The X-Ray Structure of the Haloalcohol Dehalogenase HheA from
            <i>Arthrobacter</i>
            sp. Strain AD2: Insight into Enantioselectivity and Halide Binding in the Haloalcohol Dehalogenase Family

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Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) shows great potential in producing valuable chiral epoxides and ␤-substituted alcohols. The wild-type (WT) enzyme displays a high R-enantiopreference toward most aromatic substrates, whereas no S-selective HheC has been reported to date. To obtain more enantioselective enzymes, seven noncatalytic active-site residues were subjected to iterative saturation mutagenesis (ISM). After two rounds of screening aspects of both activity and enantioselectivity (E), three outstanding mutants (Thr134Val/Leu142Met, Leu142Phe/Asn176His, and Pro84Val/ Phe86Pro/Thr134Ala/Asn176Ala mutants) with divergent enantioselectivity were obtained. The two double mutants displayed approximately 2-fold improvement in R-enantioselectivity toward 2-chloro-1-phenylethanol (2-CPE) without a significant loss of enzyme activity compared with the WT enzyme. Strikingly, the Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutant showed an inverted enantioselectivity (from an E R of 65 [WT] to an E S of 101) and approximately 100-fold-enhanced catalytic efficiency toward (S)-2-CPE. Molecular dynamic simulation and docking analysis revealed that the phenyl side chain of (S)-2-CPE bound at a different location than that of its R-counterpart; those mutations generated extra connections for the binding of the favored enantiomer, while the eliminated connections reduced binding of the nonfavored enantiomer, all of which could contribute to the observed inverted enantiopreference.T he application of biocatalysts in the production of optically pure compounds has attracted much attention during the past few decades. Enantioselectivity (E) is one of the key parameters that define the usefulness of enzymes in the corresponding industrial synthesis of fine chemicals. Thus, identification of enzymes with high enantioselectivity for a desired transformation is important. In nature, biocatalysts with high enantioselectivity for specific industrial applications are not readily available. Directed evolution has become a routine approach for developing such novel biocatalysts, and a plethora of successful examples have been reported (1-6). A recent survey regarding the location of mutations that improve enzyme properties shows that to manipulate the enantioselectivity of enzymes, closer mutations are more effective than distant mutations (7). With the availability of structural information, the methodology of CASTing (combinatorial activesite saturation test) used in an iterative manner has been particularly successful in tailoring enzyme enantioselectivity (8-11).Microbial halohydrin dehalogenases attract a great deal of attention for their ability to produce optically pure epoxides and halohydrins (12, 13). The enzymes which are involved in the biodegradation pathway of halopropanols catalyze the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins, producing epoxide and HCl. Halohydrin dehalogenase HheC has been studied extensively because of its high enzymatic act...

Section: Guo Et Alsupporting

confidence: 51%

Exploring the Enantioselective Mechanism of Halohydrin Dehalogenase from Agrobacterium radiobacter AD1 by Iterative Saturation Mutagenesis

Guo

Chen

Zheng

et al. 2015

Self Cite

“…AD2 (HheA) was resolved (PDB code 1ZMO). The enzyme has 34% amino acid sequence similarity to and shares a similar quaternary and tertiary structure with HheC, but with a much more open substrate-binding pocket (2). This correlates well with the fact that HheA prefers C4-C5 halohydrins as substrates, whereas HheC prefers short-chain substrates (C2-C3).…”

supporting

confidence: 49%

Key Residues for Controlling Enantioselectivity of Halohydrin Dehalogenase from Arthrobacter sp. Strain AD2, Revealed by Structure-Guided Directed Evolution

