Phylogenetics-based identification and characterization of a superior 2,3-butanediol dehydrogenase for Zymomonas mobilis expression

Abstract Background Zymomonas mobilis has recently been shown to be capable of producing the valuable platform biochemical, 2,3-butanediol (2,3-BDO). Despite this capability, the production of high titers of 2,3-BDO is restricted by several physiological parameters. One such bottleneck involves the conversion of acetoin to 2,3-BDO, a step catalyzed by 2,3-butanediol dehydrogenase (Bdh). Several Bdh enzymes have been successfully expressed in Z. mobilis, although a highly active enzyme is yet to be identified for expression in this host. Here, we report the application of a phylogenetic approach to identify and characterize a superior Bdh, followed by validation of its structural attributes using a mutagenesis approach. Results Of the 11 distinct bdh genes that were expressed in Z. mobilis, crude extracts expressing Serratia marcescens Bdh (SmBdh) were found to have the highest activity (8.89 µmol/min/mg), when compared to other Bdh enzymes (0.34–2.87 µmol/min/mg). The SmBdh crystal structure was determined through crystallization with cofactor (NAD+) and substrate (acetoin) molecules bound in the active site. Active SmBdh was shown to be a tetramer with the active site populated by a Gln247 residue contributed by the diagonally opposite subunit. SmBdh showed a more extensive supporting hydrogen-bond network in comparison to the other well-studied Bdh enzymes, which enables improved substrate positioning and substrate specificity. This protein also contains a short α6 helix, which provides more efficient entry and exit of molecules from the active site, thereby contributing to enhanced substrate turnover. Extending the α6 helix to mimic the lower activity Enterobacter cloacae (EcBdh) enzyme resulted in reduction of SmBdh function to nearly 3% of the total activity. In great contrast, reduction of the corresponding α6 helix of the EcBdh to mimic the SmBdh structure resulted in ~ 70% increase in its activity. Conclusions This study has demonstrated that SmBdh is superior to other Bdhs for expression in Z. mobilis for 2,3-BDO production. SmBdh possesses unique structural features that confer biochemical advantage to this protein. While coordinated active site formation is a unique structural characteristic of this tetrameric complex, the smaller α6 helix and extended hydrogen network contribute towards improved activity and substrate promiscuity of the enzyme.

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Role of Citric Acid in Preparing Highly Active CoMo/Al2O3 Catalyst: From Aqueous Impregnation Solution to Active Site Formation

Industrial & Engineering Chemistry Research ◽

10.1021/acs.iecr.7b02877 ◽

2017 ◽

Vol 56 (48) ◽

pp. 14172-14181 ◽

Cited By ~ 14

Author(s):

Jianjun Chen ◽

Jinxing Mi ◽

Kezhi Li ◽

Xiqin Wang ◽

Elizabeth Dominguez Garcia ◽

...

Keyword(s):

Citric Acid ◽

Active Site ◽

Site Formation ◽

Highly Active ◽

Al2o3 Catalyst ◽

Impregnation Solution

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Functional Roles of Streptokinase C-Terminal Flexible Peptide in Active Site Formation and Substrate Recognition in Plasminogen Activation†

Biochemistry ◽

10.1021/bi026746m ◽

2003 ◽

Vol 42 (1) ◽

pp. 114-120 ◽

Cited By ~ 17

Author(s):

Peng Zhai ◽

Nancy Wakeham ◽

Jeffrey A. Loy ◽

Xuejun C. Zhang

Keyword(s):

Active Site ◽

Substrate Recognition ◽

Site Formation ◽

Plasminogen Activation ◽

Functional Roles

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Structural insights into dihydroxylation of terephthalate, a product of polyethylene terephthalate degradation

Journal of Bacteriology ◽

10.1128/jb.00543-21 ◽

2022 ◽

Author(s):

Jai Krishna Mahto ◽

Neetu Neetu ◽

Monica Sharma ◽

Monika Dubey ◽

Bhanu Prakash Vellanki ◽

...

Keyword(s):

Crystal Structure ◽

Substrate Specificity ◽

Polyethylene Terephthalate ◽

Active Site ◽

Catalytic Domain ◽

Structural Features ◽

Comamonas Testosteroni ◽

Plastic Pollution ◽

Microbial Utilization ◽

Steady State Kinetics

Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this Polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis -diol is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDO KF1 ). The TPDO KF1 exhibited the substrate specificity for TPA ( k cat / K m = 57 ± 9 mM −1 s −1 ). The TPDO KF1 structure harbors characteristics RO features as well as a unique catalytic domain that rationalizes the enzyme’s function. The docking and mutagenesis studies reveal that its substrate specificity to TPA is mediated by Arg309 and Arg390 residues, two residues positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, its substitution to alanine decreases the activity ( k cat ) by 80%. Together, this study delineates the structural features that dictate the substrate recognition and specificity of TPDO. Importance The global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential in tackling this. Microbial utilization of this released product, TPA is an emerging and promising strategy for waste-to-value creation. Research from the last decade has discovered terephthalate dioxygenase (TPDO), as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we have determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

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Recent insights into lytic polysaccharide monooxygenases (LPMOs)

Biochemical Society Transactions ◽

10.1042/bst20170549 ◽

2018 ◽

Vol 46 (6) ◽

pp. 1431-1447 ◽

Cited By ~ 39

Author(s):

Tobias Tandrup ◽

Kristian E. H. Frandsen ◽

Katja S. Johansen ◽

Jean-Guy Berrin ◽

Leila Lo Leggio

Keyword(s):

