G1P concentrations too low for substrate-bound E-Glc to form, the
product ratio increases to a value consistent with reaction exclusively
through E-Glc, independent of the donor substrate used. Besides
explaining kinetic behavior of SucP not previously accounted for (Goedl,
Sawangwan, et al., 2008; Renirie, Pukin, van Lagen, & Franssen, 2010),
these results are useful to facilitate model-based optimization of GG
synthesis. In addition, our findings expand the theoretical basis for
analyzing kinetically analogous group transfer reactions by hydrolytic
enzymes (e.g. (Adlercreutz, 2017; Ishikawa et al., 2002; Kasche, 1986)).