Structure-based conversion of coenzyme specificity inpolysaccharide-metabolizing reductases and their applicationin biofuel production

Wataru HASHIMOTO

Graduate School of Agriculture, Kyoto University

Research aims
To build a sustainable society and reduce dependence on fossil fuels such as
petroleum and coal, biofuels are now being investigated as an alternative energy source.
An issue in Japan is that that there is limited land available for biomass production. To
overcome this, the Japanese government encourages the use of marine biomass (e.g.
seaweed) for energy production. Acidic polysaccharides, alginate, and fucoidan, allabundant in brown algae, are likely to be used for biofuel production in the future1). To
highlight the potential of such technology, we have previously demonstrated bacterial
production of bioethanol from alginate2). NADH or NADPH-dependent reductases are
essential for the metabolism of uronate-containing acidic polysaccharides such asalginateandpectin.

These

reductases

areclassified

asshort-chain

dehydrogenase/reductases (SDR) and have a common scaffold (three layer α/β/α).
Cellular coenzyme balance is also important for efficient microbial production ofvaluable products. Since NADPH is ten times more expensive than NADH, adding
NADH dependence to NADPH specific enzymes is a valuable cost saving option.
Although NADH and NADPH are structurally similar, there has been limited success inconverting coenzyme specificity while maintaining high enzyme activity. Our study
therefore aimed to clarify structural determinants for coenzyme specificity in bacterial
reductases using X-ray crystallography. These included compounds crucial for themetabolism of acidic polysaccharides, such as alginate, fucoidan, and pectin.
Understanding these structural determinants will contribute to improvements inbiotechnology by modifying coenzyme specificity in reductases, increasing productivity,and reducing costs in genetically engineered microbes or enzymes.
Methods
Bacterial reductases for the metabolism of acidic polysaccharides were crystallized in
the presence of coenzymes by multiple screening in 96-well plates. Diffraction data
were collected at SPring-8 (Hyogo, Japan) and processed using HKL2000 software.
Molecular replacements for structural determinations were calculated using Molrep,
using coordinates from coenzyme-free enzymes to create the initial models. Structuralrefinement was performed using Refmac5.
Structure-based conversion of coenzyme specificity was performed using proteinengineering.
Results(1) Alginate-metabolizing reductases
Two alginate-metabolizing reductases (NADPH-specific A1-R and NADH-specificA1-R′) are present in the alginate-assimilating Sphingomonas sp. strain A1. The
enzymes are similar in primary structure. The crystal structure of A1-R′ in complex with
NAD+ was determined and compared to NADP+-bound A1-R3). Significant differencesin space (A1-R, 44.0 Å3 and A1-R′, 1.15
Å3) and charge (A1-R, positive andA1-R′, negative) were observed. Theresidues

importantfor

structural

determinants were located in two loops,
one short and one long (Fig. 1).
Coenzyme specificity in both enzymes
was successfully converted through theexchange of the two loops.
Fig. 1. Two loops for coenzyme recognition(2) Fucoidan-metabolizing reductase
Fucoidan contains several glucuronic acid residues and reductases acting on α-ketoacid derived from glucuronic acid. They are required for metabolism ofpolysaccharides4). We determined the crystal structure of the NADH-specific reductase
DhuD from Streptococcus pyogenes (Fig. 2, left), both in and out of complex withcoenzyme. Although no crystal structure in complex could be obtained, DhuD showed ascaffold similar to NADPH-specific A1-R and NADH-specific A1-R′. Comparisons ofcoenzyme-binding sites across DhuD, A1-R, and A1-R′ revealed that DhuD has twoA1-R′-type loops (space, 1.05 Å3; charge, negative) for NADH specificity.
(3) Pectin-metabolizing reductase
Thereductase
KduD
involvedinmetabolizing pectin can utilize both NADH andNADPH. The crystal structure of KduD fromPectobacterium

carotovorum

subsp.
+

carotovorum in complex with NAD

wasdetermined (Fig. 2, right)5). This demonstrated
that KduD features a space of 15.9 Å3 locatedatthecoenzyme(2ʹsiteofadenylate

Fig. 2．Structure of DhuD and KduDribose)-binding site. This space has no charge
at pH 7.0.
Conclusions
The structural determinants (space volume andsurface charge) of coenzyme specificity inbacterial reductases were identified throughanalysis of their structural and functionalrelationships (Fig. 3). These enzymes are central
to the metabolism of acidic polysaccharides.
Understanding these determinants will contribute
totheestablishmentofstructural-based

Fig. 3. Coenzyme-binding site

conversion of coenzyme specificity in reductases
to be used in biotechnology.
References
1) Stokstad, E. (2012) Biofuels. Engineered superbugs boost hopes of turning seaweed
into fuel. Science 335: 273.
2) Takeda, H., Yoneyama, F., Kawai, S., Hashimoto, W., and Murata, K. (2011)
Bioethanol production from marine biomass alginate by metabolically engineeredbacteria. Energy Environ. Sci. 4: 2575-2581.
3) Takase, R., Mikami, B., Kawai, S., Murata, K., and Hashimoto, W. (2014)
Structure-based conversion of the coenzyme requirement of a short-chain
dehydrogenase/reductase involved in bacterial alginate metabolism. J. Biol. Chem.
289: 33198-33214.
4) Maruyama, Y., Oiki, S., Takase, R., Mikami, B., Murata, K., and Hashimoto, W.
(2015)

Metabolicfateofunsaturated

glucuronic/iduronic

acids

from

glycosaminoglycans: Molecular identification and structure determination of
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5) Takase, R., Maruyama, Y., Oiki, S., Mikami, B., Murata, K., and Hashimoto, W.
(2016)

Structural

determinantsinbacterial

2-keto-3-deoxy-D-gluconate

dehydrogenase KduD for dual-coenzyme specificity. Proteins 84: 934-947.