Active Site ◽

Room Temperature ◽

Experimental Studies ◽

Binding Mode ◽

Structural Features ◽

Oxygen Species ◽

Animal Kingdom ◽

X Ray ◽

Lytic Polysaccharide Monooxygenases ◽

Polysaccharide Monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are copper enzymes discovered within the last 10 years. By degrading recalcitrant substrates oxidatively, these enzymes are major contributors to the recycling of carbon in nature and are being used in the biorefinery industry. Recently, two new families of LPMOs have been defined and structurally characterized, AA14 and AA15, sharing many of previously found structural features. However, unlike most LPMOs to date, AA14 degrades xylan in the context of complex substrates, while AA15 is particularly interesting because they expand the presence of LPMOs from the predominantly microbial to the animal kingdom. The first two neutron crystallography structures have been determined, which, together with high-resolution room temperature X-ray structures, have putatively identified oxygen species at or near the active site of LPMOs. Many recent computational and experimental studies have also investigated the mechanism of action and substrate-binding mode of LPMOs. Perhaps, the most significant recent advance is the increasing structural and biochemical evidence, suggesting that LPMOs follow different mechanistic pathways with different substrates, co-substrates and reductants, by behaving as monooxygenases or peroxygenases with molecular oxygen or hydrogen peroxide as a co-substrate, respectively.

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Mechanistic investigation of propylene epoxidation with H2O2 over TS-1: Active site formation, intermediate identification, and oxygen transfer pathway

Molecular Catalysis ◽

10.1016/j.mcat.2017.08.011 ◽

2017 ◽

Vol 441 ◽

pp. 150-167 ◽

Cited By ~ 20

Author(s):

Xiaowa Nie ◽

Xiaojing Ji ◽

Yonggang Chen ◽

Xinwen Guo ◽

Chunshan Song

Keyword(s):

Active Site ◽

Oxygen Transfer ◽

Site Formation ◽

Propylene Epoxidation ◽

Mechanistic Investigation

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Structure Function Studies of the Phenylalanine Hydroxylase Active Site and a Summary of Structural Features

Advances in Experimental Medicine and Biology - Chemistry and Biology of Pteridines and Folates ◽

10.1007/978-1-4615-2960-6_10 ◽

1993 ◽

pp. 55-57 ◽

Cited By ~ 1

Author(s):

R. G. H. Cotton ◽

D. W. Howells ◽

J. A. Saleeba ◽

I. Dianzani ◽

P. M. Smooker ◽

...

Keyword(s):

Structure Function ◽

Active Site ◽

Phenylalanine Hydroxylase ◽

Structural Features

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Mitochondrial ClpX activates an essential biosynthetic enzyme through partial unfolding

eLife ◽

10.7554/elife.54387 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 3

Author(s):

Julia R Kardon ◽

Jamie A Moroco ◽

John R Engen ◽

Tania A Baker

Keyword(s):

Active Site ◽

Autonomous System ◽

Aminolevulinic Acid ◽

Structural Features ◽

Biosynthetic Enzyme ◽

Cofactor Binding ◽

Partial Unfolding ◽

Chaperone Interactions ◽

Aaa Protein ◽

Insight Into

Mitochondria control the activity, quality, and lifetime of their proteins with an autonomous system of chaperones, but the signals that direct substrate-chaperone interactions and outcomes are poorly understood. We previously discovered that the mitochondrial AAA+ protein unfoldase ClpX (mtClpX) activates the initiating enzyme for heme biosynthesis, 5-aminolevulinic acid synthase (ALAS), by promoting cofactor incorporation. Here, we ask how mtClpX accomplishes this activation. Using S. cerevisiae proteins, we identified sequence and structural features within ALAS that position mtClpX and provide it with a grip for acting on ALAS. Observation of ALAS undergoing remodeling by mtClpX revealed that unfolding is limited to a region extending from the mtClpX-binding site to the active site. Unfolding along this path is required for mtClpX to gate cofactor binding to ALAS. This targeted unfolding contrasts with the global unfolding canonically executed by ClpX homologs and provides insight into how substrate-chaperone interactions direct the outcome of remodeling.

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Structural Features of a Bacteroidetes-Affiliated Cellulase Linked with a Polysaccharide Utilization Locus

Scientific Reports ◽

10.1038/srep11666 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 14

Author(s):

A.E. Naas ◽

A.K. MacKenzie ◽

B. Dalhus ◽

V.G.H. Eijsink ◽

P.B. Pope

Keyword(s):

Active Site ◽

Three Dimensional ◽

Structural Features ◽

Structure And Function ◽

Dimensional Structure ◽

Active Site Cleft ◽

Polysaccharide Utilization Locus ◽

And Function ◽

Rumen Metagenome

Abstract Previous gene-centric analysis of a cow rumen metagenome revealed the first potentially cellulolytic polysaccharide utilization locus, of which the main catalytic enzyme (AC2aCel5A) was identified as a glycoside hydrolase (GH) family 5 endo-cellulase. Here we present the 1.8 Å three-dimensional structure of AC2aCel5A and characterization of its enzymatic activities. The enzyme possesses the archetypical (β/α)8-barrel found throughout the GH5 family and contains the two strictly conserved catalytic glutamates located at the C-terminal ends of β-strands 4 and 7. The enzyme is active on insoluble cellulose and acts exclusively on linear β-(1,4)-linked glucans. Co-crystallization of a catalytically inactive mutant with substrate yielded a 2.4 Å structure showing cellotriose bound in the −3 to −1 subsites. Additional electron density was observed between Trp178 and Trp254, two residues that form a hydrophobic “clamp”, potentially interacting with sugars at the +1 and +2 subsites. The enzyme’s active-site cleft was narrower compared to the closest structural relatives, which in contrast to AC2aCel5A, are also active on xylans, mannans and/or xyloglucans. Interestingly, the structure and function of this enzyme seem adapted to less-substituted substrates such as cellulose, presumably due to the insufficient space to accommodate the side-chains of branched glucans in the active-site cleft.

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