Biology subjects

Katsuyama, Y.

Publications and source records attributed to Katsuyama, Y..

3 recordsLinked to original sources

bioRxiv2022

Engineering the substrate specificity of toluene degrading enzyme XylM using biosensor XylS and machine learning

Enzyme engineering using machine learning has been developed in recent years. However, to obtain a large amount of data on enzyme activities for training data, it is necessary to develop a high-throughput and accurate method for evaluating enzyme activities. Here, we examined whether a biosensor-based enzyme engineering method can be applied to machine learning. As a model experiment, we aimed to modify the substrate specificity of XylM, a rate-determining enzyme in a multistep oxidation reaction catalyzed by XylMABC in Pseudomonas putida. XylMABC naturally converts toluene and xylene to benzoic acid and toluic acid, respectively. We aimed to engineer XylM to improve its conversion efficiency to a non-native substrate, 2,6-xylenol. Wild-type XylMABC slightly converted 2,6-xylenol to 3-methylsalicylic acid, which is the ligand of the transcriptional regulator XylS in P. putida. By locating a fluorescent protein gene under the control of the Pm promoter to which XylS binds, a XylS-producing Escherichia coli strain showed higher fluorescence intensity in a 3-methylsalicylic acid concentration-dependent manner. We evaluated the 3-methylsalicylic acid productivity of XylM variants using the fluorescence intensity of the sensor strain as an indicator. The obtained data provided the training data for machine learning for the directed evolution of XylM. Two cycles of machine learning-assisted directed evolution resulted in the acquisition of XylM-D140E-V144K-F243L-N244S with 15 times higher productivity than wild-type XylM. These results demonstrate that an indirect enzyme activity evaluation method using biosensors is sufficiently quantitative and high-throughput to be used as training data for machine learning. The findings expand the versatility of machine learning in enzyme engineering.

bioengineering↗

bioRxiv2022

Mechanism-based crosslinking probes capture E. coli ketosynthase FabB in conformationally-distinct catalytic states.

Ketosynthases (KS) catalyse essential carbon-carbon bond forming reactions in fatty acid biosynthesis using a two-step, ping-pong reaction mechanism. In E. coli, there are two homodimeric elongating KSs, FabB and FabF, both of which possess overlapping substrate selectivity. However, FabB is essential for the biosynthesis of unsaturated fatty acids (UFAs) required for cell survival in the absence of exogenous UFAs. Additionally, FabB has reduced activity towards substrates longer than 14 carbons, whereas FabF efficiently catalyses the elongation of saturated C14 and unsaturated C16:1 acyl-acyl carrier protein (ACP) complexes. In this study, we solved two crosslinked crystal structures of FabB in complex with ACPs functionalized with long-chain fatty acid crosslinking probes that approximate catalytic steps. Both homodimeric structures possess asymmetric substrate binding pockets, suggestive of cooperative relationships between the two FabB monomers when engaged with C14 and C16 acyl chains. In addition, these structures capture an unusual rotamer of the active site gating residue, F392, potentially representative of the catalytic state prior to substrate release. These structures demonstrate the utility of mechanism-based crosslinking methods to capture and elucidate at near atomic resolution conformational transitions accompanying KS-mediated catalysis. SynopsisCrystal structures of KS-ACP crosslinked complex elucidate chain length preference and substrate processing mechanism of E. coli FabB.

biochemistry↗

bioRxiv2020

Reelin haploinsufficiency affects skilled motor performance associated with suppression of training-induced gene enrichment, synaptic function and activity-dependent cortical plasticity in mice.

RELN (Reelin) is one of the genes implicated in neurodevelopmental psychiatric vulnerability. Patients with neurodevelopmental disorders can experience impairments in fine motor skills. While Reelin modulates synaptic function, whether Reelin haploinsufficiency affects activity-dependent cortical plasticity which supports development of skilled movement is unclear. Here, heterozygous Reeler mutant (HRM) and Dab1 floxed/ +; Emx1-Cre mice both displayed learning improvements measured by the reach-to-grasp task, but their performance levels of the forelimb motor skill were lower, compared with controls. The level of skilled motor performance was correlated with the area of cortical representations of the trained forelimb, examined after 10 days of training. Furthermore, we hypothesized that the genetic haploinsufficiency also alters changes that occur during the early phase of the training. Examined on day 3, the training induced synaptic modifications of the layer III cortical neurons in (wild-type) WT mice, which were contributed by synaptic potentiation and increase in spontaneous action-potential driven glutamatergic-transmission. On the other hand, the basal excitatory and inhibitory synaptic function were depressed, affected both by presynaptic and postsynaptic synaptic impairments in naive HRM; and thus, no further training-induced synaptic plasticity occurred in HRM. Lastly, examined after 3 days of training, the gene enrichment observed in trained WT mice was absent in trained HRM mice. The finding suggests the Reelin haploinsufficiency alters the skilled motor function; and we propose the suppression of gene enrichment, and synaptic abnormality led by the genetic insufficiency may contribute to impede the occurrence of activity-dependent cortical plasticity. Significance StatementImpairments in fine motor skills occur in subjects with neurodevelopmental disorders. We report a mutation relevant to the neurodevelopmental disorders can impact the cortical plasticity associated with skilled motor function. In wild-type mice, the motor training induced extensive activity-dependent cortical map plasticity, synaptic modifications through synaptic potentiation and excitatory-transmission increase, as well as enrichments in certain gene expressions. On the other hand, mice with Reelin haploinsufficiency (presumed mouse model of neurodevelopmental disorders) exhibited lower level of skilled motor performance, and the underlying correlates shown in wild-type mice were found suppressed. We conclude the suppression of gene enrichment, and synaptic abnormality due to Reelin haploinsufficiency may underlie the limited development of activity-dependent cortical plasticity, contributing to impairments in motor skills.

neuroscience↗