Tang

Zhu

Zheng

et al. 2012

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bHalohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) is a valuable tool in the preparation of R enantiomers of epoxides and ␤-substituted alcohols. In contrast, the halohydrin dehalogenase from Arthrobacter sp. AD2 (HheA) shows a low S enantioselectivity toward most aromatic substrates. Here, three amino acids (V136, L141, and N178) located in the two neighboring active-site loops of HheA were proposed to be the key residues for controlling enantioselectivity. They were subjected to saturation mutagenesis aimed at evolving an S-selective enzyme. This led to the selection of two outstanding mutants (the V136Y/L141G and N178A mutants). The double mutant displayed an inverted enantioselectivity (from S enantioselectivity [E S ] ‫؍‬ 1.7 to R enantioselectivity [E R ] ‫؍‬ 13) toward 2-chloro-1-phenylethanol without compromising enzyme activity. Strikingly, the N178A mutant showed a large enantioselectivity improvement (E S > 200) and a 5-to 6-fold-enhanced specific activity toward (S)-2-chloro-1-phenylethanol. Further analysis revealed that those mutations produced some interference for the binding of nonfavored enantiomers which could account for the observed enantioselectivities. Our work demonstrated that those three active-site residues are indeed crucial in modulating the enantioselectivity of HheA and that a semirational design strategy has great potential for rapid creation of novel industrial biocatalysts.

“…Specifically, the large residue F12 in both HheA2 and HheC is essential for the formation of the HHDH anion binding pocket and replaces a central small Gly or Ala in the T-G-X 3 -(G/A)-X-G nucleotide binding motif of classical SDR enzymes (24)(25)(26). In the first ClustalW alignments of all known HHDHs (21), residue R7 of both known B-type enzymes aligned with residue F12 of the other HHDHs.…”

Section: Resultsmentioning

confidence: 99%

“…The concerted activity of Ser-Tyr-Lys in classical SDR enzymes abstracts a proton from the substrate's hydroxyl group, and an enzyme-bound NAD(P) ϩ cofactor is responsible for hydride abstraction. In contrast, HHDHs possess a catalytic triad composed of Ser-Tyr-Arg, and instead of a cofactor-binding site, a spacious anion binding pocket is present in the structures of HheA2 and HheC (25,26). As a consequence, all known HHDH sequences form only a minute but well-defined fraction within the SDR superfamily, which comprises more than 163,000 SDR enzymes that can be retrieved from UniProt (27).…”

mentioning

confidence: 99%

“…For example, all known HHDHs make use of a catalytic triad and share the commonly found homomultimeric quaternary assembly, and both crystallized HHDHs, namely, HheA2 (25) and HheC (26), possess a tertiary structure similar to those of other Rossmann fold proteins. Aside from these overall similarities to SDR enzymes, HHDHs can be distinguished from them by a combination of mechanistic and sequence/structure characteristics.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Expanding the Halohydrin Dehalogenase Enzyme Family: Identification of Novel Enzymes by Database Mining

Schallmey

Koopmeiners

Wells

et al. 2014

bHalohydrin dehalogenases are very rare enzymes that are naturally involved in the mineralization of halogenated xenobiotics. Due to their catalytic potential and promiscuity, many biocatalytic reactions have been described that have led to several interesting and industrially important applications. Nevertheless, only a few of these enzymes have been made available through recombinant techniques; hence, it is of general interest to expand the repertoire of these enzymes so as to enable novel biocatalytic applications. After the identification of specific sequence motifs, 37 novel enzyme sequences were readily identified in public sequence databases. All enzymes that could be heterologously expressed also catalyzed typical halohydrin dehalogenase reactions. Phylogenetic inference for enzymes of the halohydrin dehalogenase enzyme family confirmed that all enzymes form a distinct monophyletic clade within the short-chain dehydrogenase/reductase superfamily. In addition, the majority of novel enzymes are substantially different from previously known phylogenetic subtypes. Consequently, four additional phylogenetic subtypes were defined, greatly expanding the halohydrin dehalogenase enzyme family. We show that the enormous wealth of environmental and genome sequences present in public databases can be tapped for in silico identification of very rare but biotechnologically important biocatalysts. Our findings help to readily identify halohydrin dehalogenases in ever-growing sequence databases and, as a consequence, make even more members of this interesting enzyme family available to the scientific and industrial community.